Broadly neutralizing antibodies against sars-cov-2 and sars-cov variants

Abstract

Disclosed are monoclonal antibodies, antigen-binding fragments, and multispecific antibodies that specifically bind to a coronavirus spike protein (e.g., SARS-CoV-2). Also disclosed are uses of these antibodies and multispecific antibodies for inhibiting a coronavirus infection (e.g., a SARS-CoV-2 infection). In addition, disclosed are methods of using the disclosed antibodies and multispecific antibodies to detect a coronavirus (e.g., SARS-CoV-2) in a biological sample.

Description

Cross-references to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 613,327, filed December 21, 2023, which is incorporated herein by reference in its entirety.

[0002] field This disclosure relates to monoclonal antibodies and antigen-binding fragments that specifically bind to the spike protein of severe acute respiratory syndrome coronavirus (SARS-CoV)-2, and their use in inhibiting SARS-CoV-2 infection and detecting SARS-CoV-2 in biological samples.

[0003] sequence list The sequence list was submitted as an XML file named "Sequence.xml" (204,472 bytes), created on October 30, 2024, and incorporated into this paper by reference.

[0004] background SARS-CoV-2 (the virus that causes COVID-19) continues to infect millions and is a leading cause of death among the elderly. Monoclonal antibodies targeting the SARS-CoV-2 spike protein are available in clinical use, both prophylactically to prevent infection and as a treatment to prevent severe illness. The former is particularly important for immunocompromised individuals, including cancer patients, as vaccines may be ineffective or contraindicated for them. However, SARS-CoV-2 variants emerge frequently and often carry amino acid sequence changes in the spike protein, allowing them to evade these monoclonal antibodies. In fact, as of November 2023, all previously FDA-authorized monoclonal products, including the therapeutic antibodies bamlaminivab and bebtelovimab, and the prophylactic formulation EVUSHELD™ (a mixture of tixagevimab and cilgavimab), are no longer authorized because recent variants cannot be neutralized by these products. Therefore, new monoclonal antibodies with broader cross-reactivity to viral variants are needed for clinical use.

[0005] Overview Isolated monoclonal antibodies or antigen-binding fragments thereof that specifically bind to the coronavirus spike protein are disclosed. These monoclonal antibodies or antigen-binding fragments are capable of neutralizing at least one coronavirus.

[0006] In some embodiments, the antigen or antigen-binding fragment includes a heavy chain variable region (V0). H ) and light chain variable region (V L The heavy chain variable region (V)H ) and light chain variable region (V L V includes any of the following: H and V L Heavy chain complementarity determinants (HCDRs) 1, HCDR 2, and HCDR 3, and light chain complementarity determinants (LCDRs) 1, LCDR 2, and LCDR 3: a) These are SEQ ID NO: 1 and 5 (A18-618-448.1), respectively; b) These are SEQ ID NO: 9 and 13 (F768-104_B2), respectively; c) These are SEQ ID NO: 17 and 21 (E184-105_F3), respectively; d) These are SEQ ID NOs: 25 and 29 (A63-652-32.1), respectively; e) These are SEQ ID NO: 33 and 37 (F768-104_G7), respectively; g) These are SEQ ID NO: 41 and 45 (E184-105_H1), respectively; f) These are SEQ ID NOs: 49 and 53 (F768-104_D10), respectively; h) are SEQ ID NO: 57 and 61 (A63-652-1.4), respectively; i) These are SEQ ID NO: 65 and 69 (E184-105_E5), respectively; j) are SEQ ID NO: 73 and 77 (E184-105_H9), respectively; k) are SEQ ID NO: 81 and 85 (E184-105_H2), respectively; l) are SEQ ID NO: 89 and 93 (A80-102+103_G1), respectively; m) are SEQ ID NO: 97 and 101 (A80-103_E2), respectively; n) are SEQ ID NO: 105 and 109 (A80-103_B6), respectively; o) are SEQ ID NO: 113 and 117 (A18-618-452.1), respectively; p) are SEQ ID NO: 121 and 125 (A80-102+103_C1), respectively; q) are SEQ ID NO: 129 and 133 (A43-d321.10.4), respectively; r) are SEQ ID NO: 137 and 141 (F768-100-pt2_A05), respectively; s) are SEQ ID NO: 145 and 149 (A80-102_G5), respectively; t) are SEQ ID NO: 153 and 157 (A18-338-19.5), respectively; u) are SEQ ID NO: 161 and 165 (A18-338-19.6), respectively; v) are SEQ ID NO: 169 and 173 (A18-338-19.8) respectively; or w) are SEQ ID NO: 177 and 181 (A18-338-19.9), respectively.

[0007] In some implementations, nucleic acid molecules encoding these antibody and antigen-binding fragments, vectors containing these nucleic acid molecules, and host cells containing these vectors are disclosed.

[0008] Pharmaceutical compositions comprising these antibodies, antigen-binding fragments, nucleic acid molecules, and carriers are also disclosed. Further embodiments disclose the use of these pharmaceutical compositions in inhibiting coronavirus infection in subjects.

[0009] In other embodiments, the use of the disclosed antibody and antigen-binding fragments for detecting coronaviruses in biological samples is disclosed.

[0010] The above and other features and advantages of the present invention will become more apparent from the following detailed description of numerous embodiments with reference to the accompanying drawings.

[0011] Brief description of the attached diagram Figure 1A-1D Cryo-electron microscopy (cryo-EM) structure of Fab A18-448.1 combined with EG.5 spikes. Figure 1A Cryo-electron microscopy density analysis of the FabA18-448.1 complex with the EG.5 spike shows antibody binding to RBDs in both RBD-up and RBD-down conformations. The complex between the antibody and the up conformation RBD is shown in the boxed image. Figure 1B . Figure 1B .Fab A18-448.1 targets class IV epitopes on the RBD. The antibody is shown with a light gray surface, and the EG.5 RBD is shown with a black surface. Figure 1CThe epitope of A18-448.1 on the RBD is relatively small, consisting only of residues 404-405 and rings 499-504. The RBD is shown in black, and the epitope is colored gray. Figure 1D A18-448.1 utilizes CDR H3 and CDR L3 to bind to RBD residues 499-504. Omicron mutation hotspots surrounding this region, such as P445, S446, and H505, do not interact with antibody A18-448.1.

[0012] Figures 2A-2D Cryo-electron microscopy structure of Fab F768-104_B2 and XBB.1.5 spike composite. Figure 2A Cryo-electron microscopy density analysis of the .FabF768-104_B2 complex with the XBB.1.5 spike shows that F768-104_B2 binds to the RBD in both RBD-up and RBD-down conformations. Details of the antibody-RBD are shown in the boxed image. Figure 2B . Figure 2B Fab F768-104_B2 targets a class III epitope on the XBB.1.5 RBD. The RBD is rendered with a gray surface, and specific XBB.1.5 mutations (relative to the original strain) are shown with a dark surface. The heavy and light chains of the antibody are shown in cartoon form. Figure 2C Epitopes of the F768-104_B2 glycan. The antibody is shown on the surface, with the heavy and light chains represented by different shades. Epitope residues are shown as bars and labeled with the interacting XBB.1.5 mutation. The spike amino acid K440 is located in the center of the epitope, while two other mutations, P373 and P445, are located at the edges. Figure 2D F768-104_B2 utilizes the heavy / light chain interface cavity to tolerate mutations at the RBD residue 440 mutation hotspot located in the center of the epitope. K440 points towards the central cavity, while the other two XBB.1.5 mutations, P373 and P445, are located at the edge of the F768-104_B2 epitope and therefore do not produce resistance.

[0013] Figures 3A-3H Provides tables showing the genetic origin of antibodies and their neutralizing activity against SARS-CoV-2 variants and sarbecoviruses.

[0014] Figures 4A-4B Provides a table for epitope plotting using the competing ELISA.

[0015] Figures 5A-5C Provide a table for the selected antibody mutation analysis.

[0016] Figure 6A table showing the synergistic neutralization of SARS-CoV-2 variants by a combination of two monoclonal antibodies (mAbs).

[0017] Figure 7 A table showing the neutralizing effect of monoclonal antibodies against SARS-CoV-2 variants is provided. Lentiviral viruses pseudotyped with the SARS-CoV-2 spike protein of the indicated variants were incubated with serially diluted antibodies, and IC50 was determined on 293-TMPRSS2-ACE2 cells. 50 and IC 80 Value. The value is IC. 50 and IC 80 , measured in micrograms per milliliter.

[0018] Figures 8A-8C Comparison of binding modes and epitopes of class IV monoclonal antibodies. Figure 8A The structure of the A80-102_G5 antibody complex with the SARS-CoV S-2P spike protein. Left image: Cryo-electron microscopy rendering of the antibody-spike complex. Right image: Antibody binding to the RBD, both presented as bands. Figure 8B Epitopes of A18-448.1, A80-102_G5, and CR3022. RBD is shown on a light gray surface, and antibody binding footprint is shown on a black surface. Figure 8C Comparison of RBD binding modes among A18-448.1, A80-102_G5, and CR3022. RBDs are shown in gray, and each antibody is shown as a cartoon.

[0019] Figures 9A-9F Antibodies targeting class I-IV epitopes protect Syrian hamsters from SARS-CoV-2 variant attacks. Figure 9A Monoclonal antibody passive transfer and viral challenge protocol. Monoclonal antibodies targeting multiple different epitopes are used in a 1×10-1 ratio. 5 Syrian hamsters were passively transferred intraperitoneally with PFU containing SARS-CoV-2 EG.5.1 24 hours prior to challenge. Another group of hamsters received an equal volume of PBS in the same manner as a control. All animals were weighed daily to monitor weight loss. Figure 9C and Figure 9E (See the image above). Four animals in each group were euthanized on days 2, 4, 6, and 10 to assess lung function. Figure 9C and Figure 9E (see image below) and nostrils ( Figure 9D and Figure 9F Viral load in the hamsters was monitored daily for weight loss over 10 days. Figure 9BIn vitro neutralizing efficacy predicts protective effect. Post-challenge (day 4) viral load in the lungs (TCID50 / g) was highly correlated with the IC80 (ng / ml) of the antibody against the challenge virus. Each point represents either an antibody or a PBS (saline) control. The dashed line indicates the limit of detection. Figure 9C-9F Class I-IV monoclonal antibodies protect hamsters from EG.5.1 attack. Monitor body weight daily. Figure 9C and Figure 9E The above figure shows the average data from 4-16 animals. The symbols and error bars represent the mean and SEM, respectively. Lung data from EG.5.1-challenged hamsters were quantified by TCID50 per gram. Figure 9C and Figure 9E (see image below) or nostrils ( Figure 9D and Figure 9F Viral load in the figure. Each symbol represents a single animal; identical values ​​may overlap. The box and horizontal line represent the IQR and median, respectively; the endpoints of the whiskers are equal to the maximum and minimum values. The dashed line indicates the detection limit for this test. One-way ANOVA was used to determine the significance between treated and untreated animals at each time point. The significance p-value is indicated on the graph. p The value is indicated as: (≤0.05) (≤0.01) (≤0.001).

[0020] Detailed description The International Committee on Taxonomy of Viruses (ICTV, 2019) describes the Coronaviridae family (…). Coronaviridae Coronavirus subfamily ( Orthocoronavirinae Several viruses in the genome are pathogenic to humans. The most common human coronaviruses cause the common cold, including alpha coronaviruses 229E and NL63, and beta coronaviruses OC43 and HKU1. In addition to coronaviruses that cause common cold symptoms, three beta coronaviruses have been shown to be highly pathogenic to humans. These viruses, namely Middle East Respiratory Syndrome Coronavirus (MERS) and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV, also known as SARS1), can produce severe symptoms and lead to death in human patients.

[0021] In late 2019, a novel coronavirus was identified as the cause of the severe respiratory distress syndrome (ARDS) outbreak. The virus was subsequently sequenced and identified as highly similar to SARS-CoV; based on this result, the novel coronavirus was renamed SARS-CoV-2. The incubation period is typically 4 to 14 days, but can be as short as 1 day. Infection is characterized by fever, fatigue, cough, dyspnea, and diarrhea. Some patients develop significant respiratory distress, requiring hospitalization and supplemental oxygen. These patients may deteriorate rapidly, requiring intensive care unit admission and intubation. Severe illness is also characterized by multiple organ dysfunction syndrome, thrombosis, and pronounced systemic inflammatory response syndrome. The elderly are at particularly high risk of developing severe illness.

[0022] The genome of coronaviruses is a large, enveloped, positive-sense, single-stranded RNA. Its length varies by species and it encodes a variety of structural and non-structural proteins in several reading frames. The spike protein (S) is expressed on the surface of the viral particle and is responsible for viral entry and infection of target cells. Coronaviruses can be transmitted in multiple ways, including through respiratory droplets, aerosols, the fecal-oral route, and contaminated surfaces.

[0023] Both humoral and cellular immunity were detected in COVID-19 survivors and vaccine recipients. While their relative contributions to protection are unclear, humoral responses, including binding and neutralization, are associated with protection. Humoral immunity was measured using assays of serum immunoglobulin binding to viral antigens, serum neutralization of viral particles, and binding of memory B cells to viral spike-based fluorescent probes.

[0024] In late 2021, the first member of the Omeprón lineage, BA.1, was identified, and its high level of resistance to clinically used monoclonal antibodies was noted. Since then, many members of the Omeprón lineage have been identified, including variants BA.1, BA.2, BA.4 / BA.5, BQ.1.1, XBB.1.5, EG.5.1, and FL.1.5.1. By the end of 2024, almost all circulating strains are descendants of Omeprón JN.1, including KP.3.1.1 and XEC, and are resistant to all previously available monoclonal antibodies under EUA (except Pemgarda). Therefore, there is a need for novel, active monoclonal antibodies against these variants and against sabeviruses that could cause future outbreaks.

[0025] I. Terminology Summary Unless otherwise stated, technical terms are used according to convention. Definitions of many commonly used molecular biology terms can be found in Krebs. et al. (eds.), Lewin's genes XIIJones & Bartlett Learning, 2017. Unless the context clearly indicates otherwise, as used herein, the singular forms “a,” “an,” and “the” refer to both the singular and the plural. For example, the term “antigen” includes both singular and plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprising” means “including.” It should be further understood that, unless otherwise stated, any and all base sizes or amino acid sizes given with respect to nucleic acids or polypeptides, as well as all molecular weight or molecular mass values, are approximate and provided for descriptive purposes. Although many methods and materials similar to or equivalent to those described and used herein may be used, particularly suitable methods and materials are described herein. In case of conflict, this specification (including the explanations of terms) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. For ease of review of the various aspects, the following explanations of terms are provided: Approximately: Unless the context otherwise indicates, “approximately” means plus or minus 5% of a reference value. For example, “approximately” 100 means 95 to 105.

[0026] Administration: The reagent (e.g., a disclosed antibody) is introduced into the subject via a selected route. Administration can be local or systemic. For example, if the selected route is intravascular, the reagent (e.g., an antibody) is administered by introducing the composition into the subject's blood vessels. Exemplary routes of administration include, but are not limited to, oral, injectable (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, percutaneous (e.g., topical), intranasal, vaginal, and inhalation routes.

[0027] Amino acid substitution: One amino acid in a polypeptide is replaced by a different amino acid.

[0028] Antibody and antigen-binding fragment: Immunoglobulin, antigen-binding fragment, or derivative thereof, which specifically bind to and recognize an analyte (antigen), such as the spike protein of a coronavirus, for example, the spike protein from SARS-CoV-2. The term "antibody" is used in the broadest sense herein and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, provided they exhibit the desired antigen-binding activity.

[0029] Non-limiting examples of antibodies include, for example, intact immunoglobulins and their variants and fragments that retain binding affinity for antigens. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments that are either generated by modifying whole antibodies or synthesized de novo using recombinant DNA methods (see, for example, Kontermann and Dübel (Eds.)). Antibody Engineering Vols. 1-2, 2 nd ed., Springer-Verlag, 2010).

[0030] Antibodies also include genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugated antibodies (e.g., bispecific antibodies).

[0031] Antibodies can have one or more binding sites. If there are more than one binding site, these binding sites can be the same as each other or they can be different from each other. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific antibodies or bifunctional antibodies have two different binding sites.

[0032] Naturally occurring immunoglobulins typically consist of heavy (H) chains and light (L) chains linked together by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable domain genes. Two types of light chains exist: lambda (λ) and kappa (κ). Five major heavy chain classes (or isotypes) exist, which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0033] Each heavy and light chain contains constant regions (or constant domains) and variable regions (or variable domains). The variable regions of the heavy and light chains combine to specifically bind antigens.

[0034] Mentioning "V" H "VH" or "VH" refers to the variable region of the antibody heavy chain, including the variable region of antigen-binding fragments (such as Fv, scFv, dsFv, or Fab). Mentioning "V"... L "VL" or "VL" refers to the variable domain of the antibody light chain, including the variable domains of Fv, scFv, dsFv, or Fab.

[0035] V H and VL A "frame" region containing three high-variable regions, also known as a "complementary determination region" or "CDR" (see, for example, Kabat). et al. , Sequences of Proteins of Immunological Interest , 5 th (ed., NIHP Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 1991). The framework region sequences of different light or heavy chains are relatively conserved within species. The antibody's framework region, i.e., the combined framework region that makes up the light and heavy chains, is used for localization and alignment of CDRs in three-dimensional space.

[0036] CDRs are primarily responsible for binding to epitopes of antigens. The amino acid sequence boundaries of a given CDR can be readily determined using any of many well-known methods, including Kabat. et al. ( Sequences of Proteins of Immunological Interest , 5 th ed., NIH Publication No. 91-3242, Public HealthService, National Institutes of Health, US Department of Health and HumanServices, 1991; “Kabat” numbering scheme), Al-Lazikani et al. , (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio. , 273(4):927-948, 1997; “Chothia” numbering scheme) and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-likedomains,” Dev. Comp. Immunol. , 27(1):55-77, 2003; those described in the “IMGT” numbering scheme). The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3 (from the N end to the C end), and are usually also identified by the chain in which the specific CDR is located. Therefore, V HCDR3 is the V of the antibody it belongs to. H The CDR3 sequence, while V L CDR1 is the V of the antibody it belongs to. L CDR1. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0037] In some respects, the disclosed antibody includes a heterologous constant domain. For example, the antibody includes a constant domain that differs from the natural constant domain, such as a constant domain containing one or more modifications (e.g., an "LS" mutation) to extend the half-life.

[0038] “Monoclonal antibody” is an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies comprising that group are identical and / or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or variants generated during the preparation of the monoclonal antibody, which are typically present in very small amounts. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier “monoclonal” indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared using a variety of techniques, including but not limited to hybridoma methods, recombinant DNA protocols, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, which are described herein, along with other exemplary methods for preparing monoclonal antibodies. In some instances, monoclonal antibodies are isolated from a subject. Monoclonal antibodies may have conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function. (See, for example, Greenfield (Ed.)) Antibodies: A Laboratory Manual , 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014.) "Humanized" antibody or antigen-binding fragments consist of a human framework region and one or more core-receiving regions (CDRs) derived from non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragments. The non-human antibody or antigen-binding fragment providing the CDR is called the "donor," and the human antibody or antigen-binding fragment providing the framework is called the "acceptor." In one respect, in humanized immunoglobulins, all CDRs are derived from the donor immunoglobulin. Constant regions are not necessarily present, but if present, they can be substantially identical to the constant regions of human immunoglobulins, for example, at least about 85-90% identical, for example, about 95% or higher. Therefore, apart from the possible CDRs, all portions of the humanized antibody or antigen-binding fragment are substantially identical to the corresponding portions of the natural human antibody sequence.

[0039] A "chimeric antibody" is an antibody that comprises sequences derived from two different antibodies, typically from different species. In some instances, a chimeric antibody includes one or more CDRs and / or frame regions from one human antibody and CDRs and / or frame regions from another human antibody.

[0040] A "fully human antibody" or "human antibody" is an antibody that includes sequences from (or derived from) the human genome but excludes sequences from other species. In some respects, human antibodies include the CDR, frame region, and (if present) Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for constructing antibodies based on sequences derived from the human genome (e.g., via phage display or using transgenic animals) (see, for example, Barbas et al.). Phage display: A Laboratory Manuel 1 st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0041] Neutralizing antibodies or antigen-binding fragments of SARS-CoV-2: Antibodies or antigen-binding fragments that specifically bind to SARS-CoV-2 antigens (e.g., spike proteins) inhibit SARS-CoV-2-related biological functions to suppress infection. This antibody can neutralize the activity of SARS-CoV-2. SARS-CoV-2 can be Omega-3 or a variant thereof. In some respects, SARS-CoV-2 is BA.1, BA.2, BA.4 / BA.5, BQ.1.1, XBB.1.5, EG.5.1, and FL.1.5.1, JN.1, KP.3.1.1, XEC, or related strains. For example, neutralizing antibodies or antigen-binding fragments of SARS-CoV-2 can interfere with the virus by directly binding to it and restricting its entry into cells. Alternatively, the antibody can interfere with one or more post-attachment interactions between the pathogen and a receptor, for example, by interfering with viral entry using the receptor. In some instances, antibodies specific to the coronavirus spike protein neutralize the infectious titer of SARS-CoV-2.

[0042] In some respects, antibodies or antigen-binding fragments that specifically bind to and neutralize SARS-CoV-2 inhibit cell infection by, for example, at least 50%, compared to control antibodies or antigen-binding fragments.

[0043] "Broad-spectrum neutralizing antibodies" are antibodies that bind to and inhibit the function of relevant antigens, such as antigens with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the surface of the antigen. For antigens from pathogens (e.g., viruses), antibodies can bind to and inhibit the function of antigens from more than one class and / or subclass of that pathogen. For example, for coronaviruses, antibodies can bind to and inhibit the function of antigens such as the spike protein from multiple SARS-CoV-2 Omeprón variants.

[0044] Biological samples: Samples obtained from a subject. Biological samples include all clinical samples that can be used to detect disease or infection in a subject, including but not limited to cells, tissues, and body fluids such as blood, blood derivatives and fractions (e.g., serum), cerebrospinal fluid; and tissues obtained through biopsy or surgical removal, such as tissues that have not been fixed, frozen, or fixed in formalin or paraffin. In specific instances, biological samples are obtained from subjects who have or are suspected of having a coronavirus infection (e.g., but not limited to SARS-CoV-2 infection).

