Antibodies targeting coronavirus spike protein

Abstract

Disclosed are monoclonal antibodies, antigen-binding fragments, and bispecific antibodies that specifically bind to the spike protein of coronaviruses, such as SARS-CoV-2. Use of these antibodies to inhibit coronavirus infection, e.g., SARS-CoV-2 infection, is also disclosed. Additionally, disclosed are methods for detecting coronaviruses, such as SARS-CoV-2, in biological samples using the disclosed antibodies. TIFF2024506315000029.tif42137

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Application No. 63 / 147,419, filed February 9, 2021, which is incorporated herein by reference.

[0002] Field of the Disclosure The present disclosure relates to monoclonal antibodies and antigen-binding fragments that specifically bind to coronavirus spike proteins and their use to inhibit beta coronavirus infection, e.g., severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, in a subject, and are useful for detecting coronaviruses such as SARS-CoV-2. [Background technology]

[0003] background In 2019, the International Committee on Taxonomy of Viruses (ICTV) described the Orthocoronavirinae subfamily of the Coronaviridae family, which includes several viruses pathogenic to humans. The most common human coronaviruses cause the common cold and include the alpha-coronaviruses 229E and NL63 and the beta-coronaviruses OC43 and HKU1. In addition to the coronaviruses that cause common cold symptoms, three beta-coronaviruses have been shown to be highly pathogenic in humans. These viruses, namely, Middle East Respiratory Syndrome Coronavirus (MERS), Severe Acute Respiratory Syndrome Coronavirus 1 (SARS-CoV-1), and SARS-CoV-2, can cause severe symptoms in human patients that can lead to death.

[0004] The coronavirus genome is a large, enveloped, positive-sense single-stranded RNA whose length varies depending on the species and encodes multiple structural and nonstructural proteins encoded in several reading frames. The spike protein (S) is expressed on the surface of the virus particle and is involved in viral entry and infection of target cells. Coronavirus transmission can occur through multiple routes, including respiratory droplets, aerosols, fecal-oral, and fomite routes.

[0005] In late 2019, a novel coronavirus was identified as the cause of the SARS respiratory distress syndrome outbreak in Wuhan, China. The virus was later sequenced and found to be highly similar to SARS-CoV-1. Based on these findings, the novel coronavirus was renamed SARS-CoV-2. The incubation period typically ranges from 4 to 14 days, but can be as short as 1 day. Infection is characterized by fever, fatigue, cough, dyspnea, and diarrhea. Some patients experience significant respiratory distress, requiring hospitalization and supplemental oxygen. These patients may rapidly deteriorate and require intensive care unit admission and intubation. Severe disease is also characterized by multiple organ failure, blood clots, and apparent systemic inflammatory response syndrome abnormalities.

[0006] Both humoral and cellular immunity are detectable in COVID-19 survivors, but their relative contributions to protection are unclear. Humoral immunity includes memory immunoglobulin responses, which can be measured using assays for binding to viral antigens and neutralization of viral particles by sera from recovered patients. There remains a need for antibodies that are highly potent at binding to coronaviruses and can be used as therapeutic and diagnostic agents. Summary of the Invention

[0007] Summary of the Disclosure Disclosed are isolated monoclonal antibodies or antigen-binding fragments that specifically bind to the coronavirus spike protein and neutralize SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment comprises one of the following: a) V, set forth as SEQ ID NOs: 1 and 5, respectively H and V L a heavy chain variable region (V) comprising heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 of H ) and the light chain variable region (V L ); b) V, set forth as SEQ ID NOs: 9 and 13, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; c) V, set forth as SEQ ID NOs: 17 and 21, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; d) V, set forth as SEQ ID NOs: 25 and 29, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; e) V as set forth as SEQ ID NOs: 33 and 37, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; f) V, set forth as SEQ ID NOs: 41 and 45, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; g) V, set forth as SEQ ID NOs: 49 and 53, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; h) V, set forth as SEQ ID NOs: 57 and 61, respectively H and V LV, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; i) V, set forth as SEQ ID NOs: 65 and 69, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; j) V as set forth as SEQ ID NOs: 73 and 77, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L , k) V as set forth in SEQ ID NO: 81 and 85 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; l) V as set forth in SEQ ID NO: 89 and 93 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; m) V as set forth in SEQ ID NO: 97 and 101 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; n) V as set forth in SEQ ID NO: 105 and 109 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V Lthe monoclonal antibody specifically binds to the coronavirus spike protein and neutralizes SARS-CoV-2; or o) V, which are set forth as SEQ ID NOs: 143 and 5, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L .