[0045] Bispecific antibodies are recombinant molecules consisting of two distinct antigen-binding domains, which thus bind to two different antigenic epitopes. Bispecific antibodies comprise molecules with two antigen-binding domains chemically or genetically linked. The antigen-binding domains can be linked using adapters. The antigen-binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. Bispecific antibodies may include one or more constant domains, but do not necessarily include constant domains.

[0046] Conditions sufficient for immune complex formation: conditions that allow an antibody or antigen-binding fragment to bind to its homologous epitope to a detectable degree, and / or substantially exclude its binding to substantially all other epitopes. The conditions sufficient for immune complex formation depend on the form of the binding reaction and are generally those utilized in the immunoassay protocol or encountered in vivo. For a description of immunoassay forms and conditions, see Greenfield (Ed.). Antibodies: A Laboratory Manual , 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014. The conditions used in these methods are “physiological conditions,” which include references to typical conditions within living mammals or mammalian cells (e.g., temperature, osmotic pressure, pH). Although it is recognized that some organs are subjected to extreme conditions, the in vivo and intracellular environment is generally at around pH 7 (e.g., pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the primary solvent, and exists at temperatures above 0°C and below 50°C. Osmotic pressure is within the range supporting cell viability and proliferation.

[0047] The formation of immune complexes can be detected by conventional methods, such as immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blotting), magnetic resonance imaging (MRI), computed tomography (CT), radiography, and affinity chromatography.

[0048] Conjugates: Complexes of two molecules linked together (e.g., covalently). On one hand, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to SARS-CoV-2 is covalently linked to an effector molecule (e.g., a detectable label). This linking can be achieved through chemical or recombinant means. On the other hand, this linking is chemical, where a reaction between the antibody portion and the effector molecule creates a covalent bond between the two molecules, thus forming a single molecule. Optionally, a peptide linker (a short peptide sequence) may be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with independent functions (e.g., an antibody and an effector molecule), they are sometimes also called "chimeric molecules."

[0049] Conservative variants: "Conservative" amino acid substitutions are substitutions that substantially do not affect or reduce protein function (e.g., the protein's ability to interact with target proteins). For example, coronavirus-specific antibodies may include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conserved amino acid substitutions compared to a reference antibody sequence, while maintaining specific binding activity and / or neutralizing activity against the spike protein. The term conservative variant also includes the use of substituted amino acids in place of unsubstituted parental amino acids.

[0050] The individual substitutions, deletions, or additions of a single amino acid or a small subset of amino acids (e.g., less than 5%, less than 1% in some respects) in the coding sequence are conserved variations in which these changes result in the substitution of the amino acid with a chemically similar amino acid.

[0051] The following six groups are examples of amino acids that are considered to be conserved substitutes for each other: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagine (N), glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), leucine (L), methionine (M), valine (V); and 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0052] Non-conserved substitutions are substitutions that reduce antibody activity or function (such as the ability to bind specifically to the coronavirus spike protein). For example, if an amino acid residue is essential for the function of a protein, even a seemingly conserved substitution may disrupt that activity. Therefore, conserved substitutions do not alter the fundamental function of the protein of interest.

[0053] Contact: This refers to direct physical association; it includes both solid and liquid forms, and can occur in vivo or in vitro. Contact includes the contact between one molecule and another, for example, the contact of an amino acid on the surface of one polypeptide (e.g., an antigen) with another polypeptide (e.g., an antibody). Contact can also include contact with cells, for example, by placing an antibody in direct physical association with a cell.

[0054] Control: Reference standard. In some respects, a control is a negative control, such as a sample obtained from a healthy patient who has never been infected with a coronavirus (such as SARS-CoV-2, e.g., BA.1, BA.2, BA.4 / BA.5, BA.2.75, BA.4.6, BA.2.75.2, BQ.1.1, BJ.1, XBB.1, XBB.1.5, XBB.1.16, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, or XEC). In other respects, a control is a positive control, such as a tissue sample obtained from a patient diagnosed with a coronavirus (such as SARS-CoV-2 infection). In still other respects, a control is a historical control or a standard reference value or numerical range (e.g., a control sample previously tested, such as a patient population with a known prognosis or outcome, or a sample group representing baseline or normal values).

[0055] The difference between the test sample and the control can be an increase or conversely a decrease. The difference can be qualitative or quantitative, such as a statistically significant difference. In some instances, the difference is an increase or decrease of at least about 5% relative to the control, for example, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.

[0056] Coronaviruses: A family of positive-sense single-stranded RNA viruses known to cause severe respiratory illness. Currently, viruses infecting humans are known to belong to the genera *Alphacoronavirus* and *Betacoronavirus*. Furthermore, the genera *Gammacoronavirus* and *Deltacoronavirus* are considered potential future sources of human infection.

[0057] Non-limiting examples of β-coronaviruses include SARS-CoV-2, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), human coronavirus HKU1 (HKU1-CoV), human coronavirus OC43 (OC43-CoV), mouse hepatitis virus (MHV-CoV), bat SARS-like coronavirus WIV1 (WIV1-CoV), and human coronavirus HKU9 (HKU9-CoV). Non-limiting examples of α-coronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and transmissible gastroenteritis coronavirus (TGEV). A non-limiting example of δ-coronavirus is porcine δ-coronavirus (SDCV).

[0058] The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. Coronavirus particles include a viral envelope containing a type I fusion glycoprotein called the spike (S) protein. Most coronaviruses share a genome organization with the replicase gene.

[0059] Degenerate variants: In the context of this disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide (e.g., an antibody heavy or light chain) that includes sequences degenerate due to the genetic code. There are 20 naturally occurring amino acids, most of which are designated by more than one codon. Therefore, all degenerate nucleotide sequences encoding peptides are included, provided that the amino acid sequence of the peptide encoded by the nucleotide sequence remains unchanged.

[0060] Detectable biomarkers: Detectable molecules (also called markers) are directly or indirectly conjugated to a second molecule (e.g., an antibody) to facilitate the detection of the second molecule. For example, detectable biomarkers can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., CT scans, MRI, ultrasound, fiber optic examination, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). Methods of using detectable biomarkers and guidelines for selecting suitable detectable biomarkers for various purposes are provided, for example, in Green and Sambrook (…). Molecular Cloning: A Laboratory Manual , 4 th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) ( Current Protocols in Molecular BiologyThis is discussed in *New York: John Wiley and Sons, including the supplement, 2017*.

[0061] Detection: Identifying the existence, appearance, or fact of something.

[0062] Effective amount: The amount of a substance sufficient to achieve the desired effect in a subject who has been given the substance. For example, this could be the amount necessary to inhibit coronavirus infection (e.g., SARS-CoV-2 infection) or to measurably alter the extrinsic symptoms of such infection. SARS-CoV-2 could be BA.1, BA.2, BA.4 / BA.5, BA.2.75, BA.4.6, BA.2.75.2, BQ.1.1, BJ.1, XBB.1, XBB.1.5, XBB.1.16, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, or XEC.

[0063] In one instance, the desired response is to suppress, reduce, or prevent SARS-CoV-2 infection. Effectiveness does not necessarily require complete elimination, reduction, or prevention of SARS-CoV-2 infection. For example, administration of an effective dose may reduce SARS-CoV-2 infection (e.g., by cell infection or by the number or percentage of subjects infected with SARS-CoV-2) by a desired amount, such as at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infection) compared to a suitable control. The antibodies disclosed herein can also suppress other coronavirus infections.

[0064] In some respects, administering an effective amount of the disclosed antibody or antigen-binding fragment that binds to the SARS-CoV-2 spike protein can reduce or inhibit the desired amount of infection (e.g., by cellular infection, or by the number or percentage of subjects infected with SARS-CoV-2, or by an increase in the survival time of infected subjects, or a reduction in infection-related symptoms), for example, by a reduction or inhibition of at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infection) compared to a suitable control.

[0065] The effective amount of a specific antibody or antigen-binding fragment binding to the SARS-CoV-2 spike protein administered to a subject to inhibit infection will vary depending on a number of factors relevant to the subject, such as the subject's overall health and / or weight. The effective amount can be determined by altering the dose and measuring the resulting response, for example, a decrease in pathogen titer. The effective amount can also be determined by a variety of in vitro, in vivo, or in situ immunoassays.

[0066] An effective dose encompasses a fraction of the dose that, in combination with previous or subsequent administrations, contributes to achieving an effective response. For example, an effective dose of the reagent may be administered as a single dose or multiple doses (e.g., daily) over a course of treatment lasting several days or weeks. However, the effective dose can vary depending on the subject being treated, the severity and type of the condition being treated, and the method of administration. The reagent may be packaged in unit dose form in the amount of the effective dose or multiples thereof, for example, in vials with sterile components (e.g., with a puncture cap) or syringes.

[0067] Effector molecules: Molecules designed to have or produce a desired effect (e.g., a desired effect on cells targeted by the effector molecule); or detectable markers. Effector molecules can include, for example, peptides and small molecules. Some effector molecules may have or produce more than one desired effect.

[0068] Epitopes: Antigenic determinants. These are specific chemical groups or peptide sequences on a molecule that are antigenic, causing them to elicit a specific immune response. For example, an epitope is an antigenic region that triggers a B cell and / or T cell response. Antibodies can bind to specific antigenic epitopes (e.g., epitopes on the SARS-CoV-2 spike protein).

[0069] Expression: The transcription or translation of a nucleic acid sequence. For example, when DNA encoding a nucleic acid sequence (e.g., a gene) is transcribed into RNA or RNA fragments, that sequence can be expressed; in some instances, these RNAs or RNA fragments are processed into mRNA. The nucleic acid sequence encoding a nucleic acid sequence (e.g., a gene) can also be expressed when mRNA is translated into an amino acid sequence (e.g., a protein or protein fragment). In a specific instance, a heterologous gene is expressed when it is transcribed into RNA. In another instance, the heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include control over transcription, translation, RNA transport and processing, degradation of intermediate molecules (e.g., mRNA), or activation, inactivation, compartmentalization, or degradation of specific protein molecules after their production.

[0070] Expression control sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is operatively linked. The expression control sequence is operatively linked to the nucleic acid sequence in controlling and regulating transcription and (if applicable) translation of the nucleic acid sequence. Therefore, the expression control sequence may include a suitable promoter, enhancer, transcription terminator, start codon (ATG) preceding a protein-coding gene, splicing signals for introns, and maintenance of the correct reading frame of the gene to allow correct translation of the mRNA and stop codon. The term "control sequence" is intended to include at least the components whose presence can affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion coupler sequences. The expression control sequence may include a promoter.

[0071] Expression vectors: Vectors comprising recombinant polynucleotides containing an expression control sequence operatively linked to a nucleotide sequence to be expressed. Expression vectors include sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Non-limiting examples of expression vectors include clomiphenes, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0072] Polynucleotides can be inserted into expression vectors containing promoter sequences that facilitate efficient transcription of the inserted gene sequence by the host. Expression vectors typically contain an origin of replication, a promoter, and specific nucleic acid sequences that allow for phenotypic selection of transformed cells.

[0073] Fc region: The antibody constant region excluding the first heavy chain constant domain. The Fc region typically refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. The Fc region may also include part or all of the flexible hinge located at the N-terminus of these domains. For IgA and IgM, the Fc region may or may not include a tailpiece and may or may not bind to the J chain. For IgG, the Fc region is generally understood to include immunoglobulin domains Cγ2 and Cγ3, and optionally the lower portion of the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as including residues from C226 or P230 to the C-terminus of the Fc group, numbered according to EU designations. For IgA, the Fc region includes immunoglobulin domains Cα2 and Cα3, and optionally the lower portion of the hinge between Cα1 and Cα2.

[0074] Heterologous: Derived from a different genetic source. A nucleic acid molecule heterologous to a cell originates from a genetic source other than the cell in which the nucleic acid molecule is expressed. In a specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein (e.g., scFv) is expressed in a cell (e.g., a mammalian cell). Methods for introducing heterologous nucleic acid molecules into cells or organisms are well known in the art, such as nucleic acid transformation, including electroporation, liposome transfection, gene gun acceleration, and homologous recombination.

[0075] Host cell: The cell in which the vector can reproduce and express its DNA. The cell can be a prokaryotic or eukaryotic cell. The term also includes any progeny of the host cell. It should be understood that all progeny may not be identical to the parent cell due to mutations that can occur during replication. However, when using the term "host cell," these progeny are included.

[0076] IgA: A polypeptide belonging to a class of antibodies essentially encoded by the well-known immunoglobulin α gene. In humans, this class, or isotype, includes IgA1 and IgA2. IgA antibodies can exist as monomers, polymers primarily in the form of dimers (called pIgA), and secreted IgA. The constant chain of wild-type IgA contains an 18-amino acid extension at its C-terminus, called the tail fragment (tp). Polymeric IgA is secreted by plasma cells and has a 15-kDa peptide (called the J chain) linking two IgA monomers via conserved cysteine ​​residues in the tail fragment.

[0077] IgG: A class of antibodies or isotypes of polypeptides that are essentially encoded by the well-known immunoglobulin γ gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.

[0078] Immune complexes are formed when antibodies or antigen-binding fragments (e.g., scFv) bind to soluble antigens. The formation of immune complexes can be detected by conventional methods such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, Western blotting (e.g., Western blotting), magnetic resonance imaging, CT scans, radiographic imaging, and affinity chromatography.

[0079] Suppressing or treating disease: Suppressing the complete development of a disease or condition in subjects at risk of developing it (e.g., SARS-CoV-2 infection). "Treatment" refers to a therapeutic intervention that reduces the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "reduction," in relation to a disease or condition, refers to any observable beneficial effect of treatment. Suppressing disease can include preventing or reducing the risk of disease, such as preventing or reducing the risk of viral infection. For example, its beneficial effect can be demonstrated by delayed onset of clinical symptoms of the disease in susceptible subjects, a reduction in the severity of some or all of the clinical symptoms of the disease, a slowing of disease progression, a reduction in viral load, an improvement in the overall health or well-being of the subject, or by other parameters specific to the disease. "Preventative" treatment is a treatment administered to subjects who do not exhibit signs of disease or only exhibit early signs, with the aim of reducing the risk of developing the disease.

[0080] The term "reduction" is a relative term, meaning that the agent reduces a disease or condition if the disease or condition is quantitatively reduced after application, or if the disease or condition is reduced after application of the agent compared to a reference agent. Similarly, the term "prevention" does not necessarily mean that the agent completely eliminates a disease or condition, but only eliminates at least one characteristic of the disease or condition. Therefore, a composition that reduces or prevents infection can (but does not necessarily completely) eliminate such infection, provided that the infection is measurably reduced, for example, by at least about 50% in the absence of the agent or compared to a reference agent, such as by at least about 70%, or about 80%, or even about 90%.

[0081] Isolated: A biological component (e.g., nucleic acid, peptide, protein, or protein complex, such as an antibody) that has been substantially isolated, produced, or purified from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins) naturally present in the somatic cells of an organism. Therefore, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids. Isolated nucleic acids, peptides, or proteins (e.g., antibodies) may be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

[0082] Kabat position: The position of a residue in the amino acid sequence, following the Kabat sequence. et al. ( Sequences of Proteins of Immunological Interest , 5 thThe numbering convention established by the Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIHP Publication No. 91-3242, 1991.

[0083] Linkers: Bifunctional molecules that can be used to link two molecules into a single continuous molecule, for example, linking a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0084] The terms "conjugation," "linking," "bonding," or "connection" can refer to the formation of a single continuous molecule from two molecules; for example, linking two polypeptides into a single continuous polypeptide, or covalently attaching an effector molecule, a detectable biomarker, a radionuclide, or other molecule to a polypeptide, such as scFv. This connection can be achieved through chemical or recombination methods. "Chemical methods" refer to the reaction between an antibody moiety and an effector molecule, resulting in a covalent bond between the two molecules to form a single molecule.

[0085] Multispecific antibodies: Recombinant molecules containing two or more distinct variable fragments (Fv). The valence of a multispecific antibody is equal to the number of Fv fragments in the molecule. The specificity of a multispecific antibody is equal to the number of unique antigen-binding domains in that multispecificity. In multispecific antibodies, specificity is limited by the total number of available Fv domains. Monospecific antibodies can be prepared using multispecific antibodies with valences of 3, 4, 5, or higher, and all Fv are identical. Bispecific antibodies can be prepared using multispecific antibodies with valences of 2, 3, 4, or higher, where the Fv domain is composed of two distinct antigen-binding domains. In this case, the number of each antigen-binding domain can be equal or different, and their position and localization can vary. Similarly, trispecific antibodies can be prepared using multispecific antibodies with valences of 3, 4, 5, or higher, where the Fv domain is composed of three distinct antigen-binding domains. In this case, the number of each antigen-binding domain can be equal or different, and their position and localization can vary. Similarly, tetraspecific antibodies can be prepared using multispecific antibodies with valences of 4, 5, 6, or higher, where the Fv domain is composed of four different antigen-binding domains. In this case, the number of each antigen-binding domain can be equal or different, and their positions and localization can vary. Multispecific antibodies comprise chemical or genetic linkers of the antigen-binding domains. The antigen-binding domains can be linked using adapters. The antigen-binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. Multispecific antibodies may contain one or more constant domains, but do not necessarily contain constant domains.

[0086] Nucleic acid (molecule or sequence): deoxyribonucleotides or ribonucleotide polymers or combinations thereof, including but not limited to cDNA, mRNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA or RNA. Nucleic acids can be double-stranded (ds) or single-stranded (ss). In the case of single-stranded nucleic acids, the nucleic acid can be a sense strand or an antisense strand. Nucleic acids can include natural nucleotides (e.g., A, T / U, C, and G) and can include analogues of natural nucleotides, such as labeled nucleotides.

[0087] "cDNA" refers to single-stranded or double-stranded DNA that is complementary to or identical to mRNA.

[0088] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) as a template for the synthesis of other polymers and macromolecules in biological processes. It has a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological characteristics. Therefore, if the transcription and translation of mRNA produced by a gene produces a protein in a cell or other biological system, then that gene encodes a protein. Both the coding strand of a gene or cDNA (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (used as a transcription template) can be considered to encode a protein or other product of that gene or cDNA. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0089] Operable ligation: The first and second nucleic acid sequences are operably ligated when they are functionally related. For example, if a promoter (e.g., the CMV promoter) affects the transcription or expression of a coding sequence, then the promoter and coding sequence are operably ligated. Typically, operably ligated DNA sequences are contiguous and located within the same reading frame when it is necessary to link two protein-coding regions.

[0090] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carrier used is conventional. Remington: The Science and Practice of Pharmacy, 22 nd ed. , London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for drug delivery of the disclosed reagents.

[0091] Generally, the nature of the carrier will depend on the specific route of administration. For example, parenteral preparations typically include injectable solutions containing pharmaceutically and physiologically acceptable liquids such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, etc., as solvents. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, added preservatives (e.g., non-natural preservatives), and pH buffers, such as sodium acetate or sorbitan monolaurate. In certain instances, pharmaceutically acceptable carriers are sterile and suitable for parenteral administration to subjects, for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in unit dosage forms (e.g., pills) or in selected amounts in vials. Unit dosage forms may include one dose or multiple doses (e.g., in a vial from which a dose of reagent can be selectively dispensed).

[0092] Polypeptide: A polymer in which monomers are linked together by an amide bond of amino acid residues. When the amino acid is an α-amino acid, either an L-optical isomer or a D-optical isomer may be used, with the L-isomer being preferred. As used herein, the terms “polypeptide” or “protein” are intended to cover any amino acid sequence and include modified sequences, such as glycoproteins. Polypeptides include naturally occurring proteins as well as proteins produced through recombinant or synthetic processes. Polypeptides have an amino terminus (N-terminus) and a carboxyl terminus. In some respects, a polypeptide is a disclosed antibody or a fragment thereof.

[0093] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide formulation is one in which peptides or proteins (e.g., antibodies) are more enriched than in their native intracellular environment. In one aspect, the formulation is purified such that proteins or peptides constitute at least 50% of the total peptide or protein content in the formulation.

[0094] Recombination: Recombinant nucleic acids are nucleic acids having a non-naturally occurring sequence or having a sequence artificially combined from two originally separate sequence segments. This artificial combination can be achieved through chemical synthesis, or more commonly, through artificial manipulation of the separated nucleic acid segments, for example, through genetic engineering techniques. Recombinant proteins are proteins having a non-naturally occurring sequence or having a sequence artificially combined from two originally separate sequence segments. In several respects, recombinant proteins are encoded by heterologous (e.g., recombinant) nucleic acids that have been introduced into a host cell (e.g., bacteria or eukaryotic cells). For example, the nucleic acid can be introduced into an expression vector having a signal capable of expressing the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into the host cell chromosome.

[0095] SARS-CoV-2, also known as the 2019 novel coronavirus, is a positive-sense single-stranded RNA virus belonging to the β-coronavirus genus and has become a highly lethal cause of severe acute respiratory infection. The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. The SARS-CoV-2 viral particle includes a viral envelope containing a large spike glycoprotein. Like most coronaviruses, the SARS-CoV-2 genome has a common genomic organization, including replicase genes at the 5' two-thirds of the genome and structural genes at the 3' one-third. The SARS-CoV-2 genome encodes a typical set of structural protein genes in the order 5'-spike (S)-enveloping (E)-membrane (M) and nucleocapsid (N)-3'. Symptoms of SARS-CoV-2 infection include fever and respiratory illness such as dry cough and shortness of breath. Severe cases can progress to severe pneumonia, multiple organ failure, and death. The time from exposure to symptom onset is approximately 2 to 14 days.

[0096] SARS-CoV-2 infection can be detected using standard methods for detecting viral infection, including, but not limited to, assessing patient symptoms and background, and genetic testing such as reverse transcription polymerase chain reaction (rRT-PCR). This test can be performed on patient samples (e.g., respiratory or blood samples).

[0097] Variant B.1.1.529, also known as the Omekkaron variant BA.1, is a variant of the original SARS-CoV-2 that was first reported to the World Health Organization on November 21, 2021. Compared to the original SARS-CoV-2 strain, this variant has a total of 60 mutations, specifically 50 non-synonymous mutations, 8 synonymous mutations, and 2 non-coding mutations. Thirty-two mutations affect the spike protein (A67V, Δ69-70, T95I, G142D, Δ143-145, Δ211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F), about half of which are located in the receptor-binding domain (319-541).

[0098] New subvariants with enhanced transmissibility (derived from BA.2 or BA.4 / BA.5) emerged and became prevalent in November 2022. Their geographical distribution is uneven, but they carry an additional limited mutant set in their spikes.

[0099] BA.2.75.2, derived from BA.2, was first observed in India and Singapore and contains R346T, F486S, and D1199N substitutions 17–19. BA.4.6 has been detected in multiple countries, carrying the R346T and N658S mutations. As of November 2022, BQ.1.1 has become the dominant prevalent lineage in many countries. It also carries the R346T mutation found in BA.2.75.2, as well as K444T and N460K substitutions (see Plantas et al., bioRXiv, available online, doi.org / 10.1101 / 2022.11.17.516888, November 17, 2022). BQ.1.1 contains amino acid substitutions relative to WA-1 (note that "-" indicates deletion): T19I, L24-, P25-, P26-, A27S, H69-, V70-, G142D, V213G, G339D, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, K444T, L452R, N460K_S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. BQ1.1 is disclosed, for example, by Miller et al., / doi.org / 10.1101 / 2022.11.01.514722, and became available on November 2, 2022. The BJ.1 variant is believed to have evolved from the Omecron BA.2 background. BJ.1 contains the following amino acid substitutions relative to WA-1, where “-” indicates deletion: T19I, L24-, P25-, P26-, A27S, V83A, G142D, Y144, H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N 440K, V445P, G446S, S477N, T478K, V483A, E484A, F490V, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, G798D, Q954H, N969K and S1003I, see Roemer et al., SARS-CoV-2 post-Omicron, virological.org / t / sars-cov-2-evolution-post-omicron / 911, November 25, 2022.XBB is a SARS-CoV-2 variant believed to have originated from interlineage recombination between BJ.1 and BA.2.75. XBB contains the following amino acid substitutions relative to WA-1, where "-" indicates deletion: T19I, L24-, P25-, P26-, A27S, V83A, G142D, Y144-, H146Q, Q183E, V213E, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S. K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486S, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K, see Roemer et al., SARS-CoV-2 post-Omicron, virological.org / t / sars-cov-2-evolution-post-omicron / 911, November 25, 2022.

[0100] BA.2.12.1 contains the following amino acid substitutions relative to WA-1, where “-” indicates deletion: T19I, L24S, P25-, P26-, A27-, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452Q, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, S704L, N764K, D796Y, Q954H, and N969K. BA2.75 contains the following amino acid substitutions relative to WA-1, where “-” indicates deletion: T19I, L24-, P25-, P26-, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, Q498R, N501Y, Y505H, H655Y, N679K, P681H, N764K, D796Y, Q954H, and N969K. The BA.4 and BA.5 spikes contain the following amino acid substitutions relative to WA-1, where “-” indicates deletion: T19I, L24S, P25-, P26-, A27-, H69-, V70-, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K.

[0101] The BA.4 and BA.5 sublineages of B.1.1.529 (whose spike sequences are indistinguishable from each other) were also detected for the first time by genome surveillance in South Africa. BA.4 and BA.5 have variations relative to B.1.1.529, including L452R and F486V mutations and an R493Q reversion mutation in the spike receptor-binding domain (RBD). BA.4 and BA.5 also differ from the BA.2 sublineage in the deletion of spike residues 69 and 70 (Khan et al., Nature Comm. 13, Article number 4686, doi.org / 10.1038 / s41467-022-32396-9, (2022)).

[0102] XBB.1.5 contains the following amino acid substitutions relative to WA-1, where "-" indicates deletion: T19I, L24-, P25-, P26-, A27S, V83A, G142D, Y144-, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T37 6A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F 490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K. XBB.1.16 contains the following amino acid substitutions relative to WA-1, where "-" indicates deletion: T19I, L24-, P25-, P26-, A27S, V83A, G142D, Y144-, H146Q, E180V, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F. T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478R, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K. CH.1.1 contains the following amino acid substitutions relative to WA-1, where "-" indicates deletion: T19I, L24-, P25-, P26-, A27S, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, S371F, S373P, S375F, T376A. D405N, R408S, K417N, N440K, K444T, G446S, L452R, N460K, S477N, T478K, E484A, F48 6S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K.XBC.1 contains the following amino acid substitutions relative to WA-1, where "-" indicates deletion: T19I, P25S, G142D, Y144-, E156-, F157-, R158G, P209L, L212S, D215H, A222V, A243-, L244-, S256L, S371F, S373P, S375F, T3 76A, D405N, R408S, K417N, N440K, G446S, L452M, S477N, T478K, E484A, F486P, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N703I, N764K, D796Y, Q954H, N969K. XBB.2.3.2 contains amino acid substitutions of G184V, D253G, F486P, and P521S from XBB.1.5. EG.5.1 contains amino acid substitutions of Q52H and F456L from XBB.1.5. XBB.1.16.6 contains amino acid substitutions of E180V, F456L, and T478R from XBB.1.5. FL.1.5.1 contains amino acid substitutions of F456L, T478R, and A701V on XBB.1.5.

[0103] BA.2.86 has two versions: BA.2.86_V670 and BA.2.86_I670. BA.2.86_I670 is the major version, containing the following amino acid substitutions relative to WA-1, where "-" indicates deletion: ins16MPLF, T19I, R21T, L24-, P25-, P26-, A27S, S50L, H69-, V70-, V127F, G142D, Y144-, F157S, R158G, N211-, L212I, V213G, L216F, H245N, A264D, I332V, G339H, K356T, S371F, S373P, S375 F, T376A, R403K, D405N, R408S, K417N, N440K, V445H, G446S, N450D, L452W, N460K, S477N, T478K, N481K, V483-, E484K, F486P, Q498R, N501Y, Y505H, E554K, A570V, D614G, P621S, H655Y, N679K, P681R, N764K, D796Y, S939F, Q954H, N969K, P1143L. JN.1 contains the L455S amino acid substitution at BA.2.86. KP.2 contains the R346T and F456L amino acid substitutions at JN.1. KP.3 contains the F456L, Q493E, and V1104L amino acid substitutions found on JN.1. LB.1 contains the S31 deletion and the Q183H, R346T, and F456L amino acid substitutions found on JN.1. KP.2.3 contains the S31 deletion and the H146Q, R346T, F456L, and V1104L amino acid substitutions found on JN.1. KP.3.1.1 contains the S31 deletion and the F456L, Q493E, and V1104L amino acid substitutions found on JN.1. XEC contains the T22N, F59S, F456L, Q493E, and V1104L amino acid substitutions found on JN.1. As of November 23, 2024, KP.3.1.1 and XEC are the major variants worldwide.

[0104] Spike (S) protein (coronavirus): A class I fusion glycoprotein. For SARS-CoV, it is initially synthesized as a precursor protein of approximately 1256 amino acids, and for SARS-CoV-2, as a precursor protein of approximately 1273 amino acids. The individual precursor S polypeptides form a homotrimer and undergo glycosylation in the Golgi apparatus, followed by processing to remove the signal peptide. The cytokines are then cleaved by cellular proteases between approximately positions 679 / 680 of the SARS-CoV protein and approximately positions 685 / 686 of the SARS-CoV-2 protein to generate separate S1 and S2 polypeptide chains. These chains remain associated in the homotrimer as the S1 / S2 progenitors, thus forming a heterodimeric trimer. The S1 subunit is located at the distal end of the viral membrane and contains a receptor-binding domain (RBD), which is believed to mediate viral attachment to its host receptor. The S2 subunit contains fusion protein mechanisms, such as fusion peptides, two heptapeptide repeat sequences (HR1 and HR2), and the central helix, transmembrane domain, and cytoplasmic tail domain typical of fusion glycoproteins.

[0105] The numbering used in the publicly available SARS-CoV-2 S protein and its fragments is relative to the SARS-CoV-2 S protein sequence, which is deposited with NCBI reference number YP_009724390.1 and became available on February 1, 2022. It is incorporated herein by reference in its entirety.

[0106] The SARS-CoV-2 spike protein (S) is a trimer of the dimers (S1 and S2). S2 mediates viral fusion with the host cell membrane, while the S1 domain mediates attachment to the target cell and host cell receptor protein angiotensin-converting enzyme (ACE) 2. S1 consists of two main domains: the N-terminal domain (NTD) and the receptor-binding domain (RBD). The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S) contains the receptor-binding motif (RBM), which binds to the human cell receptor protein ACE2. RBM binding to ACE2 is essential for SARS-CoV-2 infection of cells. The RBD exists in two conformations, referred to as "up" or "down." When the RBD is in the "down" conformation, the RBM cannot bind ACE2. However, when the RBD is in the "up" conformation, the RBM can bind ACE2. Barnes et al. defined a functional classification scheme for antibodies targeting RBDs based on the RBD state in which the antibody can bind and whether the epitope within the RBD overlaps with the ACE2 receptor binding site. Class I and II antibodies have epitopes that at least partially overlap with the RBM site, while Class III and IV antibodies do not bind to the RBM region. Class I and IV antibodies can only bind to the RBD in the "upper" position. In contrast, Class II and III antibodies can bind to the RBD in either the "upper" or "lower" position. See Barnes et al., Nature 588(7839):682-687. doi: 10.1038 / s41586-020-2852-1, October 12, 2020 (electronic version), incorporated herein by reference. This classification is used for rapid mapping of antibody epitopes by competitive assays against antibodies of known classes and epitopes.

[0107] The receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S) contains a receptor-binding motif (RBM) that binds to the human cellular receptor protein angiotensin-converting enzyme (ACE) 2. RBM binding to ACE2 is essential for SARS-CoV-2 infection of cells. The RBD exists in two conformations, referred to as "up" or "down". When the RBD is in the "down" conformation, the RBM cannot bind to ACE2. However, when the RBD is in the "up" conformation, the RBM can bind to ACE2. Barnes et al. defined a functional classification scheme for antibodies targeting the RBD based on the RBD state that the antibody can bind to and whether the epitope within the RBD overlaps with the ACE2 receptor-binding site (pubmed.ncbi.nlm.nih.gov / 33045718 / ). Class I and II antibodies have epitopes that at least partially overlap with the RBM site, while Class III and IV antibodies do not bind to the RBM region. Class I and IV antibodies can only bind to the RBD in the "up" position. In contrast, class II and class III antibodies can bind to the RBD when it is in the "up" or "down" position.

[0108] The Omeprone variant appears to have convergent mutations at the following positions: position 486, which affects the neutralization and binding of class I antibodies (including COV2-2196, a major component of EVUSHELD™); positions 444–446, affecting class III antibodies (including LY-CoV1404); and sometimes also includes a change at position 452, which affects class II antibodies. Because the novel variants incorporate mutations at these positions, they may exhibit high resistance to clinical antibodies used to treat the original SARS-CoV-2 virus and delta, see Wang et al., doi.org / 10.1101 / 2022.11.23.517532, available at biorxiv.org / content / 10.1101 / 2022.11.23.517532v1.full, November 28, 2022.

[0109] Sequence identity: The similarity between two or more nucleic acid sequences or two or more amino acid sequences, expressed as a percentage of identity. Sequence identity can be measured as a percentage; the higher the percentage, the higher the sequence identity. The V of an antibody that specifically binds to a target antigen... L or V H Homologous and variant forms are typically characterized by having at least about 75% sequence identity, calculated based on a full-length alignment with the amino acid sequence of interest, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0110] Any suitable method can be used to align sequences for comparison. Non-limiting examples of procedures and alignment algorithms are described in: Smith and Waterman. Adv. Applied Math. 2(4):482-489, 1981; Needleman andWunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Higgins and Sharp, Gene , 73(1):237-244,1988; Higgins and Sharp, Bioinformatics 5(2):151-3, 1989; Corpet, Nucleic Acids Res. 16(22):10881-10890, 1988; Huang et al. Bioinformatics, 8(2):155-165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994., Altschul et al. , J. Mol. Biol. 215(3):403-410, 1990, details sequence alignment methods and homology calculations. NCBI Basic Local Alignment Search Tool (BLAST) (A Altschul et al. , J. Mol. Biol. (215(3):403-410, 1990) is available from multiple sources, including the National Center for Biotechnology Information (NCBI) and the Internet, for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information is available on the NCBI website.

[0111] Typically, when two sequences are compared, the number of matches can be determined by counting the number of positions in the two sequences where the same nucleotide or amino acid residues are present. The percentage of sequence identity between two sequences is determined by dividing the number of matches by the length of the sequence listed in the identified sequence, or by the hinge length (e.g., 100 consecutive nucleotide or amino acid residues from the sequence listed in the identified sequence), and then multiplying the result by 100.

[0112] Specific binding: When referring to antibody or antigen-binding fragments, this refers to a binding reaction that identifies the presence of a target protein (such as the coronavirus spike protein) in the presence of heterogeneous protein groups and other biological agents. Therefore, under specified conditions, antibodies preferentially bind to specific target proteins, peptides, or polysaccharides (e.g., antigens present on the surface of pathogens, such as the coronavirus spike protein), rather than binding significantly to other proteins present in the sample or subject. Regarding spike proteins, epitopes may be present on the spike proteins of more than one type of coronavirus, allowing antibodies to bind to the spike proteins of more than one type of virus, but not to other proteins (e.g., proteins from other viruses or other proteins of coronaviruses (non-spike proteins)). Specific binding can be determined using standard methods. For a description of the forms and conditions of immunoassays that can be used to determine specific immunoreactivity, see Harlow & Lane. Antibodies, A Laboratory Manual , 2 nd ed., ColdSpring Harbor Publications, New York (2013).

[0113] Regarding antibody-antigen complexes, the K-axis of the specific binding of antigens and antibodies... D Less than about 10 -7 moles, for example less than about 10 -8 mole, 10 -9 moles, even less than about 10 -10 Moore. K D This refers to the dissociation constant of a given interaction, such as peptide-ligand interactions or antibody-antigen interactions. For example, for a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, it is the concentration of each component in the bimolecular interaction divided by the concentration of the complex.

[0114] Antibodies that specifically bind to epitopes (e.g., RBD domains) on the coronavirus spike protein can bind to molecules / reagents including that domain, including viruses, spike protein-attached substrates, or proteins in biological specimens. Of course, it is recognized that some degree of non-specific interaction can occur between antibodies and non-targets. Typically, specific binding results in association between the antibody and the spike protein being much stronger than association between the antibody and other different coronavirus proteins (e.g., E, M, or N proteins) or non-coronavirus proteins. Specific binding typically results in a greater than 2-fold increase (per unit time) in the amount of antibody binding to a protein containing that epitope or to cells or tissues expressing the target epitope compared to proteins or tissues lacking that epitope. Under these conditions, specific binding to the protein requires selection of antibodies specific to that particular protein. Various immunoassays are suitable for selecting antibodies or other ligands that have a specific immune response to a specific protein. For example, solid-phase enzyme ELISA is commonly used to select monoclonal antibodies that have a specific immune response to a protein.

[0115] Subjects: Live multicellular vertebrate organisms, including humans and non-human mammals such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, etc. In this example, the subject is a human. In a specific instance, the subject is a human. In another instance, subjects requiring suppression of SARS-CoV-2 infection are selected. For example, subjects who are not infected but at risk of SARS-CoV-2 infection, or who are already infected and require treatment.

[0116] Transformed: Transformed cells are cells into which nucleic acid molecules have been introduced using molecular biology techniques. As used herein, the terms transformed (e.g., transformation, transfection, transduction, etc.) encompass all techniques that can introduce nucleic acid molecules into cells, including viral vector transduction, plasmid vector transformation, and accelerated DNA introduction via electroporation, liposome transfection, and gene guns.

[0117] Vector: An entity containing a nucleic acid molecule (e.g., a DNA or RNA molecule) carrying a promoter operatively linked to and capable of expressing a coding sequence of a protein of interest. Non-limiting examples include naked DNA or packaged (lipid and / or protein) DNA, naked RNA or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may not be capable of replication, or a virus or bacterium or other microorganism that may be capable of replication. Vectors are sometimes referred to as constructs. A recombinant DNA vector is a vector containing recombinant DNA. A vector may include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. A vector may also include one or more optional marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector having at least some nucleic acid sequences derived from one or more viruses. In some aspects, a viral vector includes a nucleic acid molecule encoding a publicly disclosed antibody or antigen-binding fragment that specifically binds to and neutralizes the coronavirus spike protein. In some aspects, a viral vector may be an adeno-associated virus (AAV) vector.

[0118] Under conditions sufficient to…: A phrase used to describe any environment that allows the desired activity.

[0119] II. Description of various implementation schemes Monoclonal antibodies with activity against SARS-CoV-2 and SARS-CoV-related viruses from bats, pangolins, etc. (collectively known as sarbecoviruses) are valuable in pandemic preparedness. These zoonotic coronaviruses can enter host cells using various ACE2 orthologs and exhibit binding affinity similar to SARS-CoV-2. Therefore, these viruses can cause cross-species transmission and lead to human infection. Although many monoclonal antibodies have been identified and shown protective efficacy against SARS-CoV-2, most of them have no or only weak cross-neutralizing activity against these zoonotic coronaviruses. Therefore, there is a need to develop broad-spectrum monoclonal antibodies or antiviral agents against sarbecoviruses to address the future threat of zoonotic coronaviruses.

[0120] Monoclonal antibodies neutralizing the Omeprone variant are also useful. This article discloses 23 monoclonal antibodies with high and medium neutralizing titers against the current major variant lineage XBB and its relatives. All 23 monoclonal antibodies target the receptor-binding domain (RBD) of the spike protein. These antibodies possess unique neutralizing breadth and potency. Compared to the previously leading clinical monoclonal antibody Ly-CoV1404 (bertronib), all of these antibodies exhibit broader cross-reactivity, and most are more potent; neutralizing all current variants; and also neutralizing several related sabevirins.

[0121] An isolated monoclonal antibody and antigen-binding fragment that specifically bind to the coronavirus spike protein are provided. This monoclonal antibody and antigen-binding fragment specifically bind to the coronavirus spike protein and neutralize SARS-CoV-2. In some respects, the antibody specifically binds to the spike protein of XBB.1, XBB1.5, and XBB1.16, as well as at least one SARS-CoV-2-related virus from bats or pangolins. The antibody and antigen-binding fragment can be fully human.

[0122] The antibody and antigen-binding fragment can neutralize SARS-CoV-2. In some respects, the disclosed antibody can inhibit SARS-CoV-2 infection in vivo and can be administered before or after SARS-CoV-2 infection.

[0123] Multispecific antibodies, such as bispecific antibodies, containing variable domains of these antibodies are also provided. Furthermore, compositions comprising the antibody, an antigen-binding fragment, and a pharmaceutically acceptable carrier are disclosed herein. Nucleic acids encoding the antibody, the antigen-binding fragment, the variable domains, and expression vectors (e.g., adeno-associated virus (AAV) viral vectors) containing these nucleic acids are also provided. The antibody, antigen-binding fragment, nucleic acid molecule, host cell, and composition can be used for research, diagnostic, therapeutic, and preventative purposes. For example, the disclosed antibody and antigen-binding fragment can be used to diagnose subjects with SARS-CoV-2 infection or can be administered to inhibit SARS-CoV-2 infection in subjects.

[0124] A. Monoclonal antibodies that specifically bind to the coronavirus spike protein and their antigen-binding fragments The following discussion of monoclonal antibodies refers to isolated monoclonal antibodies containing heavy and / or light chain variable domains (or antigen-binding fragments thereof) containing CDR1, CDR2, and / or CDR3 according to the IMGT numbering scheme (unless the context otherwise indicates). Various CDR numbering schemes (e.g., Kabat, Chothia, or the IMGT numbering scheme) can be used to determine CDR positions. The amino acid sequences of the heavy and light chains and CDRs of the disclosed monoclonal antibodies according to the IMGT numbering scheme are provided in the sequence listing, but these are merely exemplary. The disclosed monoclonal antibodies specifically bind to the spike protein of SARS-CoV-2.

[0125] 1. Exemplary monoclonal antibody In some aspects, monoclonal antibodies are provided that comprise the heavy and light chain CDRs of any of the antibodies described herein. In other aspects, monoclonal antibodies are provided that comprise the heavy and light chain variable regions of any of the antibodies described herein. Antigen-binding fragments of these monoclonal antibodies are also provided. In some aspects, the antibody or its antigen-binding fragment is conjugated with SARS-CoV-2BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 and / or SHC-014.

[0126] In some respects, A18-448.1 and its antigen-binding fragment are disclosed. In other respects, the antibody or its antigen-binding fragment neutralizes BA.1, BA.2, BA.4 / 5, EG.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In still other respects, the antibody or its antigen-binding fragment neutralizes SARS-CoV-related sabeviruses from humans, bats, and / or civet cats.

[0127] In other aspects, F768-104_B2 and its antigen-binding fragment are disclosed. In some aspects, the antibody or its antigen-binding fragment neutralizes SARS-CoV-2-related viruses of animal origin. In some aspects, the antibody or its antigen-binding fragment specifically binds to spike proteins from BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC.

[0128] In another aspect, a third group of antibodies and their antigen-binding fragments are disclosed. These antibodies include A80-102_G5, A80-338-19.5, A80-338-19.6, A80-338-19.8, and A80-338-19.9. In some aspects, these antibodies and antigen-binding fragments have neutralizing activity against SARS-CoV-2 variants and / or SARS-CoV-associated sabezioviruses. In many other aspects, these antibodies and antigen-binding fragments have neutralizing activity against Omeprone BA.1, BA.2, BA.5, EG.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In another respect, these antibodies and antigen-binding fragments have neutralizing activity against SARS-CoV-2-related viruses from animals (including the pangolin GX variant) and SARS-CoV-related sabeviruses from humans, bats, and civets.