[0008] In a further embodiment, multispecific antibodies comprising combinations of two or more of these antibodies and / or antigen-binding fragments are disclosed.

[0009] In more embodiments, methods for inhibiting SARS-CoV-2 infection in a subject are disclosed.

[0010] In a further aspect, a method for detecting SARS-CoV-2 in a biological sample is disclosed.

[0011] The foregoing and other features and advantages of the present invention will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings. [Brief explanation of the drawings]

[0012] [Figure 1A]Figures 1A-1E. Antibody binding to coronavirus spike protein and neutralization of pseudotyped virus. (A) SARS-CoV2 variants tested for binding and neutralization. (B) Cell surface binding of the indicated antibodies to SARS-CoV2 spike variants. White indicates no change in binding compared to the D614G variant; darker red indicates increased antibody binding to the variant; darker blue indicates decreased antibody binding to the variant. (C) Neutralization of pseudotyped lentivirus by the Wuhan 1 (wt) spike (S) or the indicated SARS-CoV2 variants with the mutations shown in panel A. Neutralization was determined by incubating virus with various dilutions of antibody before adding it to cells. % infection was used to generate values ​​for 50% (IC50) and 80% (IC80) inhibitory concentrations. These values ​​represent the amount of antibody in μg / mL required to reduce infection by 50% and 80%, respectively. (D) Protein domains from the spike protein of SARS-CoV2, including the stabilized diproline (S2P), N-terminal domain (NTD), receptor-binding domain (RBD), and S1 domain, or SARS-CoV S2P, were coated onto an ELISA plate, and the indicated antibodies were tested for reactivity. Positive reactivity for the SARS-CoV-2 ELISA against the S2P, NTD, RBD, or S1 domain is indicated with a †, no reactivity is indicated with a -, and low reactivity is indicated with a †-. The classification of binding domains based on the results is shown in the column labeled "Target." Binding to SARS-CoV-1 (also known as SARS1) or SARS-CoV2 S2P in the ELISA is shown. For each antibody, the reactivity level is indicated as -, +, ++, +++, ++++, or +++++. (E) Initial variant neutralization data. See also Figure 28 for additional neutralization data. [Figure 1B] See legend to Figure 1A. [Figure 1C] See legend to Figure 1A. [Figure 1D] See legend to Figure 1A. [Figure 1E] See legend to Figure 1A. [Figure 2] Determination of competitor groups by BLI for A23-58.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 3] Cryo-EM structure of the A23-58.1 Fab complexed with the SARS-CoV-2 spike. Three Fabs were shown to bind to the RBD on the spike protein. The epitope is located at the tip of the RBD, with residues 417, 453, 455, 456, 473, 475-480, 483-488, 489, and 493 contributing to antibody binding. Residues 417 and E484 are located at the edge of the epitope and contribute approximately 8% of the antibody-binding surface. [Figure 4A] Determination of competition groups by BLI for A19-61.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. [Figure 4B] Epitope mapping by negative staining EM. [Figure 5] Determination of competitor groups by BLI for A19-46.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 6]Determination of competitor groups by BLI for A23-105.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 7] Determination of competitor groups by BLI for A789-1.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 8] Determination of competitor groups by BLI for A20-29.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 9] Determination of competitor groups by BLI for A19-30.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 10]Competitor group determination by BLI for A20-36.1 and A20-9.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 11] Determination of competitor groups by BLI for A23-97.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 12] Determination of competitor groups by BLI for A23-113.