[0129] Furthermore, a fourth group of antibodies and their antigen-binding fragments were disclosed. These antibodies include E184-105_F3, A63-652-32.1, F768-104_G7, E184-105_H1, F768-104_D10, and A63-652-1.4. In some respects, these antibodies and antigen-binding fragments exhibit neutralizing activity against Omeprón lineage pseudoviruses. In other respects, these antibodies and antigen-binding fragments exhibit neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, and BA.2.86. In other respects, these antibodies and antigen-binding fragments neutralize SARS-CoV-2-related coronaviruses from bats and pangolins. F768-104_D10 and F768-104_G7 also neutralize the D614G strain carrying the F456L mutation. A63-652-1.4 has neutralizing activity against JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC.

[0130] In other aspects, a fifth group of antibodies and their antigen-binding fragments were disclosed. These antibodies include E184-105_E5, E184-105_H9, E184-105_H2, and A80-102+103_G1. In some aspects, these antibodies and antigen-binding fragments exhibit neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In many other aspects, these antibodies and antigen-binding fragments exhibit neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, and BA.2.86. In still other aspects, these antibodies and antigen-binding fragments neutralize SARS-CoV-2-related coronaviruses from bats and pangolins. E184-105_E5 and A80-102+103_G1 also neutralize D614G strains carrying multiple mutations known to reduce the activity of other class I antibodies (including the F456L mutation).

[0131] In another aspect, a sixth group of antibodies and their antigen-binding fragments were disclosed. These antibodies include A80-103_E2, A80-103_B6, A18-618-452.1, A80-102+103_C1, A43-d321.10.4, and F768-100-pt2_A05. In some respects, these antibodies and antigen-binding fragments exhibit neutralizing activity against SARS-CoV-2 Omeprón lineage pseudoviruses. In many other respects, these antibodies and antigen-binding fragments exhibit neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB. In other respects, these antibodies and antigen-binding fragments exhibit neutralizing activity against BA EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. Furthermore, these antibodies and antigen-binding fragments neutralize SARS-CoV-2-related coronaviruses and SARS-CoV from animals.

[0132] Table A provides the antibody names and V values ​​of the antibodies disclosed in this paper. H HCDR1, HCDR2, HCDR3, V L The sequences contained in LCDR1, LCDR2 and LCDR3, as well as some positional information.

[0133] Table A. IMGT CDR and SEQ ID NO of the antibody

[0134] a. Monoclonal antibody A18-618-448.1 In some respects, the antibody or antigen-binding fragment is based on or derived from the A18-618-448.1 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0135] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A18-618-448.1 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus, which can be SARS-CoV-2. In some respects, the antibody or antigen-binding fragment neutralizes BA.1, BA.2 and BA.4 / 5, EG.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In other respects, the antibody or antigen-binding fragment neutralizes SARS-CoV-related sabeviruses from humans, bats, and / or civets.

[0136] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 1, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 5, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 1 and 5, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0137] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 2, 3, and 4, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, 7(AAS), and 8, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0138] In some respects, the antibody or antigen-binding fragment contains V H (It contains HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 2, 3, and 4, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, 7(AAS), and 8, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 1 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1), wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 5 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 5), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0139] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 1 and specifically binds to the coronavirus spike protein, and neutralizes the coronavirus. In further respects, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 5 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V LIt contains the amino acid sequences shown in SEQ ID NO: 1 and 5, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0140] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0141] b. Monoclonal antibody F768-104_B2 In some respects, the antibody or antigen-binding fragment is based on or derived from the F768-104_B2 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the F768-104_B2 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0142] In some respects, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related viruses of animal origin. In other respects, this antibody or antigen-binding fragment specifically binds to spike proteins from BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC.

[0143] In some respects, the antibody or antigen-binding fragment contains V H The V H The antibody or antigen-binding fragment contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 9, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, the antibody or antigen-binding fragment contains V... L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 13, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 9 and 13, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0144] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, 11, and 12 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14, 15(GAS), and 16, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0145] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, 11, and 12, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14, 15(GAS), and 16, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 9 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 9), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:13 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:13), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0146] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 9 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 13 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and VL It contains the amino acid sequences shown in SEQ ID NO:9 and 13, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0147] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0148] c. Monoclonal antibody E184-105_F3 In some respects, the antibody or antigen-binding fragment is based on or derived from the E184-105_F3 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0149] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the E184-105_F3 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0150] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against Omeprón lineage pseudoviruses. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, and KP.2.3. Furthermore, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins.

[0151] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 17, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 21, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and VL They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 17 and 21, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0152] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 18, 19, and 20 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 22, 23(GAS), and 24, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0153] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 22, 23(GAS), and 24, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 17 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 17), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:21 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:21), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0154] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 17 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 21 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains VH and V L It contains the amino acid sequences shown in SEQ ID NO:17 and 21, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0155] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0156] d. Monoclonal antibody A63-652-32.1 In some respects, the antibody or antigen-binding fragment is based on or derived from the A63-652-32.1 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0157] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A63-652-32.1 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0158] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, and BA.2.86. Furthermore, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins.

[0159] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 25, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 29, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 25 and 29, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0160] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 26, 27, and 28 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 30, 31(AAS), and 32, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0161] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 26, 27, and 28, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 30, 31(AAS), and 32, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 25 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 25), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:29 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:29), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0162] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 25 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 29 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and VL It contains the amino acid sequences shown in SEQ ID NO:25 and 29, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0163] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0164] e. Monoclonal antibody F768-104_G7 In some respects, the antibody or antigen-binding fragment is based on or derived from the F768-104_G7 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0165] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the F768-104_G7 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0166] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against Omeprón lineage pseudotypes. In other respects, it exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, and BA.2.86. Furthermore, it neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins. In yet another respect, it neutralizes the D614G strain carrying the F456L mutation.

[0167] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 33, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 37, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V...H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 33 and 37, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0168] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 34, 35, and 36, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, 39(AAS), and 40, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0169] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 34, 35, and 36, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, 39(AAS), and 40, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 33 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 33), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:37 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:37), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0170] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 33 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... LIt contains the amino acid sequence shown in SEQ ID NO: 37 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:33 and 37, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0171] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0172] f. Monoclonal antibody E184-105_H1 In some respects, the antibody or antigen-binding fragment is based on or derived from the E184-105_H1 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0173] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the E184-105_H1 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0174] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, BA.2.86, and JN.1. In still other respects, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins.

[0175] In some respects, the antibody or antigen-binding fragment contains V H The V H The antibody or antigen-binding fragment contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 41, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, the antibody or antigen-binding fragment contains V... L The V LIt contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 45, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 41 and 45, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0176] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 42, 43, and 44 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 46, 47(DAS), and 48, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0177] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 42, 43, and 44, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 46, 47(DAS), and 48, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 41 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 41), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:45 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:45), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0178] In some respects, the antibody or antigen-binding fragment contains V HIt contains the amino acid sequence shown in SEQ ID NO: 41 and specifically binds to the coronavirus spike protein, and neutralizes the coronavirus. In further respects, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 45 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:41 and 45, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0179] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0180] g. Monoclonal antibody F768-104_D10 In some respects, the antibody or antigen-binding fragment is based on or derived from the F768-104_D10 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0181] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the F768-104_D10 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0182] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against Omeprón lineage pseudoviruses. In other respects, it exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, BA.2.86, and JN.1. Furthermore, it neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins. In yet another respect, it neutralizes the D614G strain carrying the F456L mutation.

[0183] In some respects, the antibody or antigen-binding fragment contains V H The V HContaining an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 49, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V... L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 53, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 49 and 53, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0184] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 50, 51, and 52 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 54, 55(GAS), and 56, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0185] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 50, 51, and 52, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 54, 55(GAS), and 56, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 49 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 49), and wherein the V LIt contains an amino acid sequence that has at least 90% identity with SEQ ID NO:53 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:53), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0186] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 49 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 53 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:49 and 53, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0187] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0188] h. Monoclonal antibody A63-652-1.4 In some respects, the antibody or antigen-binding fragment is based on or derived from the A63-652-1.4 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0189] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A63-652-1.4 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0190] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against Omeprón lineage pseudoviruses. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. Furthermore, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins.

[0191] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 57, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 61, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 57 and 61, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0192] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 58, 59, and 60 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 62, 63(DAS), and 64, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0193] In some respects, the antibody or antigen-binding fragment contains V H(It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 58, 59, and 60, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 62, 63(DAS), and 64, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 57 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 57), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:61 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:61), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0194] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 57 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 61 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:57 and 61, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0195] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0196] i. Monoclonal antibody E184-105_E5 In some respects, the antibody or antigen-binding fragment is based on or derived from the E184-105_E5 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0197] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the E184-105_E5 antibody, respectively. H and V LIt specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0198] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, BA.2.86, and JN.1. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins. In still another respect, this monoclonal antibody or antigen-binding fragment neutralizes the D614G strain carrying the F456L mutation.

[0199] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 65, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 69, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 65 and 69, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0200] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 66, 67, and 68, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 70, 71(AAS), and 72, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0201] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 66, 67, and 68, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 70, 71(AAS), and 72, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 65 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 65), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:69 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:69), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0202] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 65 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 69 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:65 and 69, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0203] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0204] j. Monoclonal antibody E184-105_H9 In some respects, the antibody or antigen-binding fragment is based on or derived from the E184-105_H9 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0205] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the E184-105_H9 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0206] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, BA.2.86, and JN.1. In still other respects, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins.

[0207] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 73, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 77, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 73 and 77, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0208] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 74, 75, and 76 respectively), and / or V L(It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 78, 79(AAS), and 80, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0209] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 74, 75, and 76, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 78, 79(AAS), and 80, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 73 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 73), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:77 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:77), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0210] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 73 and specifically binds to the coronavirus spike protein, and neutralizes the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 77 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:73 and 77, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0211] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0212] k. Monoclonal antibody E184-105_H2 In some respects, the antibody or antigen-binding fragment is based on or derived from the E184-105_H2 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0213] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the E184-105_H2 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0214] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, and BA.2.86. In still other respects, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins.

[0215] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 81, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 85, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 81 and 85, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0216] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 82, 83, and 84 respectively), and / or V L(It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 86, 87(AAS), and 88, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0217] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 82, 83, and 84, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 86, 87(AAS), and 88, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 81 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 81), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:85 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:85), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0218] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 81 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 85 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:81 and 85, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0219] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0220] l. Monoclonal antibody A80-102+103_G1 In some respects, the antibody or antigen-binding fragment is based on or derived from the A80-102+103_G1 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0221] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A80-102+103_G1 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0222] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, XBB, EG.5.1, and BA.2.86. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from bats and pangolins. Furthermore, this monoclonal antibody or antigen-binding fragment neutralizes the D614G strain carrying the F456L mutation.

[0223] In some respects, the antibody or antigen-binding fragment contains V H The V H Containing an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 89, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 93, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 89 and 93, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0224] In some respects, the antibody or antigen-binding fragment contains V H(which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 90, 91, and 92 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 94, 95(NNN), and 96, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0225] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 90, 91, and 92, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 94, 95(NNN), and 96, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 89 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 89), and wherein the V L It contains an amino acid sequence that has at least 90% identity with SEQ ID NO:93 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO:93), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0226] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 89 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 93 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO:89 and 93, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0227] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0228] m. Monoclonal antibody A80-103_E2 In some respects, the antibody or antigen-binding fragment is based on or derived from the A80-103_E2 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0229] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A80-103_E2 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0230] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB. In still other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BAEG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from animals, as well as SARS-CoV.

[0231] In some respects, the antibody or antigen-binding fragment contains V H The V H Containing an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 97, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 101, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 97 and 101, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0232] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 98, 99, and 100 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 102, 103 (SDS), and 104, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0233] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 98, 99, and 100, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 102, 103 (SDS), and 104, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 97 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 97), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 101 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 101), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0234] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 97 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 101 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains VH and V L It contains the amino acid sequences shown in SEQ ID NO:97 and 101, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0235] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0236] n. Monoclonal antibody A80-103_B6 In some respects, the antibody or antigen-binding fragment is based on or derived from the A80-103_B6 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0237] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A80-103_B6 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0238] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB. In still other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BAEG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from animals, as well as SARS-CoV.

[0239] In some respects, the antibody or antigen-binding fragment contains V H The V H Containing an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 105, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V... L The V LIt contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 109, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 105 and 109, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0240] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 106, 107, and 108, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 110, 111(SYN), and 112, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0241] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 106, 107, and 108, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 110, 111(SYN), and 112, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 105 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 105), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 109 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 109), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0242] In some respects, the antibody or antigen-binding fragment contains V HIt contains the amino acid sequence shown in SEQ ID NO: 105 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 109 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 105 and 109, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0243] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0244] o. Monoclonal antibody A18-618-452.1 In some respects, the antibody or antigen-binding fragment is based on or derived from the A18-618-452.1 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0245] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A18-618-452.1 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0246] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB. In still other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BAEG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from animals, as well as SARS-CoV.

[0247] In some respects, the antibody or antigen-binding fragment contains V H The V HContaining an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 113, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 117, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 113 and 117, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0248] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 114, 115, and 116, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 118, 119(AAS), and 120, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0249] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 114, 115, and 116, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 118, 119(AAS), and 120, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 113 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 113), and wherein the V LIt contains an amino acid sequence that has at least 90% identity with SEQ ID NO: 117 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 117), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0250] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 113 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 117 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 113 and 117, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0251] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0252] p. Monoclonal antibody A80-102+103_C1 In some respects, the antibody or antigen-binding fragment is based on or derived from the A80-102+103_C1 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0253] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A80-102+103_C1 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0254] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB. In still other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BAEG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from animals, as well as SARS-CoV.

[0255] In some respects, the antibody or antigen-binding fragment contains V H The V H Containing an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 121, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 125, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 121 and 125, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0256] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 122, 123, and 124, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 126, 127(DAS), and 128, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0257] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 122, 123, and 124, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 126, 127(DAS), and 128, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 121 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 121), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 125 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 125), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0258] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 121 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 125 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 121 and 125, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0259] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0260] q. Monoclonal antibody A43-d321.10.4 In some respects, the antibody or antigen-binding fragment is based on or derived from the A43-d321.10.4 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0261] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the A43-d321.10.4 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0262] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudotypes. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB. In still other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BAEG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from animals, as well as SARS-CoV.

[0263] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 129, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 133, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 129 and 133, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0264] In some respects, the antibody or antigen-binding fragment contains V H(which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 130, 131, and 132, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 134, 135(DAS), and 136, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0265] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 130, 131, and 132, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 134, 135(DAS), and 136, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 129 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 129), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 133 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 133), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0266] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 129 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 133 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 129 and 133, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0267] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0268] r. Monoclonal antibody F768-100-pt2_A05 In some respects, the antibody or antigen-binding fragment is based on or derived from the F768-100-pt2_A05 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0269] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the F768-100-pt2_A05 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0270] In some respects, this antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 Omeprón lineage pseudoviruses. In other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB. In still other respects, this antibody or antigen-binding fragment exhibits neutralizing activity against BAEG.5.1, FL.1.5.1, and BA.2.86. In yet another respect, this antibody or antigen-binding fragment neutralizes SARS-CoV-2-related coronaviruses from animals, as well as SARS-CoV.

[0271] In some respects, the antibody or antigen-binding fragment contains V H The V H Containing an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 137, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 141, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 137 and 141, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0272] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 138, 139, and 140 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 142, 143(WAS), and 144, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0273] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 138, 139, and 140, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 142, 143 (WAS), and 144, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 137 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 137), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 141 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 141), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0274] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 137 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... LIt contains the amino acid sequence shown in SEQ ID NO: 141 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 137 and 141, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0275] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0276] s. Monoclonal antibody A80-102_G5 In some respects, the antibody or antigen-binding fragment is based on or derived from the A80-102_G5 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0277] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A80-102_G5 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0278] In some respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 variants and / or SARS-CoV-associated sabefoviruses. In other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against Omeprone BA.1, BA.2, BA.5, EG.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In still other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2-associated viruses from animals (including the pangolin GX variant) and SARS-CoV-associated sabefoviruses from humans, bats, and civets.

[0279] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 145, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The VL It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 149, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 145 and 149, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0280] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 146, 147, and 148, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 150, 151(DAS), and 152, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0281] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 146, 147, and 148, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 150, 151(DAS), and 152, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 145 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 145), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 149 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 149), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0282] In some respects, the antibody or antigen-binding fragment contains VH It contains the amino acid sequence shown in SEQ ID NO: 145 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 149 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 145 and 149, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0283] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0284] t. Monoclonal antibody A18-338-19.5 In some respects, the antibody or antigen-binding fragment is based on or derived from the A18-338-19.5 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0285] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A18-338-19.5 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0286] In some respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 variants and / or SARS-CoV-associated sabefoviruses. In other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against Omeprone BA.1, BA.2, BA.5, EG.5.1, and BA.2.86. In still other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2-associated viruses from animals (including the pangolin GX variant) and SARS-CoV-associated sabefoviruses from humans, bats, and civets.

[0287] In some respects, the antibody or antigen-binding fragment contains V H The V HContaining an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 153, and specifically binding to and neutralizing the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V... L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 157, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 153 and 157, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0288] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 154, 155, and 156, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 158, 159(DAS), and 160, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0289] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 154, 155, and 156, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 158, 159(DAS), and 160, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 153 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 153), and wherein the V LIt contains an amino acid sequence having at least 90% identity with SEQ ID NO: 157 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 157), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0290] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 153 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 157 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 153 and 157, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0291] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0292] u. Monoclonal antibody A18-338-19.6 In some respects, the antibody or antigen-binding fragment is based on or derived from the A18-338-19.6 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0293] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A18-338-19.6 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0294] In some respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 variants and / or SARS-CoV-associated sabefoviruses. In other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against Omeprone BA.1, BA.2, BA.5, EG.5.1, and BA.2.86. In still other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2-associated viruses from animals (including the pangolin GX variant) and SARS-CoV-associated sabefoviruses from humans, bats, and civets.

[0295] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 161, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 165, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 161 and 165, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0296] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 162, 163, and 164, respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 166, 167(DAS), and 168, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0297] In some respects, the antibody or antigen-binding fragment contains V H(It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 162, 163, and 164, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 166, 167(DAS), and 168, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 161 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 161), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 165 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 165), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0298] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 161 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 165 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 161 and 165, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0299] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0300] v. Monoclonal antibody A18-338-19.8 In some respects, the antibody or antigen-binding fragment is based on or derived from the A18-338-19.8 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0301] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A18-338-19.8 antibody, respectively. H and VL It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0302] In some respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 variants and / or SARS-CoV-associated sabefoviruses. In other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against Omeprone BA.1, BA.2, BA.5, EG.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. In still other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2-associated viruses from animals (including the pangolin GX variant) and SARS-CoV-associated sabefoviruses from humans, bats, and civets.

[0303] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 169, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 173, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 169 and 173, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0304] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 170, 171, and 172, respectively), and / or V L(It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 174, 175(DAT), and 176, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0305] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 170, 171, and 172, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 174, 175(DAT), and 176, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 169 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 169), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 173 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 173), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0306] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 169 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 173 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 169 and 173, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0307] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0308] w. Monoclonal antibody A18-338-19.9 In some respects, the antibody or antigen-binding fragment is based on or derived from the A18-338-19.9 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus.

[0309] In some instances, the antibody or antigen-binding fragment comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) containing the A18-338-19.9 antibody, respectively. H and V L It specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus could be SARS-CoV-2.

[0310] In some respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2 variants and / or SARS-CoV-associated sabefoviruses. In other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against Omeprone BA.1, BA.2, BA.5, EG.5.1, and BA.2.86. In still other respects, the antibody or antigen-binding fragment exhibits neutralizing activity against SARS-CoV-2-associated viruses from animals (including the pangolin GX variant) and SARS-CoV-associated sabefoviruses from humans, bats, and civets.

[0311] In some respects, the antibody or antigen-binding fragment contains V H The V H It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 177, and specifically binds to and neutralizes the coronavirus spike protein. In further respects, this antibody or antigen-binding fragment contains V L The V L It contains an amino acid sequence having at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequence shown in SEQ ID NO: 181, and specifically binds to and neutralizes the coronavirus spike protein. In another aspect, the antibody or antigen-binding fragment contains V... H and V L They each independently contain an amino acid sequence that has at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identity with the amino acid sequences shown in SEQ ID NO: 177 and 181, and specifically bind to and neutralize the coronavirus spike protein. This coronavirus may be SARS-CoV-2.

[0312] In some respects, the antibody or antigen-binding fragment contains V H (which includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 178, 179, and 180 respectively), and / or V L (It contains LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 182, 183(DAS), and 184, respectively), and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0313] In some respects, the antibody or antigen-binding fragment contains V H (It includes HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 178, 179, and 180, respectively), V L (It includes LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 182, 183(DAS), and 184, respectively), wherein the V H Contains an amino acid sequence having at least 90% identity with SEQ ID NO: 177 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 177), and wherein the V L It contains an amino acid sequence having at least 90% identity with SEQ ID NO: 181 (e.g., 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 181), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations arising from sequence identity are located outside the CDR. The coronavirus may be SARS-CoV-2.

[0314] In some respects, the antibody or antigen-binding fragment contains V H It contains the amino acid sequence shown in SEQ ID NO: 177 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. Furthermore, this antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 181 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, this antibody or antigen-binding fragment contains V H and V L It contains the amino acid sequences shown in SEQ ID NO: 177 and 181, respectively, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. This coronavirus may be SARS-CoV-2.

[0315] In some respects, the disclosed antibodies inhibit viral entry and / or replication.

[0316] 2. Additional description of antibody-antigen binding fragments The antibodies or antigen-binding fragments of antibodies disclosed herein may be human antibodies or fragments thereof. Chimeric antibodies are also provided. The antibody or antigen-binding fragment may contain any suitable frame region, such as (but not limited to) human frame regions from another source, or optimized frame regions. Alternatively, heterologous frame regions (such as, but not limited to, mouse or monkey frame regions) may be contained in the heavy or light chain of the antibody.

[0317] The antibody can be any isotype. It can be, for example, IgA, IgM, or IgG antibodies, such as IgG1, IgG2, IgG3, or IgG4. The class of antibodies that specifically bind to the coronavirus spike protein can be converted to another class. In one aspect, antibodies encoding V... L or V H The nucleic acid molecules were isolated so that they did not contain any nucleic acid sequences encoding the light or heavy chain constant regions. Then, the sequences encoding V... L or V H The nucleic acid molecule B8 is operatively linked to C, which encodes molecules from different classes of immunoglobulins. L Or C H The nucleic acid sequence. This can be, for example, using a sequence containing C. L Or C H This can be achieved using carriers or nucleic acid molecules. For example, an antibody that initially binds specifically to the spike protein and is IgG can be class-converted to IgA. Class conversion can be used to convert one IgG subclass to another, such as from IgG1 to IgG2, IgG3, or IgG4.

[0318] In some instances, the disclosed antibodies are antibody oligomers, such as dimers, trimers, tetramers, pentamers, hexamers, heptamers, octamers, etc.

[0319] The antibody or antigen-binding fragment can be derivatized or linked to another molecule (e.g., another peptide or protein). Typically, the antibody or antigen-binding fragment is derivatized so that binding to the spike protein is not adversely affected by derivatization or labeling. For example, the antibody or antigen-binding fragment can be functionally linked (through chemical coupling, genetic fusion, non-covalent binding, or other means) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable biomarker, an effector molecule, or a protein or peptide that mediates the binding of the antibody or antibody portion to another molecule (e.g., the streptavidin core region or a multihistidine tag).

[0320] (a) Combining affinity In several respects, the antibody or antigen-binding fragment has a size not exceeding 1.0 x 10⁻⁶. -8 M, not greater than 5.0 x 10 -8 M, not greater than 1.0 x 10 -9 M, not greater than 5.0 x 10 -9 M, not greater than 1.0 x 10 -10 M, not greater than 5.0 x 10 -10 M or not greater than 1.0 x 10 -11 The affinity of M (e.g., through K) D (Measurement) specifically binds to the coronavirus spike protein. K D This can be measured, for example, by using a Fab version of the antibody of interest and its antigen in a radiolabeled antigen-binding assay (RIA). In one assay, the solution-binding affinity of Fab to the antigen is measured by mixing Fab with the lowest concentration of ( 125 I) Labeled antigens are equilibrated in the presence of a titration series of unlabeled antigens, and then the bound antigens are captured on a plate coated with anti-Fab antibody (see, for example, Chen et al.). J. Mol. Biol. 293(4):865-881, 1999). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23°C). In non-adsorption plates (NUNC™ catalog number 269620), 100 μM or 26 pM [ 125 I]-Antigen is mixed with a series of dilutions of the Fab of interest (e.g., with Presta, etc.). Cancer Res. (Consistent with the evaluation of the anti-VEGF antibody Fab-12 in 57(20):4593-4599, 1997). The Fab of interest was then incubated overnight; however, incubation could be prolonged (e.g., about 65 hours) to ensure equilibration. The mixture was then transferred to a capture plate and incubated at room temperature (e.g., one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. After the plate was dried, 150 μl / well of scintillation buffer (MICROSCINT™-20; PerkinElmer) was added and the plate was run on a TOPCOUNT™ γ counter (PerkinElmer) for several tens of minutes. Each Fab concentration that gave a maximum binding of less than or equal to 20% was selected for the competitive binding assay.

[0321] In another measurement, K D Surface plasmon resonance assays can be performed using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C, using an immobilized antigen CM5 chip (approximately 10 response units (RU)). In short, the carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 l / min to achieve approximately 10 response units (RU) of conjugated protein. After antigen injection, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, serially diluted Fab (0.78 nM to 500 nM) was injected into PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at a flow rate of approximately 25 l / min at 25 °C. Binding rate (k on ) and dissociation rate (k off The equilibrium dissociation constant (K0.05) was calculated by simultaneously fitting binding and dissociation sensor maps using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2). D ) is calculated as k off / k on The ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the binding rate measured by the above surface plasmon resonance method exceeds 10... 6 M 1 s 1 The binding rate can be determined using fluorescence quenching, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of progressively increasing antigen concentrations, as measured in a spectrometer such as an Aviv Instruments 8000 Series SLM-AMINCO™ spectrophotometer with a stop-flow function or a ThermoSpectronic 8000 Series SLM-AMINCO™ spectrophotometer with a stirred cuvette. Affinity can also be measured using high-throughput SPR from a Carterra LSA.

[0322] (b) Multispecific antibodies In some respects, multispecific or bispecific antibodies, such as dual variable domain antibodies (DVD-IG™), are provided that comprise an antibody or antigen-binding fragment that specifically binds to the coronavirus spike protein, as provided herein. Multispecific antibody forms produced using the antibodies and antigen-binding fragments of this disclosure are disclosed, for example, in Misasi et al., doi.org / 10.1101 / 2022.07.29.502029, biorxiv.org / content / 10.1101 / 2022.07.29.502029v3, November 21, 2022, which are incorporated herein by reference.

[0323] The multispecific antibody can be, for example, a bispecific or trispecific antibody. In some respects, a multivalent antibody is a monospecific antibody (e.g., trivalent but with only one specificity). A multispecific antibody may contain A18-448.1 or its antigen-binding fragment. A multispecific antibody may contain F768-104_B2 or its antigen-binding fragment. A multispecific antibody may contain a group 3 antibody or its antigen-binding fragment. A multispecific antibody may contain a group 4 antibody or its antigen-binding fragment. A multispecific antibody may contain a group 5 antibody or its antigen-binding fragment. A multispecific antibody may contain a group 6 antibody or its antigen-binding fragment.

[0324] The multispecific antibody, such as a bispecific antibody, may comprise an A18-618-448.1 antibody or its antigen-binding fragment, and a fourth group antibody or its antigen-binding fragment. The fourth group antibody may be, for example, the F768-104_G7 antibody. The multispecific antibody, such as a bispecific antibody, may comprise an F768-104_B2 antibody or its antigen-binding fragment, combined with a third group antibody or its antigen-binding fragment. The third group antibody may be, for example, A80-102_G5. The multispecific antibody, such as a bispecific antibody, may comprise an F768-104_B2 antibody or its antigen-binding fragment, and an A18-448.1 antibody or its antigen-binding fragment.

[0325] Other antibodies that specifically bind to SARS-CoV-2 may be included in this multispecific antibody, such as a bispecific antibody. In some aspects, the multispecific antibody, such as a bispecific antibody, may comprise F768-104_B2 or its antigen-binding fragment and F769-E12 or its antigen-binding fragment, disclosed in U.S. Provisional Application No. 63 / 433,719, which is incorporated herein by reference. In other aspects, the multispecific antibody, such as a bispecific antibody, may comprise A18-448.1 or its antigen-binding fragment and F769-E12, F770-G11, or F770-E8 or their antigen-binding fragments, disclosed in U.S. Application No. 63 / 433,719, which is incorporated herein by reference.

[0326] In some respects, the antibody can be a bispecific antibody. Bispecific antibodies can be designed and generated using any suitable method, such as crosslinking two or more antibodies of the same or different types, or antigen-binding fragments (e.g., scFv). Exemplary methods for preparing multispecific antibodies (e.g., bispecific antibodies) include those described in PCT Publication No. WO2013 / 163427, which is incorporated herein by reference in its entirety. Non-limiting examples of suitable crosslinking agents include heterobifunctional crosslinking agents (having two different reactive groups separated by a suitable spacer, such as m-maleimide benzoyl-N-hydroxysuccinimide ester) or homobifunctional crosslinking agents (e.g., disuccinimide octanoate).

[0327] This multispecific antibody can have any suitable form that allows the antibody or antigen-binding fragment provided herein to bind to the coronavirus spike protein. Bispecific single-chain antibodies can be encoded by a single nucleic acid molecule. Non-limiting examples of bispecific single-chain antibodies and methods for constructing such antibodies are provided in U.S. Patent Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, and 6,723,538. Other examples of bispecific single-chain antibodies can be found in PCT application No. WO 99 / 54440; Mack et al. J. Immunol. , 158(8):3965-3970, 1997; Mack et al, Proc. Natl. Acad. Sci. USA , 92(15):7021-7025, 1995; Kufer et al., Cancer Immunol. Immunother. , 45(3-4):193-197, 1997; Löffler et al., Blood, 95(6):2098-2103, 2000; and Brühl et al., J. Immunol. , 166(4):2420-2426, 2001. The generation of bispecific Fab-scFv (“bibody”) molecules is described, for example, by Schoonjans et al. ( J. Immunol. , 165(12):7050-7057, 2000) and Willems et al. ( J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1-2):161-176, 2003). For bibody, scFv molecules can be fused to V. L -CL (L) or V H - On one of the CH1 chains, for example, to produce a bibody, an scFv is fused to the C end of the Fab chain.

[0328] A bispecific tetravalent immunoglobulin, known as a dual variable domain immunoglobulin or DVD-immunoglobulin molecule, is disclosed in Wu et al., MAbs. 2009;1:339–47, doi: 10.4161 / mabs.1.4.8755, which is incorporated herein by reference. See also Nat Biotechnol. 2007 Nov;25(11):1290-7. doi: 10.1038 / nbt1345, electronically published on 14 October 2007, which is also incorporated herein by reference. The DVD-immunoglobulin molecule comprises two heavy chains and two light chains. However, unlike IgG, both the heavy and light chains of the DVD-immunoglobulin molecule contain V-type components via adapter sequences in existing monoclonal antibodies (mAbs). H and V L The N-terminus is linked to an additional variable domain (VD). Therefore, when the heavy and light chains are combined, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites (see Jakob et al.). Mabs 5: 358-363, 2013, which is incorporated herein by reference; see Figure 1 for schematic diagram and space-filled diagram. DVD-immunoglobulin molecules are capable of binding two different antigens simultaneously on each DFab.

[0329] The outermost or N-terminal variable domain is called VD1, and the innermost variable domain is called VD2; VD2 is located near the C-terminus CH1 or CL. As disclosed by Jakob et al. (see above), DVD-immunoglobulin molecules can be mass-produced and purified to homogeneity, exhibiting pharmacological properties similar to conventional IgG1 and demonstrating in vivo potency. Any disclosed monoclonal antibody can be included in the form of DVD-immunoglobulin.

[0330] (c) Antigen-binding fragments This disclosure covers antigen-binding fragments such as Fab, F(ab')2, and Fv, which contain heavy chains and V L They specifically bind to the coronavirus spike protein. These antibody fragments retain the ability to selectively bind to antigens and are "antigen-binding" fragments. Non-limiting examples of such fragments include: (1) Fab, which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digesting the whole antibody with papain to obtain the complete light chain and a portion of the heavy chain; (2) Fab', this antibody fragment can be obtained by treating the whole antibody with pepsin and then reducing it to obtain the complete light chain and part of the heavy chain; (3) (Fab')2, this antibody fragment can be obtained by treating the whole antibody with pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing V expressed as two strands. L and V L ;as well as (5) Single-chain antibodies (such as scFv) are defined as containing V linked by a suitable polypeptide linker. H and V L Genetically engineered molecules, as single-stranded molecules for gene fusion (see, for example, Ahmad et al.), Clin. Dev. Immunol. , 2012,doi:10.1155 / 2012 / 980250; Marbry and Snavely, IDrugs , 13(8):543-549, 2010). scFv in V H Domain and V L The intramolecular orientation of the domains is not decisive for the antibody provided (e.g., for the provided multispecific antibody). Therefore, domains with two possible arrangements (V...) can be used. H Structural domain - joint structural domain - V L Domain; V L Structural domain - joint structural domain - V H scFv of structural domain.

[0331] (6) Single-chain antibody dimer (scFV2), defined as a dimer of scFV. It is also known as a “miniantibody”.

[0332] Any suitable method can be used to generate the antigen-binding fragments discussed above. Non-limiting examples are provided by Harlow and Lane. Antibodies:A Laboratory Manual , 2 nd, Cold Spring Harbor Laboratory, New York, 2013.

[0333] Antigen-binding fragments can be prepared by hydrolyzing antibodies through proteolysis or by expressing DNA encoding the fragment in host cells (such as *E. coli* cells). Antigen-binding fragments can also be obtained by digesting whole antibodies with pepsin or papain using conventional methods. For example, an antigen-binding fragment can be generated by digesting an antibody with pepsin to produce a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the thiol group generated by disulfide bond cleavage, to produce a 3.5S Fab' monovalent fragment.

[0334] Other methods of antibody cleavage can also be used, such as separating the heavy chain to form monovalent light-heavy chain fragments, further cleaving the fragments, or other enzymatic, chemical, or genetic techniques, as long as these fragments bind to the antigen recognized by the intact antibody.

[0335] (d) variant In some respects, amino acid sequence variants of the antibodies and multispecific antibodies (e.g., bispecific antibodies) provided herein are offered. For example, it may be necessary to improve the binding affinity and / or other biological properties of the antibody or bispecific antibody. Amino acid sequence variants of the antibody can be introduced by introducing appropriate modifications into the encoding antibody V. H Domain and / or V L The modification can be performed within the nucleotide sequence of the domain, or via peptide synthesis. Such modifications include, for example, residue deletions, and / or insertions and / or substitutions within the antibody amino acid sequence. Any combination of deletions, insertions, and substitutions can be performed to obtain the final construct, provided that the final construct possesses the desired characteristics, such as antigen binding.

[0336] In some respects, variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutagenesis include the CDR and frame regions. Amino acid substitutions can be introduced into antibodies of interest, and products can be screened to obtain desired activities, such as preserved / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0337] This variant typically retains the correct folding and V-shape. H With V L The amino acid residues necessary for stabilization between regions will retain the charge properties of the residues to maintain the molecule's low pI and low toxicity. This can be achieved in V... H and V L Amino acid substitutions are performed in the region to increase yield.

[0338] In some aspects, compared with the amino acid sequence shown in SEQ ID NO: 1, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some aspects, compared with the amino acid sequence shown in SEQ ID NO: 5, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0339] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 9, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 13, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0340] In another aspect, compared with the amino acid sequence shown in SEQ ID NO: 17, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some aspects, compared with the amino acid sequence shown in SEQ ID NO: 21, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0341] In other aspects, compared with the amino acid sequence shown in SEQ ID NO: 29, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In other aspects, compared with the amino acid sequence shown in SEQ ID NO: 29, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0342] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 33, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 37, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0343] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 41, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 45, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0344] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 49, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 53, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0345] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 57, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 61, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0346] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 65, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 69, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0347] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 73, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 77, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0348] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 81, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 85, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0349] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 89, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 93, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0350] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 97, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 101, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0351] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 105, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 109, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0352] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 113, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 117, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0353] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 121, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 125, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0354] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 129, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 133, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0355] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 137, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 141, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0356] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 145, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 149, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0357] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 153, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 157, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0358] In other respects, compared with the amino acid sequence shown in SEQ ID NO: 161, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 165, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0359] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 169, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 173, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0360] In many respects, compared with the amino acid sequence shown in SEQ ID NO: 177, the heavy chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions). In some respects, compared with the amino acid sequence shown in SEQ ID NO: 181, the light chain of this antibody contains up to 10 (e.g., up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (e.g., conserved amino acid substitutions).

[0361] In some respects, compared to known frame regions or to the frame regions of antibodies, the antibody or antigen-binding fragment may contain up to 10 (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions (e.g., conserved amino acid substitutions) in the frame region of the heavy chain of an antibody / multispecific antibody, or the frame region of the heavy and light chains of an antibody / bispecific antibody, and maintain specific binding activity to spike protein epitopes. In these respects, the substitutions are not in the CDR.

[0362] In some respects, substitution, insertion, or deletion can occur within one or more CDRs, as long as such changes do not substantially reduce the antibody's ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (such as the conserved substitutions presented herein) can be made in CDRs. Variant V presented above... H and V L In some aspects of the sequence, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions. Variant V provided above... H and V L In some aspects of the sequence, only the framework residues are modified, so that the CDR remains unchanged.

[0363] To increase antibody binding affinity, V L and V H The region can be randomly mutated, for example through a process similar to the in vivo somatic mutation process responsible for antibody affinity maturation during the innate immune response, by randomly mutating within the HCDR3 or LCDR3 region. Therefore, in vitro affinity maturation can be achieved by amplifying V using PCR primers complementary to HCDR3 or LCDR3, respectively. H and V L The process is completed in a specific region. During this process, the primers have been "spiked" with a random mixture of four nucleotide bases at certain positions, resulting in the PCR product encoding V... H and V L In the section, its V H and / or V L Random mutations have been introduced into the CDR3 region. These random mutations can be tested on V. H and V L The segment is used to determine the binding affinity to the spike protein. In a specific instance, V H The amino acid sequence is one of SEQ ID NO: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, 105, 113, 121, 129, 137, 145, 153, 161, 169, or 177. In other instances, V L The amino acid sequences are one of SEQ ID NO: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, 109, 117, 125, 133, 141, 149, 157, 165, 173 or 181.

[0364] In some respects, the antibodies, antigen-binding fragments, or bispecific antibodies disclosed herein are modified to increase or decrease the degree of glycosylation of the antibody or antigen-binding fragment. The addition or deletion of glycosylation sites can be conveniently achieved by altering the amino acid sequence to create or remove one or more glycosylation sites.

[0365] In the case of an antibody containing an Fc region, the carbohydrates to which it is attached can be altered. Naturally occurring antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region via N-linking. See, for example, Wright et al. Trends Biotechnol. 15(1):26-32, 1997. This oligosaccharide can contain various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some respects, oligosaccharides in antibodies can be modified to produce antibody variants with certain improved properties.

[0366] In one aspect, variants with carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region are provided. For example, the fucose content in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The fucose content is determined by calculating the average fucose content within the glycan chain at Asn297 relative to the sum of all sugar structures attached to Asn297 (e.g., complex, heterozygous, and high-mannose structures), as described, for example, in WO 2008 / 077546, measured by MALDI-TOF mass spectrometry. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region; however, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants can possess improved ADCC function. See, for example, U.S. Patent Publication No. US2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003 / 0157108; WO2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO 2002 / 031140; Okazaki et al., J. Mol. Biol. ,336(5):1239-1249, 2004; Yamane-Ohnuki et al., Biotechnol. Bioeng. 87(5):614-622, 2004. Examples of cell lines capable of producing defucosylation antibodies include Lec 13 CHO cells with protein fucosylation defects (Ripka et al., Arch. Biochem. Biophys. 249(2):533-545,1986; US Patent Application Nos. US 2003 / 0157108 and WO 2004 / 056312, particularly Example 11), and knockout cell lines, such as CHO cells with α-1,6-fucosyltransferase gene FUT8 knocked out (see, for example, Yamane-Ohnuki et al., Biotechnol. Bioeng.,87(5): 614-622, 2004; Kanda et al, Biotechnol. Bioeng. 94(4):680-688, 2006; and WO2003 / 085107).

[0367] Antibody variants having bisected oligosaccharides are also provided, for example, wherein the biantennary oligosaccharides attached to the Fc region of the antibody are bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.); U.S. Patent No. 6,602,684 (Umana et al.); and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0368] In several respects, the constant region of this antibody or bispecific antibody contains one or more amino acid substitutions to optimize the antibody's in vivo half-life. The serum half-life of IgG Ab is regulated by the neonatal Fc receptor (FcRn). Therefore, in several respects, this antibody contains amino acid substitutions that increase binding to FcRn. Non-limiting examples of such substitutions include the substitutions T250Q and M428L at the IgG constant region (see, e.g., Hinton et al.). J Immunol., 176(1):346-356, 2006); M428L and N434S (“LS” mutations, see, for example, Zalevsky et al., Nature Biotechnol. 28(2):157-159 , 2010); N434A (see, for example, Petkova et al.) Int. Immunol ., 18(12):1759-1769, 2006); T307A, E380A and N434A (see, for example, Petkova et al.) Int. Immunol ., 18(12):1759-1769,2006); and M252Y, S254T and T256E (see, for example, Dall'Acqua et al.) J. Biol. Chem ., 281(33):23514-23524, 2006). The disclosed antibody and antigen binding fragments may be linked to or contain Fc peptides including any of the above substitutions, for example, the Fc peptide may contain M428L and N434S substitutions.

[0369] In some respects, the antibodies or multispecific (e.g., bispecific) antibodies provided herein can be further modified to include additional non-protein moieties. Suitable moieties for antibody derivatization include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, polypropylene oxide / ethylene oxide copolymers, polyoxyethyleneized polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. Typically, the quantity and / or type of polymer used for derivatization can be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, and whether the antibody derivative will be used for application under defined conditions.

[0370] B. Conjugates As disclosed herein, antibodies that specifically bind to the coronavirus spike protein, antigen-binding fragments, and multispecific (e.g., bispecific) antibodies can be conjugated to reagents such as effector molecules or detectable biomarkers. Both covalent and non-covalent attachment methods can be used. A variety of effector molecules and detectable biomarkers can be used, including but not limited to toxins and radioactive reagents. 125 I, 32 P, 14 C 3 H and 35 S, along with other markers, targeting moieties, enzymes, and ligands, etc. The selection of specific effector molecules or detectable biomarkers depends on the specific target molecule or cell and the desired biological effect.

[0371] The method of attaching a detectable biomarker to an antibody, antigen-binding fragment, or bispecific antibody varies depending on the chemical structure of the effector molecule. Peptides typically contain multiple functional groups, such as carboxyl (-COOH), free amino (-NH2), or thiol (-SH), which can be used to react with suitable functional groups on the peptide to achieve binding of the effector molecule or detectable biomarker. Alternatively, the antibody, antigen-binding fragment, or bispecific antibody can be derivatized to expose or attach additional reactive functional groups. Derivatization may involve the attachment of any suitable linker molecule. The linker is capable of forming a covalent bond with both the antibody or antigen-binding fragment and the effector molecule or detectable biomarker. Suitable linkers include, but are not limited to, straight-chain or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. When both the antibody, antigen-binding fragment, or bispecific antibody and the effector molecule or detectable biomarker are peptides, the linker can be linked to a constitutive amino acid via its side chain (e.g., via a disulfide bond of cysteine) or α-carbon, or via the amino and / or carboxyl groups of the terminal amino acid.

[0372] Given the numerous reported methods for attaching various radiodiagnostic compounds, radiotherapy compounds, labels (e.g., enzymes or fluorescent molecules), toxins, and other reagents to antibodies, suitable methods for attaching a given reagent to an antibody, antigen-binding fragment, or bispecific antibody can be determined.