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 13] Determination of competitor groups by BLI for A23-80.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 14]Determination of competitor groups by BLI for A19-82.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 15] Determination of competitor groups by BLI for B1-182.1. The order of addition to the biosensor was SARS CoV2 S2P (antigen), competitor mAb, and analyte mAb, with mAb114 being an isotype control mAb that does not bind to S2P. Red indicates >80% competition, yellow indicates 60-79.9% competition, and white indicates <60% or no competition. Black boxes are not relevant to competitor group assignment and have been removed for clarity. [Figure 16A]Figures 16A-16H. Identification and classification of highly potent antibodies from convalescent SARS-CoV-2 subjects. (A) Sera from 22 convalescent subjects were tested for neutralizing (y-axis, ID50) and binding (x-axis, S-2P ELISA AUC) antibodies. Four subjects, A19, A20, A23, and B1 (colored), with both high neutralizing and binding activity against WA-1 were selected for antibody isolation. (B) Final flow cytometry sorting gates for CD19+ / CD20+ / IgG+ or IgA+ PBMCs from four convalescent subjects (A19, A20, A23, and B1). Staining for RBD-SD1 BV421, S1 BV786, and S-2P APC or Ax647 is shown. Cells were sorted using the indicated sorting gates (pink), and the percent positive cells that were either RBD-SD1, S1, or S-2P positive are shown for each subject. (C) The overall binding epitope distribution was determined using MSD-based ELISA testing against the RBD, NTD, S1, S-2P, or HexaPro. S2 binding was inferred by S-2P or HexaPro binding and not by other antigens. Undefined epitopes exhibited mixed binding profiles. The total number of antibodies (i.e., 200) and absolute numbers of antibodies within each group are shown. (D) Neutralization curves (n = 2-3) were generated using WA-1 spike pseudotyped lentivirus and live virus neutralization assays to test the neutralizing capacity of the indicated antibodies. (E) Table showing antibody binding targets, IC50s for pseudovirus and live virus neutralization, and Fab:S-2P binding kinetics (n = 2) for the indicated antibodies. (F) SPR-based epitope binning experiment. Competitor antibodies (y-axis) bound to S-2P prior to incubation with the indicated analyte antibodies (x-axis). Percent competition range bins are shown as red (>=75%), orange (60-75%), or white (<60%) (n=2). The negative control antibody was the anti-Ebola glycoprotein antibody mAb114 (37). (G) Competition for ACE2 binding.The indicated antibodies (y-axis) compete for S-2P binding to soluble ACE2 protein using biolayer interferometry (left column, percent competition (>=75% shown as red, <60% shown as white)) or for cell surface-expressed ACE2 using cell surface staining (right column, EC50 in ng / ml). (H) Negative-stained 3D reconstruction of SARS-CoV-2 spike and Fab complexes. A19-46.1 and A19-61.1 bind to the down-positioned RBD, while A23-58.1 and B1-182.1 bind to the up-positioned RBD. Representative classes are shown with two Fabs bound, although stoichiometries of 1–3 were observed. [Figure 16B] See legend to Figure 16A. [Figure 16C] See legend to Figure 16A. [Figure 16D] See legend to Figure 16A. [Figure 16E] See legend to Figure 16A. [Figure 16F] See legend to Figure 16A. [Figure 16G] See legend to Figure 16A. [Figure 16H] See legend to Figure 16A. [Figure 17A]Figures 17A-17D. Antibody binding and neutralization of variants of interest. (A) Table showing the domains and mutations compared to WA-1 for each of the 10 variants tested in panels B-C. (B) Spike protein variants were expressed on the surface of HEK293T cells, and binding to the indicated antibodies was measured using flow cytometry. Data are shown as mean fluorescence intensity (MFI) normalized to MFI for the same antibody against the D614G parent variant. Percent change is indicated by a color gradient from red (increased binding, up to 500%) to white (no change, 100%) to blue (no binding, 0%). (C) IC50 and IC80 values ​​for the indicated antibodies against the 10 variants shown in (A). Ranges are indicated by the following colors: white (>10,000 ng / mL), light blue (1,000–10,000 ng / mL), yellow (100–1,000 ng / mL), orange (50–100 ng / mL), red (10–50 ng / mL), maroon (1–10 ng / mL), and purple (<1 ng / mL). (D) Locations of spike protein variant mutations on the spike glycoprotein for B.1.1.7, B.1.351, B.1.429, and P.1 v2. P681 and V1176 were not resolved in the structures, so their locations are not shown for B.1.1.7 and P.1 v2. [Figure 17B] See legend to Figure 17A. [Figure 17C] See legend to Figure 17A. [Figure 17D] See legend to Figure 17A. [Figure 18A]Figures 18A-18E. Structural basis for binding and neutralization for antibodies A23-58.1 and B1-182.1. (A) Cryo-EM structure of A23-58.1 Fab complexed with the SARS-CoV-2 HexaPro spike. The overall density map is shown on the left, with protomers shaded gray. One of the A23-58.1 Fabs bound to the RBD is shown. The RBD and A23-58.1 structures after local focused refinement are shown on the right. The heavy chain CDRs are confirmed for CDR H1, CDR H2, and CDR H3, respectively. The light chain CDRs are also confirmed for CDR L1, CDR L2, and CDR L3, respectively. The contour level of the cryo-EM map is 5.7 seconds. (B) Cryo-EM structure of B1-182.1 Fab complexed with the SARS-CoV-2 HexaPro spike. The overall density map is shown on the left, with the protomers indicated. One of the B1-182.1 Fabs bound to the RBD is shown. The RBD and B1-182.1 structures after localized refinement are shown on the right. The heavy chain CDRs are shown for CDR H1, CDR H2, and CDR H3, respectively. The light chain CDRs are shown for CDR L1, CDR L2, and CDR L3, respectively. The contour level of the cryo-EM map is 4.0 seconds. (C) Interaction between A23-58.1 and the RBD. All CDRs were involved in RBD binding. The epitope of A23-58.1 is shown on the light green surface. RBD mutations in currently circulating SARS-CoV-2 variants are colored red. K417 and E484 are located at the edges of the epitope. (D) Detail of interactions at the antibody-RBD interface. The edge of the RBD binds to the cavity formed by the CDRs (shown looking down into the cavity). Interactions between aromatic / hydrophobic residues are prominent at the bottom of the cavity. Hydrogen bonds at the edge of the cavity are marked with dashed lines. RBD residues are shown in italic font. (E) Paratopes of A23-58.1, B1-182.1, S2E12 (PDB ID: 7K45), and COVOX253 (PDB ID: 7BEN) from the same germline.The sequences of B1-182.1 (SEQ ID NO:1 VH, SEQ ID NO:5 VL), A23-58.1 (SEQ ID NO:25 VH and SEQ ID NO:29 VL), S2E12, and COVOX253 were aligned with variant residues underlined. Paratope residues of A23-58.1, B1-182.1, S2E12, and COVOX253 are highlighted. IGHV1-58*01 VH is SEQ ID NO:147, A23-58.1 VH is SEQ ID NO:25, B1-182.1 VH is SEQ ID NO:1, S2E1 VH is SEQ ID NO:148, COVOX253 VH is SEQ ID NO:149, IGHV1-58*01 VL is SEQ ID NO:150, A23-58.1 VH is SEQ ID NO:29, B1-182.1 VH is SEQ ID NO:5, S2E1 VL is SEQ ID NO:151, and COVOX253 VL is SEQ ID NO:152. [Figure 18B] See legend to Figure 18A. [Figure 18C] See legend to Figure 18A. [Figure 18D] See legend to Figure 18A. [Figure 18E] See legend to Figure 18A. [Figure 19A]Figures 19A-19E. The unique binding modes of A23-58.1 and B1-182.1 enable neutralization against VOCs. (A) Mapping of epitopes for A23-58.1, B1-182.1, and other antibodies on the RBD (SEQ ID NO:141). Epitope residues for various RBD-targeting antibodies are marked with an * below the RBD sequence. (B) Comparison of the binding modes of A23-58.1 and B1-182.1. Analysis showed that the axis of Fab B1-182.1 was rotated 6 degrees from the axis of A23-58.1 (left). This rotation caused a slight shift of the epitope of B1-182.1 on the RBD, thereby reducing its contact with E484 (right). The RBD mutations