[0373] Antibodies, antigen-binding fragments, or multispecific (e.g., bispecific) antibodies can be conjugated to detectable biomarkers; for example, detectable biomarkers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as computed tomography (CT), computed axial computed tomography (CAT), magnetic resonance imaging (MRI), magnetic resonance imaging (MTR), ultrasound, fiber optic examination, and laparoscopy. Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme conjugates, radioactive isotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable biomarkers include fluorescent compounds, including luciferin, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent biomarkers are also useful, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). Antibodies, antigen-binding fragments, or multispecific (e.g., bispecific) antibodies can also be conjugated to enzymes that can be used for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When an antibody or antigen-binding fragment is conjugated to a detectable enzyme, detection can be achieved by adding additional reagents that the enzyme uses to produce a distinguishable reaction product. For example, in the presence of horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine produces a colored reaction product that can be detected visually. Antibodies, antigen-binding fragments, or multispecific (e.g., bispecific) antibodies can also be conjugated to biotin and detected by indirectly measuring avidin or streptavidin binding. It should be noted that avidin itself can be conjugated to enzymes or fluorescent labels.

[0374] Antibodies, antigen-binding fragments, or bispecific antibodies can be conjugated to paramagnetic agents (e.g., gadolinium). Paramagnetic agents such as superparamagnetic iron oxide can also be used as labels. Antibodies can also be conjugated to lanthanides (e.g., europium and dysprosium) and manganese. Antibodies, antigen-binding fragments, or multispecific (e.g., bispecific) antibodies can also be labeled with predetermined peptide epitopes recognized by second reporter molecules (e.g., leucine zipper pairs, secondary antibody binding sites, metal-binding domains, epitope tags).

[0375] Antibodies, antigen-binding fragments, or multispecific (e.g., bispecific) antibodies can also be conjugated to radiolabeled amino acids, for example, for diagnostic purposes. For instance, radiolabeling can be used to detect coronaviruses via radiography, emission spectroscopy, or other diagnostic techniques. Examples of peptide labeling include, but are not limited to, the following radioisotopes: 3 H, 14 C 35 S, 90 Y、 99m Tc, 111 In、 125 I,131 I. Radioactive labels can be detected, for example, using photographic film or a scintillation counter; fluorescent labels can be detected by detecting the emitted light using a photodetector. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction products produced by the enzyme acting on the substrate; colorimetric labels are detected simply by visually observing the colored label.

[0376] The average number of detectable marker moieties on each antibody, antigen-binding fragment, or bispecific antibody in a conjugate can range, for example, from 1 to 20 moieties per antibody or antigen-binding fragment. In some aspects, the average number of effector molecules or detectable marker moieties on each antibody or antigen-binding fragment in a conjugate ranges from about 1 to about 2, about 1 to about 3, about 1 to about 8, about 2 to about 6, about 3 to about 5, or about 3 to about 4. The loading of the conjugate (e.g., the effector molecule ratio per antibody) can be controlled in various ways, for example, by: (i) restricting the molar excess of the effector molecule-linker intermediate or linker reagent relative to the antibody; (ii) restricting the conjugation reaction time or temperature; (iii) partial or restrictive reduction conditions for cysteine ​​thiol modification; (iv) engineering the amino acid sequence of the antibody using recombinant techniques such that the number and position of cysteine ​​residues are modified to control the number or position of linker-effect molecule attachments.

[0377] C. Polynucleotides and their expression Nucleic acid molecules (e.g., cDNA or RNA molecules) encoding amino acid sequences of antibodies, antigen-binding fragments, bispecific antibodies, and conjugates that specifically bind to the coronavirus spike protein disclosed herein are provided. Using the amino acid sequences provided herein (e.g., CDR sequences and V...), nucleic acid molecules (e.g., cDNA or RNA molecules) can be used. H and V L From sequences (sequences available in the art, such as frame or constant region sequences) and the genetic code, nucleic acids encoding these molecules can be readily generated. In several respects, nucleic acid molecules can encode the V of the disclosed antibody or antigen-binding fragment. H V L 、 or V H and V L Both (e.g., in bicistronic expression vectors). In some respects, the nucleic acid molecule encodes scFv. In several respects, the nucleic acid molecule can be expressed in host cells (e.g., mammalian cells) to produce the disclosed antibody or antigen-binding fragment. Nucleic acid molecules encoding scFv are provided.

[0378] The genetic code can be used to construct multiple functionally equivalent nucleic acid sequences, such as sequences that are different but encode the same antibody sequence, or sequences that encode sequences containing V. L and / or V H Nucleic acid conjugates or fusion proteins of nucleic acid sequences.

[0379] In a non-limiting example, the isolated nucleic acid molecule encodes the V of the disclosed antibody. H In another non-limiting example, the nucleic acid molecule encodes the V of the disclosed antibody. L In a further non-limiting example, the nucleic acid molecule may encode a bispecific antibody, for example, in the form of DVD-immunoglobulin.

[0380] Nucleic acid molecules encoding antibodies, antigen-binding fragments, multispecific (e.g., bispecific) antibodies, and conjugates that specifically bind to the coronavirus spike protein can be prepared by any suitable method, including, for example, cloning suitable sequences or direct chemical synthesis using standard methods. Chemical synthesis produces single-stranded oligonucleotides. These can be converted into double-stranded DNA by hybridization with complementary sequences or by polymerization with DNA polymerase using single strands as templates.

[0381] Exemplary nucleic acids can be prepared using cloning techniques. Examples of suitable cloning and sequencing techniques can be found, for example, in Green and Sambrook (…). Molecular Cloning: A Laboratory Manual , 4 th (ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (eds.) Current Protocols in Molecular Biology (New York: John Wiley and Sons, including supplements).

[0382] Nucleic acids can also be prepared through amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustaining sequence replication systems (3SR).

[0383] Nucleic acid molecules can be expressed in recombinant engineered cells such as bacteria, plants, yeast, insects, and mammalian cells. Antibodies, antigen-binding fragments, and conjugates can serve as expression bases for antibodies, antigens, and other antigens. H and / or V L The antibody and antigen-binding fragments can be expressed individually (with effector molecules or detectable markers linked as needed) or as fusion proteins. Any suitable method for expressing and purifying the antibody and antigen-binding fragments can be used; non-limiting examples are found in Al-Rubeai (ed.). Antibody Expression and Production Dordrecht; New York: Springer, 2011. Immunoadhesins can also be expressed. Therefore, in some instances, encoding V is provided. H and V LAnd the nucleic acid of immunoadhesins. The nucleic acid sequence may optionally encode a leader sequence.

[0384] To generate scFv, the encoded V can be... H and V L The DNA fragment is operatively linked to another fragment encoding a flexible adapter (e.g., a fragment encoding the amino acid sequence (Gly4-Ser)3), allowing V to... H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V H The structural domains are connected by flexible joints (see, for example, Bird et al.). Science ,242(4877):423-426, 1988; Huston et al. , Proc. Natl. Acad. Sci. USA , 85(16):5879-5883,1988; McCafferty et al. Nature , 348:552-554, 1990; Kontermann and Dübel (eds.), Antibody Engineering Vols. 1-2, 2 nd ed., Springer-Verlag, 2010; Greenfield (ed.), Antibodies: A Laboratory Manual , 2 nd (ed. New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, the adapter may contain cleavage sites, such as furin cleavage sites.

[0385] If only a single V is used H and V L Then, a single-chain antibody can be monovalent; if two V's are used... H and V L If more than two V's are used, then it is divalent; H and V L This is multivalent. It can produce bispecific or multivalent antibodies that specifically bind to the coronavirus spike protein and another antigen. The encoded V... H and V L Optionally, it can be used in V H and V L The domains contain furin cleavage sites. They can also encode adapters, for example, when nucleic acid molecules encode bispecific antibodies in DVD-IG™ form.

[0386] One or more DNA sequences encoding antibodies, antigen-binding fragments, bispecific antibodies, or conjugates can be expressed in vitro by transferring the DNA into suitable host cells. Cells can be prokaryotic or eukaryotic. Numerous expression systems can be used to express proteins, including *Escherichia coli* (E. coli). E. coli Other bacterial hosts, yeast, and various higher eukaryotic cell lines such as COS, CHO, HeLa, and myeloma cell lines can all be used to express the disclosed antibodies and antigen-binding fragments. Stable transfer methods (i.e., the foreign DNA is persistently retained in the host) can be used. Hybridomas expressing antibodies of interest are also included in this disclosure.

[0387] Expression of nucleic acids encoding antibodies, antigen-binding fragments, and multispecific antibodies (e.g., bispecific antibodies) described herein can be achieved by operatively linking DNA or cDNA to a promoter (constitutive or inducible) and then incorporating it into an expression cassette. The promoter can be any promoter of interest, including cytomegalovirus promoters. Optionally, enhancers such as cytomegalovirus enhancers are included in the construct. The cassette can be adapted for replication and integration in prokaryotes or eukaryotes. A typical expression cassette contains specific sequences that can be used to regulate the expression of DNA encoding proteins. For example, an expression cassette may include a suitable promoter, enhancer, transcription and translation terminators, a start sequence, a start codon (i.e., ATG) preceding the protein-coding gene, splicing signals for introns, sequences that maintain the correct reading frame of the gene to allow correct mRNA translation, and a stop codon. The vector may encode optional biomarkers, such as biomarkers encoding drug resistance (e.g., ampicillin or tetracycline resistance).

[0388] To achieve high-level expression of cloning genes, it is desirable to construct expression cassettes containing, for example, a strong promoter to guide transcription, a ribosome binding site for translation initiation (e.g., an internal ribosome binding sequence), and a transcription / translation terminator. For *E. coli*, this may include promoters such as T7, trp, lac, or lambda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, control sequences may include, for example, promoters and / or enhancers derived from immunoglobulin genes, HTLV, SV40, or cytomegalovirus, as well as polyadenylated sequences, and may also include splice donor and / or acceptor sequences (e.g., CMV and / or HTLV splice acceptor and donor sequences). The cassette can be transferred to selected host cells by any suitable method, such as transformation or electroporation for *E. coli*, and calcium phosphate treatment, electroporation, or lipid transfection for mammalian cells. Cells transformed by the cassette can be selected based on antibiotic resistance conferred by genes contained within the cassette (e.g., amp, gpt, neo, and hyg genes).

[0389] Nucleic acids encoding the polypeptides described herein can be modified without diminishing their biological activity. Modifications can be made to facilitate the cloning, expression, or incorporation of target molecules into fusion proteins. Such modifications include, for example, stop codons, sequences for creating positionally convenient restriction sites, and sequences that add methionine to the amino terminus to provide a start site or additional amino acids (such as polyhistidine) to aid in purification steps.

[0390] Once expressed, antibodies, antigen-binding fragments, multispecific (e.g., bispecific) antibodies, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, etc. (see generally Simpson et al., eds.). Basic methods in Protein Purification and Analysis: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 2009). Antibodies, antigen-binding fragments, and conjugates do not need to be 100% pure. After purification (partial purification or purification to homogeneity as needed), the peptides should be substantially free of endotoxins if intended for prophylactic purposes.

[0391] Methods for expressing antibodies, antigen-binding fragments, bispecific antibodies, and conjugates from mammalian cells and bacteria such as *Escherichia coli*, and / or refolding them into suitable active forms, have been described and are applicable to the antibodies disclosed herein. See, for example, Greenfield (ed.). Antibodies: A Laboratory Manual , 2 nd ed. New York: ColdSpring Harbor Laboratory Press, 2014; Simpson et al. (eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual New York: Cold Spring Harbor Laboratory Press, 2009; and Ward et al. Nature 341(6242):544-546, 1989.

[0392] D. Methods and Compositions 1. Inhibit coronavirus infection This article discloses methods for inhibiting coronavirus infection (e.g., SARS-CoV-2 infection) in subjects. This article also discloses methods for inhibiting coronavirus infection (e.g., SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1 and / or XEC, RaTG13, Pangolin GD, Pangolin GX-P2V, WIV1 and / or SHC-014). SARS-CoV-2 can be BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, or XEC. The method comprises administering to a subject at risk of coronavirus infection or who has coronavirus infection an effective amount (i.e., an amount that effectively inhibits infection in the subject) of the disclosed antibody, antigen-binding fragment, or bispecific antibody, or nucleic acid encoding such antibody, antigen-binding fragment, or bispecific antibody. The method can be used before or after exposure. The subject may be immunocompromised. The subject may be elderly, for example, older than approximately 65 years, approximately 70 years, approximately 75 years, approximately 80 years, approximately 85 years, or approximately 90 years. In some aspects, the antibody or antigen-binding fragment may be used in the form of a bispecific antibody. The antigen-binding fragment may be scFv.

[0393] For the method to be effective, complete elimination or suppression of the infection is not required. For example, the method can reduce the infection by a desired amount compared to a coronavirus infection without treatment, such as at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (eliminating or preventing detectable coronavirus infection). In some respects, the subject may also be treated with an effective amount of additional reagents (e.g., antiviral agents).

[0394] In some respects, administration of an effective amount of the disclosed antibody, antigen-binding fragment, bispecific antibody, or nucleic acid molecule can inhibit the occurrence of infection and / or subsequent disease progression in subjects, which may include any statistically significant reduction in coronavirus infection activity (e.g., growth or invasion) or symptoms in subjects.

[0395] This document discloses a method for inhibiting the replication of coronaviruses (e.g., SARS-CoV-2) in subjects. The coronavirus may be SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 and / or SHC-014. The coronavirus may be SARS-CoV-1, or a SARS-CoV-1-related virus from a non-human animal (such as a civet or pangolin). The method comprises administering an effective amount (i.e., an amount that effectively inhibits replication in the subject) of the disclosed antibody, antigen-binding fragment, bispecific antibody, or nucleic acid encoding such an antibody, antigen-binding fragment, or bispecific antibody to a subject at risk of coronavirus infection or who has coronavirus infection. The method can be used before or after exposure.

[0396] Methods for treating SARS-CoV-2 infection in subjects are disclosed. Methods for preventing SARS-CoV-2 infection in subjects are also disclosed. These methods include administering one or more antibodies, antigen-binding fragments, bispecific antibodies, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, as disclosed herein.

[0397] Antibodies, their antigen-binding fragments, and bispecific antibodies can be administered via intravenous infusion. The dosages of antibodies, antigen-binding fragments, or bispecific antibodies vary, but generally range from about 0.5 mg / kg to about 50 mg / kg, for example, about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, or about 50 mg / kg. In some instances, the dosage of antibodies, antigen-binding fragments, or bispecific antibodies may be from about 0.5 mg / kg to about 5 mg / kg, for example, about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg. Antibodies, antigen-binding fragments, or bispecific antibodies are administered according to a dosing regimen determined by a medical practitioner. In some instances, antibodies, antigen-binding fragments, or bispecific antibodies are administered weekly, bi-weekly, bi-weekly, or bi-weekly.

[0398] In some respects, methods for suppressing infection in subjects also include administering one or more additional agents to the subjects. Additional agents of interest include, but are not limited to, antiviral agents such as hydroxychloroquine, arbidol, remdesivir, favipiravir, baricitinib, lopinavir / ritonavir, zinc ions, and interferon beta-1b, or combinations thereof.

[0399] In some aspects, the method includes administering a first antibody that specifically binds to the SARS-CoV-2 spike protein as disclosed herein and a second antibody that also specifically binds to the SARS-CoV-2 protein (e.g., a different epitope of a coronavirus protein). Therefore, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of the disclosed antibodies or their antigen-binding fragments can be administered to the subject.

[0400] The method may include administering at least 2, 3, 4, or 5 of the disclosed monoclonal antibodies or antigen-binding fragments. The method may include administering an effective amount of A18-448.1 or its antigen-binding fragment. The method may include administering an effective amount of F768-104_B2 or its antigen-binding fragment. The method may include administering an effective amount of a third group antibody or its antigen-binding fragment. The method may include administering an effective amount of a fourth group antibody or its antigen-binding fragment. The method may include administering an effective amount of a fifth group antibody or its antigen-binding fragment. The method may include administering an effective amount of a sixth group antibody or its antigen-binding fragment.

[0401] Combination use includes, but is not limited to, the use of A18-618-448.1 or its antigen-binding fragment in combination with a group 4 antibody or its antigen-binding fragment. The group 4 antibody can be, for example, F768-104_G7. Another combination use is the use of F768-104_B2 or its antigen-binding fragment in combination with a group 3 antibody or its antigen-binding fragment. The group 3 antibody can be, for example, A80-102_G5. Yet another combination use is the use of F768-104_B2 or its antigen-binding fragment in combination with A18-448.1 or its antigen-binding fragment.

[0402] Other antibodies that specifically bind to SARS-CoV-2 can be used in conjunction with the antibodies disclosed herein. In some aspects, F768-104_B2 or its antigen-binding fragment can be used in combination with antibody F769-E12 or its antigen-binding fragment, disclosed in U.S. Provisional Application No. 63 / 433,719, which is incorporated herein by reference. Furthermore, A18-448.1 or its antigen-binding fragment can be used in combination with F769-E12, F770-G11, or F770-E8 or their antigen-binding fragments, disclosed in U.S. Application No. 63 / 433,719, which is incorporated herein by reference.

[0403] In some respects, subjects may be administered a combination of class I and II, class I and III, class II and III, or class I, class II and III antibodies. More than one class I, class II, or class III antibody may be used.

[0404] The disclosed methods may include administering one or more multispecific antibodies. Combinations of these multispecific antibodies, as well as combinations of bispecific antibodies, such as one, two, three, four, or five of these multispecific antibodies (e.g., bispecific antibodies), may be administered to the subject. Nucleic acid molecules are also useful in these respects.

[0405] In some aspects, DNA or RNA encoding a disclosed antibody, antigen-binding fragment, or multispecific antibody (e.g., a bispecific antibody) is administered to a subject to provide in vivo antibody production, for example, by utilizing the subject's cellular mechanisms. Any suitable nucleic acid administration method can be used; non-limiting examples are provided in U.S. Patent Nos. 5,643,578, 5,593,972, and 5,817,637. U.S. Patent No. 5,880,103 describes several methods for delivering nucleic acids encoding proteins to an organism. One method of nucleic acid administration is direct administration with plasmid DNA (e.g., using mammalian expression plasmids). The nucleotide sequence encoding the disclosed antibody, its antigen-binding fragment, or multispecific antibody (e.g., a bispecific antibody) can be placed under promoter control to increase expression. The methods include liposome delivery of nucleic acids. These methods can be applied to produce antibodies or their antigen-binding fragments. In some aspects, the pVRC8400 vector (described in Barouch et al.) is used. J. Virol .,79(14), 8828-8834, 2005 (This article is incorporated herein by reference) expresses the disclosed antibody or antigen-binding fragment in subjects.

[0406] In several respects, an effective amount of an AAV viral vector comprising one or more nucleic acid molecules encoding a disclosed antibody, antigen-binding fragment, or multispecific (e.g., bispecific) antibody can be administered to a subject (e.g., a human subject at risk of or infected with coronavirus). The AAV viral vector is designed to express a nucleic acid molecule encoding the disclosed antibody, antigen-binding fragment, or bispecific antibody, and administration of an effective amount of the AAV viral vector to a subject results in the expression of an effective amount of said antibody, antigen-binding fragment, or bispecific antibody in the subject. Non-limiting examples of AAV viral vectors that can be used to express the disclosed antibody, antigen-binding fragment, or bispecific antibody in a subject include Johnson et al. Nat. Med ., 15(8):901-906, 2009 and Gardner et al., Nature The references provided in , 519(7541):87-91,2015 are all incorporated into this paper in their entirety through citation.

[0407] In one aspect, nucleic acids encoding the disclosed antibody, antigen-binding fragment, or multispecific antibody (e.g., bispecific antibody) are directly introduced into the tissue. For example, nucleic acids can be loaded onto gold microspheres using standard methods and delivered via HELIOS, such as those from Bio-Rad. TM The gene gun device is introduced into the skin. Nucleic acids can be "naked" nucleic acids, composed of plasmids under the control of strong promoters.

[0408] DNA is typically injected into muscle, but it can also be injected directly into other sites. The injection dose is usually between about 0.5 μg / kg and about 50 mg / kg, and typically between about 0.005 mg / kg and about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0409] Compositions containing the disclosed antibodies, antigen-binding fragments, or multispecific antibodies (e.g., bispecific antibodies), conjugates, or nucleic acid molecules encoding such molecules may be administered once or multiple times, according to the patient's desired and tolerable dosage and frequency. A dose may be administered once, or it may be administered periodically until the desired result is achieved or until treatment needs to be discontinued due to side effects. Typically, the dose is sufficient to suppress coronavirus infection without causing unacceptable toxicity to the patient.

[0410] Data obtained from cell culture assays and animal studies can be used to determine dosage ranges for human use. Dosages are typically within the range of ED. 50 Within the cyclic concentration range, it exhibits almost no or minimal toxicity. Dosage can vary within this range depending on the dosage form and route of administration. Effective dosage can be determined through cell culture assays and animal studies.

[0411] SARS-CoV-2 specific antibodies, antigen-binding fragments, multispecific antibodies (e.g., bispecific antibodies), or nucleic acid molecules encoding such molecules, or compositions containing such molecules, can be administered to subjects in a variety of ways, including local and systemic administration, such as subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. In one aspect, antibodies, antigen-binding fragments, multispecific antibodies (e.g., bispecific antibodies), or nucleic acid molecules encoding such molecules, or compositions containing such molecules, can be administered via a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection once daily. Antibodies, antigen-binding fragments, multispecific antibodies (e.g., bispecific antibodies), conjugates, or nucleic acid molecules encoding such molecules, or compositions containing such molecules, can also be administered by direct injection at or near the disease site. Another method of administration is via an osmotic pump (e.g., an Alzet pump) or a micropump (e.g., an Alzet microosmotic pump), which allows for the controlled, continuous, and / or sustained-release delivery of antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding such molecules, or compositions containing such molecules, over a predetermined time period. Osmotic pumps or micropumps can be implanted subcutaneously or near the target site.

[0412] 2. Composition Compositions are provided comprising one or more coronavirus spike protein-specific antibodies, antigen-binding fragments, multispecific antibodies (e.g., bispecific antibodies), conjugates, or nucleic acid molecules encoding such molecules, and pharmaceutically acceptable carriers. In some aspects, the composition comprises two, three, four, or more antibodies, antigen-binding fragments, or bispecific antibodies that specifically bind to the coronavirus spike protein. The composition is useful, for example, for inhibiting or detecting coronavirus infection, such as, but not limited to, SARS-CoV-2 infection. The coronavirus may be SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1, and / or SHC-014. The coronavirus can be SARS-CoV-1, or a SARS-CoV-1-related virus from non-human animals (such as civets or pangolins). In some aspects, the composition comprises one or more antibodies disclosed herein: A18-448.1, F768-104_B2, A80-102_G5, A80-338-19.5, A80-338-19.6, A80-338-19.8, A80-338-19.9, E184-105_F3, A63-652-32.1, F768-104_G7, E184-105_H1, F768-10 The composition contains 4_D10, A63-652-1.4, E184-105_E5, E184-105_H9, E184-105_H2, A80-102+103_G1, A80-103_E2, A80-103_B6, A18-618-452.1, A80-102+103_C1, A43-d321.10.4, and F768-100-pt2_A05, or antigen-binding fragments thereof, or multispecific antibodies thereof. In some aspects, the composition comprises two, three, four, or more antibodies that specifically bind to the coronavirus spike protein. The composition is useful, for example, for inhibiting or detecting coronavirus infections, such as SARS-CoV-2 infection.

[0413] The composition can be prepared in unit dosage forms, such as kits, for administration to subjects. The amount and timing of administration are determined by the attending physician to achieve the intended purpose. The antibody, antigen-binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such a molecule can be formulated for systemic or local administration. In one example, the antigen-binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such a molecule is formulated for parenteral administration, such as intravenous administration.

[0414] In some aspects, the antibody, antigen-binding fragment, bispecific antibody, or conjugate thereof in the composition has a purity of at least 70% (e.g., at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%). In some aspects, the composition contains less than 10% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.

[0415] Compositions for administration may comprise solutions of antibodies, antigen-binding fragments, multispecific antibodies (e.g., bispecific antibodies), conjugates, or nucleic acid molecules encoding such molecules dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). A variety of aqueous carriers may be used, such as buffered saline solutions. These solutions are sterile and generally free of unwanted substances. These compositions may be sterilized using any suitable technique. The composition may contain pharmaceutically acceptable excipients close to physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibodies in these formulations can vary widely and will be selected primarily based on the specific administration modality chosen and the needs of the subject, taking into account factors such as liquid volume, viscosity, and body weight.

[0416] Typical compositions for intravenous administration comprise about 0.01 to about 30 mg / kg per subject per day of an antibody, antigen-binding fragment, bispecific antibody, or conjugate (or a corresponding dose of a conjugate containing such antibody or antigen-binding fragment). Any suitable method may be used to prepare the administerable composition; non-limiting examples are provided such as Remington: The Science and Practice of Pharmacy, 22 nd ed. In publications such as, London, UK: PharmaceuticalPress, 2013. In some aspects, the composition may be a liquid formulation comprising one or more antibodies, antigen-binding fragments, or bispecific antibodies in concentrations ranging from about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 1 mg / ml to about 10 mg / ml.

[0417] Antibodies, their antigen-binding fragments, multispecific antibodies (e.g., bispecific antibodies), or nucleic acids encoding such molecules can be provided in lyophilized form and rehydrated with sterile water prior to administration; however, they can also be provided as sterile solutions of known concentrations. The solution containing the antibody, antigen-binding fragment, bispecific antibody, or nucleic acid encoding such a molecule can then be added to an infusion bag containing 0.9% sodium chloride (USP) and administered typically at a dose of 0.5 mg / kg body weight to 15 mg / kg body weight. There is considerable experience in the art of administering antibody drugs, which have been marketed in the United States since rituximab was approved in 1997. Antibodies, antigen-binding fragments, conjugates, or nucleic acids encoding such molecules can be administered by slow infusion rather than intravenous bolus or bolus injection. In one instance, a higher loading dose is administered, followed by a maintenance dose at a lower level. For example, the initial loading dose is 4 mg / kg, which can be infused over approximately 90 minutes. Subsequently, if the previous dose is well tolerated, a maintenance dose of 2 mg / kg can be given weekly over 30 minutes for 4–8 weeks.

[0418] Controlled-release parenteral formulations can be formulated as implants, oil-based injectables, or granular systems. For a comprehensive overview of protein delivery systems, see Banga. Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems Lancaster, PA: Technomic Publishing Company, Inc., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain an active protein agent (such as a cytotoxin or drug) as a core. In microspheres, the active protein agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, therefore only nanoparticles can be administered intravenously. Microparticles typically have a diameter of about 100 μm and are administered subcutaneously or intramuscularly. See, for example, Kreuter, Colloidal Drug Delivery Systems , J.Kreuter (Ed.), New York, NY: Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications , A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.

[0419] Polymers can be used for the ion-controlled release of the compositions disclosed herein. Any suitable polymer can be used, such as degradable or non-degradable polymer matrices designed for controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins. In another aspect, liposomes are used for controlled release and drug targeting via lipid encapsulation of drugs.

[0420] 2. Detection and Diagnostic Methods Methods for detecting the presence of coronavirus spike protein in vitro or in vivo are also provided. In one instance, the presence of coronavirus spike protein is detected in a biological sample from a subject and can be used to identify a subject with coronavirus infection. The coronavirus can be SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 and / or SHC-014. The coronavirus can be SARS-CoV-1, or SARS-CoV-1-related viruses from non-human animals such as civets or pangolins.

[0421] The sample can be any sample, including but not limited to tissues from biopsies, autopsies, and pathological specimens. Biological samples also include tissue sections, such as frozen sections for histological purposes. Biological samples further include bodily fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. Detection methods may include contacting cells or a sample with an antibody, antigen-binding fragment, or multispecific antibody (e.g., a bispecific antibody) that specifically binds to the SARS-CoV-2 spike protein, or a conjugate thereof (e.g., a conjugate containing a detectable biomarker), under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable biomarker conjugated to the antibody or antigen-binding fragment).

[0422] In one aspect, the antibody, antigen-binding fragment, or multispecific antibody (e.g., a bispecific antibody) is directly labeled with a detectable marker. In another aspect, the antibody (or antigen-binding fragment or bispecific antibody) binding to the SARS-CoV-2 spike protein (the primary antibody) is unlabeled and detected using a secondary antibody or other molecule capable of binding the primary antibody. The secondary antibody is selected to specifically bind to the primary antibody of a particular species and class. For example, if the primary antibody is human IgG, the secondary antibody could be anti-human IgG. Other molecules capable of binding antibodies include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable markers for the antibody, antigen-binding fragment, bispecific antibody, or secondary antibody are known and described above, including a variety of enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic reagents, and radioactive materials.

[0423] In some respects, the disclosed antibodies, their antigen-binding fragments, or multispecific antibodies (e.g., bispecific antibodies) are used to test vaccines. For example, this is used to test whether a vaccine composition containing a coronavirus spike protein or a fragment thereof presents a conformation containing a disclosed antibody epitope. Therefore, this document provides a method for testing a vaccine, wherein the method includes contacting a sample containing the vaccine (e.g., a coronavirus spike protein immunogen) with the disclosed antibody, antigen-binding fragment, or bispecific antibody under conditions sufficient to form an immune complex, and detecting the immune complex to detect a vaccine containing an epitope of interest in the sample. In one example, the detection of the immune complex in the sample indicates that the vaccine component (e.g., the immunogen) presents a conformation capable of binding the antibody or antigen-binding fragment.

[0424] Overview Clause 1. An isolated monoclonal antibody or its antigen-binding fragment, comprising a heavy chain variable region (V... H ) and light chain variable region (V L The heavy chain variable region (V) H ) and light chain variable region (V L V includes any of the following: H and V LThe heavy chain complementarity determinants (HCDRs) 1, HCDR 2, and HCDR 3 and the light chain complementarity determinants (LCDRs) 1, LCDR 2, and LCDR 3 are: a) SEQ ID NO: 1 and 5 (A18-618-448.1); b) SEQ ID NO: 9 and 13 (F768-104_B2); c) SEQ ID NO: 17 and 21 (E184-105_F3); d) SEQ ID NO: 25 and 29 (A63-652-32.1); e) SEQ ID NO: 33 and 37 (F768-104_G7); g) SEQ ID NO: 41 and 45 (E184-105_H1); f) SEQ ID NO: 49 and 53 (F768-104_D10); h) SEQ ID NO: 57 and 61 (A63-652-1.4); i) SEQ ID NO: 65 and 69 (E184-105_E5); j) SEQ ID NO: 73 and 77 (E184-105_H9); k) SEQ ID NO: 81 and 85 (E184-105_H2); l) SEQ ID NO: 89 and 93 (A80-102+103_G1); m) SEQ ID NO: 97 and 101 (A80-103_E2); n) SEQ ID NO: 105 and 109 (A80-103_B6); o) SEQ ID NO: 113 and 117 (A18-618-452.1); p) SEQ ID NO: 121 and 125 (A80-102+103_C1); q) are SEQ ID NO: 129 and 133 (A43-d321.10.4); r) are SEQ ID NO: 137 and 141 (F768-100-pt2_A05); s) are SEQ ID NO: 145 and 149 (A80-102_G5); t) are SEQ ID NO: 153 and 157 (A18-338-19.5); u) are SEQ ID NO: 161 and 165 (A18-338-19.6); v) are SEQ ID NO: 169 and 173 (A18-338-19.8); or w) are SEQ ID NO: 177 and 181. (A18-338-19.9), wherein the monoclonal antibody or its antigen-binding fragment specifically binds to SARS CoV-2.

[0425] Clause 2. The isolated monoclonal antibody or antigen-binding fragment as described in Clause 1, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence represented by any one of the following: a) SEQ ID NO: 2, 3, 4, 6, 7 (AAS) and 8; b) SEQ ID NO: 10, 11, 12, 14, 15 (GAS) and 16; c) SEQ ID NO: 18, 19, 20, 22, 23 (GAS) and 24; d) SEQ ID NO: 26, 27, 28, 30, 31 (AAS) and 32; e) SEQ ID NO: 34, 35, 36, 38, 39 (AAS) and 40; f) SEQ ID NO: 42, 43, 44, 46, 47 (DAS) and 48; g) SEQ ID NO: 50, 51, 52, 54, 55 (GAS) and 56; h) SEQ ID NO: 58, 59, 60, 62, 63 (DAS) and 64; i) SEQ ID NO: 66, 67, 68, 70, 71 (AAS) and 72; j) SEQ ID NO: 74, 75, 76, 78, 79 (AAS) and 80; k) SEQ ID NO: 82, 83, 84, 86, 87 (AAS) and 88; l) SEQ ID NO: 90, 91, 92, 94, 95 (NNN) and 96; m) SEQ ID NO: 98, 99, 100, 102, 103 (SDS) and 104; n) SEQ ID NO: 106, 107, 108, 110, 111 (SYN) 112; o) SEQ ID NO: 114, 115, 116, 118, 119 (AAS) and 120; p) SEQ ID NO: 122, 123, 124, 126, 127 (DAS) and 128; q) SEQ ID NO: 130, 131, 132, 134, 135 (DAS) and 136; r) SEQ ID NO: 138, 139, 140, 142, 143 (WAS) and 144; s) SEQ ID NO: 146, 147, 148, 150, 151 (DAS) and 152; t) SEQ ID NO: 154, 155, 156, 158, 159 (DAS) and 160; u) SEQ ID NO: 162, 163, 164, 166, 167 (DAS) and 168; v) SEQ ID NO: 170, 171, 172, 174, 175 (DAT) and 176;Or w) SEQ ID NO: 178, 179, 180, 182, 183 (DAS) and 184.

[0426] Clause 3. An isolated monoclonal antibody or antigen-binding fragment as described in Clause 1 or Clause 2, wherein the V H and the V L Each sequence comprises an amino acid sequence having at least 90% identity with the amino acid sequence shown in any of the following: a) SEQ ID NO: 1 and 5; b) SEQ ID NO: 9 and 13; c) SEQ ID NO: 17 and 21; d) SEQ ID NO: 25 and 29; e) SEQ ID NO: 33 and 37; f) SEQ ID NO: 41 and 45; g) SEQ ID NO: 49 and 53; h) SEQ ID NO: 57 and 61; i) SEQ ID NO: 65 and 69; j) SEQ ID NO: 73 and 77; k) SEQ ID NO: 81 and 85; l) SEQ ID NO: 89 and 93; m) SEQ ID NO: 97 and 101; n) SEQ ID NO: 105 and 109; o) SEQ ID NO: 113 and 117; p) SEQ ID NO: 121 and 125; q) SEQ ID NO: 129 and 133; r) SEQ ID NO: 137 and 141; s) SEQ ID NO: 145 and 149; t) SEQ ID NO: 153 and 157; u) SEQ ID NO: 161 and 165; v) SEQ ID NO: 169 and 173; or w) SEQ ID NO: 177 and 181.

[0427] Clause 4. The isolated monoclonal antibody or antigen-binding fragment as described in any of the preceding clauses contains a human frame region.

[0428] Clause 5. An isolated monoclonal antibody or antigen-binding fragment as described in any of the preceding clauses, wherein the V H and the V LEach of the following amino acid sequences is contained in any one of the following: a) SEQ ID NO: 1 and 5; b) SEQ ID NO: 9 and 13; c) SEQ ID NO: 17 and 21; d) SEQ ID NO: 25 and 29; e) SEQ ID NO: 33 and 37; f) SEQ ID NO: 41 and 45; g) SEQ ID NO: 49 and 53; h) SEQ ID NO: 57 and 61; i) SEQ ID NO: 65 and 69; j) SEQ ID NO: 73 and 77; k) SEQ ID NO: 81 and 85; l) SEQ ID NO: 89 and 93; m) SEQ ID NO: 97 and 101; n) SEQ ID NO: 105 and 109; o) SEQ ID NO: 113 and 117; p) SEQ ID NO: 121 and 125; q) SEQ ID NO: 129 and 133; r) SEQ ID NO: 137 and 141; s) SEQ ID NO: 145 and 149; t) SEQ ID NO: 153 and 157; u) SEQ ID NO: 161 and 165; v) SEQ ID NO: 169 and 173; or w) SEQ ID NO: 177 and 181.

[0429] Clause 6. An isolated monoclonal antibody as described in any of the preceding clauses, wherein the monoclonal antibody comprises a human constant domain.

[0430] Clause 7. An isolated monoclonal antibody as described in any of the preceding clauses, wherein the monoclonal antibody is a human antibody.

[0431] Clause 8. An isolated monoclonal antibody as described in any of the preceding clauses, wherein the monoclonal antibody is IgG.

[0432] Clause 9. An isolated monoclonal antibody as described in any of the preceding clauses, comprising a recombinant constant domain, the recombinant constant domain comprising a modification that increases the half-life of the monoclonal antibody.

[0433] Clause 10. The isolated monoclonal antibody as described in Clause 9, wherein the modification increases binding to the neonatal Fc receptor.

[0434] Clause 11. An isolated monoclonal antibody or antigen-binding fragment as described in any one of Clauses 1-10, wherein the monoclonal antibody neutralizes SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, KP.2.3, LB.1, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 and / or SHC-014.

[0435] Clause 12. An antigen-binding fragment as described in any one of Clauses 1-5 or 11.

[0436] Clause 13. An antigen-binding fragment as described in Clause 12, wherein the antigen-binding fragment is an Fv, Fab, F(ab')2, scFV, or scFV2 ​​fragment.

[0437] Clause 14. An isolated monoclonal antibody or antigen-binding fragment as described in any one of Clauses 1-13, conjugated to a detectable biomarker.

[0438] Clause 15. A multispecific antibody comprising a monoclonal antibody or an antigen-binding fragment as described in any one of Clauses 1-14.

[0439] Clause 16. An isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment as described in any one of Clauses 1-15, or encoding a V-type of said monoclonal antibody. H or V L .

[0440] Clause 17. A nucleic acid molecule as described in Clause 16, wherein said nucleic acid molecule is encoding the V H and the V L The cDNA sequence.

[0441] Clause 18. Nucleic acid molecules as described in Clause 16 or Clause 17, which are operatively linked to a promoter.

[0442] Clause 19. A vector comprising a nucleic acid molecule as described in any one of Clauses 16-18.

[0443] Clause 20. A host cell comprising a nucleic acid molecule as described in any one of Clauses 16-20 or a vector as described in Clause 19.

[0444] Clause 21. A pharmaceutical composition for inhibiting coronavirus infection, comprising an effective amount of a monoclonal antibody or antigen-binding fragment as described in any one of Clauses 1-14, a multispecific antibody as described in Clause 15, a nucleic acid molecule as described in any one of Clauses 16-18, or a carrier as described in Clause 19; and a pharmaceutically acceptable carrier.

[0445] Clause 22. A method for generating an antibody or antigen-binding fragment that specifically binds to the spike protein of a coronavirus, comprising: expressing in a host cell one or more nucleic acid molecules encoding a monoclonal antibody or antigen-binding fragment as described in any one of Clauses 1-14; and purifying said antibody or antigen-binding fragment.

[0446] Clause 23. A method for detecting the presence of SARS CoV-2 in a biological sample from a subject, comprising: contacting the biological sample with an effective amount of a monoclonal antibody or antigen-binding fragment as described in any one of Clauses 1-14 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the biological sample, wherein the presence of the immune complex in the biological sample indicates the presence of SARS CoV-2 in the sample.

[0447] Clause 24. The method as described in Clause 23, wherein the detection of the presence of the immune complex in the biological sample indicates that the subject has a SARS-CoV-2 infection.

[0448] Clause 25. A method for inhibiting SARS-CoV-2 infection in a subject, comprising administering to the subject an effective amount of a monoclonal antibody or antigen-binding fragment as described in any one of Clauses 1-14, a multispecific antibody as described in Clause 15, a nucleic acid molecule as described in any one of Clauses 16-18, a vector as described in Clause 19, or a pharmaceutical composition as described in Clause 21, wherein the subject has SARS-CoV-2 infection or is at risk of SARS-CoV-2 infection.

[0449] Clause 26. The method as described in Clause 25, wherein the coronavirus is SARS-CoV-2 D614G, BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ.1.1, XBB.1, XBB.1.5, XBB.1.16, CH.1.1, XBB.2.3.2, XBC.1, EG.5.1, XBB.1.16.6, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, KP.2.3, LB.1, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 or SHC-014.

[0450] Clause 27. Use of any monoclonal antibody, antigen-binding fragment, multispecific antibody, nucleic acid molecule, vector or pharmaceutical composition as described in any one of Clauses 1-25 in inhibiting SARS-CoV-2 infection in a subject or in detecting the presence of SARS-CoV-2 in a biological sample.

[0451] Clause 28. Use as described in Clause 27, wherein the SARS-CoV-2 is SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, KP.2.3, LB.1, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 or SHC-014.

[0452] Example Twenty-three monoclonal antibodies used clinically for the prevention and treatment of COVID-19 have been disclosed. These antibodies possess potent neutralizing capabilities and target the spike glycoprotein of SARS-CoV-2. They are more effective than other monoclonal antibodies currently in clinical trials and exhibit higher cross-reactivity than antibodies previously available under EUA. Notably, they neutralize SARS-CoV-2-related viral strains from animals, and many also neutralize SARS-CoV and related animal coronaviruses, suggesting their applicability against potentially emerging variants and coronaviruses with pandemic potential.

[0453] Example 1 method B cell binding and sorting using flow cytometry: Based on high serum neutralizing antibody titers against Omeprón lineage variants BA.4 / 5, BQ.1.1, and XBB, SARS-CoV-2 vaccinated and / or recovered subjects were selected for antibody isolation. Memory B cells derived from peripheral blood mononuclear cells were identified by binding to fluorescently labeled SARS-CoV-2 spike RBD protein or the full-length soluble spike protein and sorted into single wells. For several subjects, SARS-CoV-2 spike-specific B cells were isolated using S-2P spike protein derived from variants BQ.1.1 and XBB. In other cases, antibodies with a higher probability of binding to class I and III RBD epitopes and also capable of neutralizing Omeprón lineage viruses were selected. In these cases, single-cell sorting of B cells was performed using RBD proteins with specific mutations.

[0454] Sequencing, cloning, and expression:Next-generation sequencing methods were used to determine the heavy and light chain nucleotide sequences of B cells. After synthesis, the heavy and light chain sequences were cloned into expression cassettes / plasmid vectors. Antibodies were expressed using the expression cassettes for initial screening; larger-scale preparations were then performed using the plasmid vectors for further characterization. Antibodies were purified using standard methods and techniques.

[0455] Epitope plotting: Using monoclonal antibodies with known RBD epitopes (including class I, II, III, and IV antibodies), global mapping was performed via ELISA-based assays, Octet / BLI, or SPR competitive assays to determine the binding characteristics and epitopes of monoclonal antibodies on S-2P or RBD. For mapping via competitive ELISA, an unlabeled competitive monoclonal antibody was added to a SARS-CoV-2 WA-1 S2P-coated plate. After incubation at room temperature for 30 minutes, biotinylated monoclonal antibody was added, and OD readings were recorded, using biotinylated monoclonal antibody alone as a binding control. The percentage of binding inhibition was calculated as follows: 100 – (reading of biotinylated monoclonal antibody in the presence of competitive monoclonal antibody) / (reading of biotinylated monoclonal antibody alone) × 100.

[0456] Neutralization: The neutralizing effect of monoclonal antibodies against viral infection was determined using pseudotyped lentiviral particles carrying the coronavirus spike protein. Infection induced by the virus was determined by measuring the expression of a luciferase reporter gene encoded by the viral genome. SARS-CoV-2 variants in the test group included pre-Omecron variants α, β, γ, and δ; Omecron variants BA.1, BA.2, BA.4 / BA.5, BA.2.75.2, BQ.1.1, XBB.1, and XBB.1.5; and recent variants XBB.1.16, CH.1., XBB.2.3.2, XBC.1, EG.5.1, XBB.1.16.6, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. SARS-CoV-2-related sabevirins include RaTG13-d21aa from bats and several strains from pangolins. In addition to the bat-related SARS-CoV virus SHC014 and the civet-related virus Civet007, standard SARS-CoV strains and their related strain Frankfurt 1 were also used. Fine epitope mapping was performed using a spike protein genome with a point mutation in the D614G background, employing neutralization assays.

[0457] Synergistic effect:Antibody 1 and Antibody 2 were diluted separately and mixed in matrix form. After incubation with pseudovirus (XBB.1.5 or EG.5.1), the antibody-virus mixture was added in triplicate to plates pre-coated with 293-TMPRSS2-ACE2 cells. The plates were harvested after 72 hours, and the neutralization percentage was calculated. The mean and maximum ZIP synergistic scores were calculated using the web-based software SynergyFinder 3.0 (http: / / synergyfinder.fimm.fi). A ZIP score <-10 indicated antagonism, -10 to 10 indicated additive effects, and >10 indicated synergistic neutralization.

[0458] structure: The molecular structures of several antibodies complexed with RBD or S-2P were determined using cryo-electron microscopy (Cryo-EM).

[0459] Example 2 Antibody Antibody 1. A18-448.1 is a highly potent and cross-reactive neutralizing antibody with activity against SARS-CoV-2 variants and SARS-CoV-associated sabeziovirus. It neutralizes all tested SARS-CoV-2 variants, including pro-Omecron SARS-CoV-2 variants, Omecron BA.1, BA.2 and BA.4 / 5, EG.5.1 and BA.2.86 (IC50 from 0.001 μg / ml to 0.01 μg / ml), and more recent JN.1 and its subvariants including KP.3.1.1 and XEC (IC50 from 0.0026 μg / ml to 0.0095 μg / ml). Figure 7 It exhibits potent neutralizing activity. It strongly neutralizes SARS-CoV-related sabeviruses from human, bat, and civet sources (IC50 of 0.001 μg / ml to 0.01 μg / ml) and several animal-derived SARS-CoV-2-related viruses (Table 1; Figures 3A-3H The mapping study of antibody A18-448.1 showed that its epitope is class I / IV: in the competitive assay using ELISA, it competes with known class I monoclonal antibodies (including CB6), class III monoclonal antibodies (including LY-CoV1404), and class IV monoclonal antibodies (including BD55-5514) for binding to S-2P, indicating that it belongs to class IV (Table 2; Figures 4A-4B The structural data is consistent with the Class IV epitopes (Figure 1).

[0460] Compared to previous monoclonal antibodies, A18-448.1 exhibits broader cross-reactivity and is more effective in most cases. In terms of its epitope and neutralization profile, it is similar to antibody SA55 (also known as BD55-5514), which is currently undergoing clinical trials in China. Notably, A18-448.1 is more effective than SA55. Both antibodies have the same binding competition profile. Neither is affected by common escape mutations (including F456L); their activity is eliminated by mutations at positions 500 and 504 of the spike protein (Table 3). Figures 5A-5C These mutations are extremely rare in the GIAID spike sequence database. Genetically, both originate from IGHV1-69 in their heavy chain genes; however, their CDRH1, CDRH2, and CDRH3 sequences differ. Furthermore, the light chain gene of A18-448.1 originates from IGKV1D-39, while that of SA55 originates from IGVK1-33. A18-448.1 differs from SA55.

[0461] Antibody 2. F768-104_B2 is a highly potent and cross-reactive neutralizing antibody with activity against SARS-CoV-2 variants and SARS-CoV. It exhibits broad and potent neutralizing activity (IC50 < 0.1 μg / mL) against SARS-CoV-2 Omeprón lineage pseudotypes (including BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, and BA.2.86), and also strongly neutralizes recent variants, including JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC (IC50 0.051–0.591 μg / mL). It also neutralizes SARS-CoV (IC50 = 0.125 μg / mL) and several animal-derived SARS-CoV-2-related viruses. This broad spectrum and potency are equivalent to or superior to the previously leading authorized therapeutic antibody LY-CoV1404 (Table 1; Figures 3A-3H ; Figure 7 ).

[0462] The competitive assay for F768-104_B2 is consistent with its class III antibody status, as it competes with known class III monoclonal antibodies, including Ly-CoV1404 (bertronib) (Table 2). Figures 4A-4B For pseudoviruses containing K444N on the D614G spike, it showed weakened but not eliminated neutralization, and still neutralized pseudoviruses with other substitutions (e.g., G446V / R, V445A / R, and P449R) on key residues of class III antibodies (Table 3; Figures 5A-5CThis demonstrates the uniqueness of the antibody. Cryo-electron microscopy analysis of the epitope footprint on the RBD showed that it contacts more highly conserved residues compared to other class III antibodies (Figure 2); this is consistent with its superior broad-spectrum activity across variants.

[0463] Example 3 Other antibodies Group 3 antibodies: A80-19 lineage Antibodies A80-102_G5, A80-338-19.5, A80-338-19.6, A80-338-19.8, and A80-338-19.9 (also known as A80-19.3, A80-19.5, A80-19.6, A80-19.8, and A80-19.9, respectively) are highly correlated antibodies derived from the same donor. These antibodies possess neutralizing activity against SARS-CoV-2 variants and SARS-CoV-associated sabevir. They are presumed to be members of a clonal lineage, originating from a common B-cell precursor in the donor. Genetically, they are derived from the heavy chain gene IGHV1-3. 01 and κ light chain IGKV1D-33 01 or IGKV1-33 01. At the amino acid level, their heavy chains share 83% to 95% identity, and their light chains share 88% to 95% identity. These antibodies neutralize all tested SARS-CoV-2 variants, including potent neutralization against pro-Omecron SARS-CoV-2 variants (IC50 < 1 μg / ml), and moderate potency against Omecron BA.1, BA.2, BA.5, EG.5.1, BA.2.86, and more recent variants including JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC (IC50 1–10 μg / ml). They also potently neutralize SARS-CoV-2-associated viruses from animals, including the pangolin GX variant, and SARS-CoV-associated sabeviruses from human, bat, and civet sources (IC50 < 0.05 μg / ml) (Table 1;). Figures 3A-3H ; Figure 7 The graphical analysis shows that the epitope is of class I / IV (Table 2; Figures 4A-4B A80-102_G5 maintained neutralizing efficacy against a group of D614G-based pseudoviruses carrying multiple point mutations that affect other class I and IV antibodies, including the F456L mutation, which is found in most variants circulating in the fall of 2023 (Table 3). Figures 5A-5C ).

[0464] Group 4 antibodies: Class I, genetically similar Antibodies E184-105_F3, A63-652-32.1, F768-104_G7, E184-105_H1, F768-104_D10, and A63-652-1.4 were classified as Class I antibodies and showed broad and potent neutralizing activity against SARS-CoV-2 Omeprón lineage pseudoviruses. Strains including BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ.1.1, and XBB, as well as more recent variants including EG.5.1 and BA.2.86, were all potently neutralized (IC50 from 0.0001 μg / mL to 0.35 μg / mL). These antibodies also neutralized SARS-CoV-2-related coronaviruses from bats and pangolins (Table 1; Figures 3A-3H F768-104_D10 and F768-104_G7 are able to strongly neutralize the D614G strain carrying the F456L mutation, which is found in most strains circulating in the fall of 2023 (Table 3). Figures 5A-5C E184-105_F3 can neutralize JN.1 and its sublineages, including KP.2, KP.3, KP.2.3, and LB.1. Figure 7 A63-652-1.4 also demonstrates potent neutralizing effects against the latest variants, including KP.3.1.1 and XEC. Figure 7 The competitive profiles and / or genetic characteristics of these antibodies are consistent with their binding to class I epitopes within the RBD (Table 2;). Figures 4A-4B Genetically, they originate from IGHV3-53 or IGVH3-66, paired with IGVK1-9 or IGVK1D-33, a common lineage among class I antibodies. Each of these antibodies exhibits broad spectrum and potency not found in LY-CoV1404 or other clinically used antibodies.

[0465] Group 5 antibodies: Class I, from different gene sources. Antibodies E184-105_E5, E184-105_H9, E184-105_H2, and A80-102+103_G1 were classified as class I antibodies and showed broad and potent neutralizing activity against SARS-CoV-2 Omeprón lineage pseudoviruses, including BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ.1.1, XBB, and more recent variants including EG.5.1 and BA.2.86 (IC50 from 0.003 μg / mL to 0.487 μg / mL). These antibodies also neutralized SARS-CoV-2-related coronaviruses from bats and pangolins (Table 1; Figures 3A-3H E184-105_E5 and E184-105_H9 also neutralize JN.1 ( Figure 7The competitive profiles of these antibodies are consistent with their binding to class I epitopes within the RBD (Table 2). Figures 4A-4B Unlike many other class I antibodies, they are genetically derived from IGVH4-39 or IGVH5-51. Each of these antibodies exhibits broad spectrum and potency not found in LY-CoV1404 or other clinically used antibodies. E184-105_E5 and A80-102+103_G1 are potent neutralizers of the D614G strain carrying several mutations known to reduce the activity of other class I antibodies, including the F456L mutation, which is found in most Omeprone variants circulating in the fall of 2023 (Table 3). Figures 5A-5C ).

[0466] Group 6 Antibodies: Diverse Genetics and Epitopes Antibodies A80-103_E2, A80-103_B6, A18-618-452.1, A80-102+103_C1, A43-d321.10.4, and F768-100-pt2_A05 showed broad and potent (IC50 < 0.1 μg / mL) neutralizing activity against SARS-CoV-2 Omeprón lineage pseudoviruses (including BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ1.1, and XBB), and moderate potency (IC50 from 1 μg / mL to 10 μg / mL) against EG.5.1, FL.1.5.1, and BA.2.86, as well as recent variants including JN.1, KP.2, KP.3, LB.1, KP.2.3, KP.3.1.1, and XEC. They also strongly neutralized SARS-CoV-2-related coronaviruses from animals as well as SARS-CoV (IC50 from 0.02 μg / mL to 2 μg / mL) (Table 1; Figures 3A-3H ; Figure 7 A80-103_B6, A80-102+103_C1, and A80-103_E2 maintained neutralizing efficacy against a group of D614G-based pseudoviruses carrying multiple point mutations that affect other class I and IV antibodies, including the F456L mutation, which is found in most variants circulating in the fall of 2023 (Table 3). Figures 5A-5C ).

[0467] This group of antibodies targets multiple antigen sites: The plotting data for A80-103_E2 indicate that it is a V site (Table 2); Figures 4A-4B ).

[0468] A80-103_B6 competes with class IV antibodies but not with class I antibodies, indicating that it has a unique binding mode (Table 2). Figures 4A-4BFurther plotting was performed using antibodies A43-d321.10.4, A80-102+103_C1, and A18-618-452.1.

[0469] The sequence information is provided as follows: Ab1. A18-618-448.1 The amino acid sequences of the heavy and light chains of expression version A18-618-448.1 are shown in Table 1. Variable heavy and light chain sequences were synthesized and cloned into immunoglobulin expression vectors containing the human constant regions of the IgG1 heavy and κ light ...

Claims

1. An isolated monoclonal antibody or its antigen-binding fragment, containing a heavy chain variable region (V... H ) and light chain variable region (V L The heavy chain variable region (V) H ) and light chain variable region (V L V includes any of the following: H and V L Heavy chain complementarity determinants (HCDRs) 1, HCDR 2, and HCDR 3 and light chain complementarity determinants (LCDRs) 1, LCDR 2, and LCDR 3: a) These are SEQ ID NO: 1 and 5 (A18-618-448.1), respectively; b) These are SEQ ID NO: 9 and 13 (F768-104_B2), respectively; c) These are SEQ ID NO: 17 and 21 (E184-105_F3), respectively; d) These are SEQ ID NOs: 25 and 29 (A63-652-32.1), respectively; e) These are SEQ ID NO: 33 and 37 (F768-104_G7), respectively; g) These are SEQ ID NO: 41 and 45 (E184-105_H1), respectively; f) These are SEQ ID NOs: 49 and 53 (F768-104_D10), respectively; h) are SEQ ID NO: 57 and 61 (A63-652-1.4), respectively; i) These are SEQ ID NO: 65 and 69 (E184-105_E5), respectively; j) are SEQ ID NO: 73 and 77 (E184-105_H9), respectively; k) are SEQ ID NO: 81 and 85 (E184-105_H2), respectively; l) are SEQ ID NO: 89 and 93 (A80-102+103_G1), respectively; m) are SEQ ID NO: 97 and 101 (A80-103_E2), respectively; n) are SEQ ID NO: 105 and 109 (A80-103_B6), respectively; o) are SEQ ID NO: 113 and 117 (A18-618-452.1), respectively; p) are SEQ ID NO: 121 and 125 (A80-102+103_C1), respectively; q) are SEQ ID NO: 129 and 133 (A43-d321.10.4), respectively; r) are SEQ ID NO: 137 and 141 (F768-100-pt2_A05), respectively; s) are SEQ ID NO: 145 and 149 (A80-102_G5), respectively; t) are SEQ ID NO: 153 and 157 (A18-338-19.5), respectively; u) are SEQ ID NO: 161 and 165 (A18-338-19.6), respectively; v) are SEQ ID NO: 169 and 173 (A18-338-19.8) respectively; or w) are SEQ ID NO: 177 and 181 (A18-338-19.9) respectively. The monoclonal antibody or antigen-binding fragment thereunder specifically binds to severe acute respiratory syndrome coronavirus (SARSCoV)-2.

2. The isolated monoclonal antibody or antigen-binding fragment as described in claim 1, wherein HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 each comprise an amino acid sequence as shown in any one of the following: a) SEQ ID NO: 2, 3, 4, 6, 7 (AAS) and 8; b) SEQ ID NO: 10, 11, 12, 14, 15 (GAS) and 16; c) SEQ ID NO: 18, 19, 20, 22, 23 (GAS) and 24; d) SEQ ID NO: 26, 27, 28, 30, 31 (AAS) and 32; e) SEQ ID NO: 34, 35, 36, 38, 39 (AAS) and 40; f) SEQ ID NO: 42, 43, 44, 46, 47 (DAS) and 48; g) SEQ ID NO: 50, 51, 52, 54, 55 (GAS) and 56; h) SEQ ID NO: 58, 59, 60, 62, 63 (DAS) and 64; i) SEQ ID NO: 66, 67, 68, 70, 71 (AAS) and 72; j) SEQ ID NO: 74, 75, 76, 78, 79 (AAS) and 80; k) SEQ ID NO: 82, 83, 84, 86, 87 (AAS) and 88; l) SEQ ID NO: 90, 91, 92, 94, 95 (NNN) and 96; m) SEQ ID NO: 98, 99, 100, 102, 103 (SDS) and 104; n) SEQ ID NO: 106, 107, 108, 110, 111 (SYN) and 112; o) SEQ ID NO: 114, 115, 116, 118, 119 (AAS) and 120; p) SEQ ID NO: 122, 123, 124, 126, 127 (DAS) and 128; q) SEQ ID NO: 130, 131, 132, 134, 135 (DAS) and 136; r) SEQ ID NO: 138, 139, 140, 142, 143 (WAS) and 144; s) SEQ ID NO: 146, 147, 148, 150, 151 (DAS) and 152; t) SEQ ID NO: 154, 155, 156, 158, 159 (DAS) and 160; u) SEQ ID NO: 162, 163, 164, 166, 167 (DAS) and 168; v) SEQ ID NO: 170, 171, 172, 174, 175 (DAT) and 176; or w) SEQ ID NOs: 178, 179, 180, 182, 183 (DAS) and 184.

3. The isolated monoclonal antibody or antigen-binding fragment as described in claim 1 or claim 2, wherein the V H and the V L Each of the following contains an amino acid sequence that is at least 90% identical to the amino acid sequence shown in any of the following: a) SEQ ID NO: 1 and 5; b) SEQ ID NO: 9 and 13; c) SEQ ID NO: 17 and 21; d) SEQ ID NO: 25 and 29; e) SEQ ID NO: 33 and 37; f) SEQ ID NO: 41 and 45; g) SEQ ID NO: 49 and 53; h) SEQ ID NO: 57 and 61; i) SEQ ID NO: 65 and 69; j) SEQ ID NO: 73 and 77; k) SEQ ID NO: 81 and 85; l) SEQ ID NO: 89 and 93; m) SEQ ID NO: 97 and 101; n) SEQ ID NO: 105 and 109; o) SEQ ID NO: 113 and 117; p) SEQ ID NO: 121 and 125; q) SEQ ID NO: 129 and 133; r) SEQ ID NO: 137 and 141; s) SEQ ID NO: 145 and 149; t) SEQ ID NO: 153 and 157; u) SEQ ID NO: 161 and 165; v) SEQ ID NO: 169 and 173; or w) SEQ ID NO: 177 and 181.

4. The isolated monoclonal antibody or antigen-binding fragment as described in any of the preceding claims, comprising a human frame region.

5. The isolated monoclonal antibody or antigen-binding fragment as described in any of the preceding claims, wherein the V H and the V L Each of the following amino acid sequences is contained in any one of them: a) SEQ ID NO: 1 and 5; b) SEQ ID NO: 9 and 13; c) SEQ ID NO: 17 and 21; d) SEQ ID NO: 25 and 29; e) SEQ ID NO: 33 and 37; f) SEQ ID NO: 41 and 45; g) SEQ ID NO: 49 and 53; h) SEQ ID NO: 57 and 61; i) SEQ ID NO: 65 and 69; j) SEQ ID NO: 73 and 77; k) SEQ ID NO: 81 and 85; l) SEQ ID NO: 89 and 93; m) SEQ ID NO: 97 and 101; n) SEQ ID NO: 105 and 109; o) SEQ ID NO: 113 and 117; p) SEQ ID NO: 121 and 125; q) SEQ ID NO: 129 and 133; r) SEQ ID NO: 137 and 141; s) SEQ ID NO: 145 and 149; t) SEQ ID NO: 153 and 157; u) SEQ ID NO: 161 and 165; v) SEQ ID NO: 169 and 173; or w) SEQ ID NO: 177 and 181.

6. The isolated monoclonal antibody as claimed in any of the preceding claims, wherein the monoclonal antibody comprises a human constant domain.

7. The isolated monoclonal antibody as claimed in any of the preceding claims, wherein the monoclonal antibody is a human antibody.

8. The isolated monoclonal antibody as claimed in any of the preceding claims, wherein the monoclonal antibody is IgG.

9. The isolated monoclonal antibody as claimed in any of the preceding claims, comprising a recombinant constant domain, said recombinant constant domain comprising a modification that increases the half-life of said monoclonal antibody.

10. The isolated monoclonal antibody of claim 9, wherein the modification increases binding to the neonatal Fc receptor.

11. The isolated monoclonal antibody or antigen-binding fragment according to any one of claims 1-10, wherein the monoclonal antibody neutralizes SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, KP.2.3, LB.1, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 and / or SHC-014.

12. The antigen-binding fragment as described in any one of claims 1-5 or 11.

13. The antigen-binding fragment of claim 12, wherein the antigen-binding fragment is an Fv, Fab, F(ab')2, scFV, or scFV2 ​​fragment.

14. The isolated monoclonal antibody or antigen-binding fragment as described in any one of claims 1-13, conjugated to a detectable biomarker.

15. A multispecific antibody comprising a monoclonal antibody or an antigen-binding fragment as described in any one of claims 1-14.

16. An isolated nucleic acid molecule encoding a monoclonal antibody or antigen-binding fragment as described in any one of claims 1-15, or encoding a V-cell of said monoclonal antibody. H or V L .

17. The nucleic acid molecule of claim 16, wherein the nucleic acid molecule encodes the V H and the V L The cDNA sequence.

18. The nucleic acid molecule of claim 16 or 17, operatively linked to a promoter.

19. A vector comprising a nucleic acid molecule as described in any one of claims 16-18.

20. A host cell comprising a nucleic acid molecule as described in any one of claims 16-20 or a vector as described in claim 19.

21. A pharmaceutical composition for inhibiting coronavirus infection, comprising an effective amount of a monoclonal antibody or antigen-binding fragment as described in any one of claims 1-14, a multispecific antibody as described in claim 15, a nucleic acid molecule as described in any one of claims 16-18, or a carrier as described in claim 19; and Pharmaceutically acceptable carrier.

22. A method for generating antibodies or antigen-binding fragments that specifically bind to the coronavirus spike protein, comprising: Expressing in host cells one or more nucleic acid molecules encoding a monoclonal antibody or antigen-binding fragment as described in any one of claims 1-14; as well as Purify the antibody or antigen-binding fragment.

23. A method for detecting the presence of SARS-CoV-2 in biological samples from subjects, comprising: Under conditions sufficient to form immune complexes, the biological sample is contacted with an effective amount of a monoclonal antibody or antigen-binding fragment as described in any one of claims 1-14; as well as The presence of the immune complex in the biological sample is detected, wherein the presence of the immune complex in the biological sample indicates the presence of SARS CoV-2 in the sample.

24. The method of claim 23, wherein detecting the presence of the immune complex in the biological sample indicates that the subject has a SARS-CoV-2 infection.

25. A method for inhibiting SARS-CoV-2 infection in a subject, comprising administering to the subject an effective amount of a monoclonal antibody or antigen-binding fragment as described in any one of claims 1-14, a multispecific antibody as described in claim 15, a nucleic acid molecule as described in any one of claims 16-18, a vector as described in claim 19, or a pharmaceutical composition as described in claim 21, wherein the subject has SARS-CoV-2 infection or is at risk of SARS-CoV-2 infection.

26. The method of claim 25, wherein the coronavirus is SARS-CoV-2 D614G, BA.1, BA.2, BA.4 / 5, BA.2.75.2, BQ.1.1, XBB.1, XBB.1.5, XBB.1.16, CH.1.1, XBB.2.3.2, XBC.1, EG.5.1, XBB.1.16.6, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, KP.2.3, LB.1, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 or SHC-014.

27. Use of any monoclonal antibody, antigen-binding fragment, multispecific antibody, nucleic acid molecule, vector, or pharmaceutical composition as described in any one of claims 1-21 in inhibiting SARS CoV-2 infection in a subject or detecting the presence of SARS CoV-2 in a biological sample.

28. The use as claimed in claim 27, wherein the SARS-CoV-2 is SARS-CoV-2 BA.1, BA.2, BA.4 / 5, BA.2.75.2, XBB.1.5, EG.5.1, FL.1.5.1, BA.2.86, JN.1, KP.2, KP.3, KP.2.3, LB.1, KP.3.1.1 and / or XEC, RaTG13, pangolin GD, pangolin GX-P2V, WIV1 or SHC-014.

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