in question are highlighted, and the epitope surface of B1-182.1, the boundary of the ACE2 binding site, and the boundary of the A23-58.1 epitope are shown. (C) Comparison of the binding modes of A23-58.1, CB6, and REGN10933. For clarity, one Fab is shown binding to the RBD on the spike. A shift in the binding site to the saddle of the RBD surrounds K417, E484, and Y453 within the CB6 (black line) and REGN10933 epitopes (surface), explaining their susceptibility to the K417N, Y453F, and E484K mutations. (D) Comparison of the binding modes of A23-58.1 and LY-CoV555. One Fab is shown binding to the RBD on the spike (left). E484 is located within the LY-CoV555 epitope (top right), and the E484K / Q mutation eliminates critical contacts between the RBD and CDRs H2 and L3. Furthermore, E484K / Q and L452R may cause clashes with the heavy chain of LY-CoV555, explaining its susceptibility to E484K / Q and L452R mutations (bottom right). (E) IGHV1-58-derived antibodies target supersites with minimal contact to mutational hotspots. The supersites defined by the common atoms on the RBD contacted by IGHV1-58-derived antibodies (A23-58.1, B1-182.1, S2E12, and COVOX253) are shown.The ACE2 binding site, epitope boundaries for class I, II, and III antibodies represented by C102 (PDB ID 7K8M), C144 (PDB ID 7K90), and C135 (PDB ID 7K8Z) are shown. [Figure 19B] See legend to Figure 19A. [Figure 19C] See legend to Figure 19A. [Figure 19D] See legend to Figure 19A. [Figure 19E] See legend to Figure 19A. [Figure 20A] Figures 20A-20B. Critical binding residues for antibodies A23-58.1 and B1-182.1. (A) The indicated spike protein mutations, predicted by structural analysis, were expressed on the surface of HEK293T cells, and binding to the indicated antibodies was measured using flow cytometry. Data are shown as mean fluorescence intensity (MFI) normalized to MFI relative to WA-1 parent binding of the same antibodies. Percent change is indicated by a color gradient from red (increased binding, up to 200%) to white (no change, 100%) to blue (no binding, 0%). (B) IC50 and IC80 values ​​for the indicated antibodies against WA-1 and nine spike mutations. Ranges are indicated by the following colors: white (>10,000 ng / mL), light blue (1,000–10,000 ng / mL), yellow (100–1,000 ng / mL), orange (50–100 ng / mL), red (10–50 ng / mL), maroon (1–10 ng / mL), and purple (<1 ng / mL). [Figure 20B] See legend to Figure 20A. [Figure 21-1]Figures 21A-21E. Reduction of escape risk using dual antibody combinations. (A) Replication-competent vesicular stomatitis virus (rcVSV) expressing the SARS-CoV-2 WA-1 genome was incubated with serial dilutions of the indicated antibodies. After 48-72 hours, wells showing cytopathic effect (CPE) were passaged for subsequent rounds (Figure S8). Total supernatant RNA was harvested, and the viral genome was shotgun sequenced to determine the frequency of amino acid changes. The amino acid / positional changes and frequency of spike protein changes are shown as logo plots. Amino acid changes observed in two independent experiments are indicated in blue and green text. (B) The indicated spike protein mutations, predicted by structural analysis (Figures 18A-18E) or observed by escape analysis (Figure 21A), were expressed on the surface of HEK293T cells, and binding to the indicated antibodies was measured using flow cytometry. Data are shown as mean fluorescence intensity (MFI) normalized to MFI for WA-1 parent binding of the same antibody. Percent change is indicated by a gradient from gray (increased binding, up to 200%) to white (no change, 100%) to gray (no binding, 0%). (C) IC50 and IC80 values ​​for the indicated antibodies against WA-1 and mutations predicted by structural analysis (Figures 18A-18E) or observed by escape analysis (Figure 21A). (D) Negative-stain 3D reconstructions of ternary complexes of Fab B1-182.1 and spike with A19-46.1 (left) or A19-61.1 (right). (E) rcVSV SARS-CoV-2 was incubated with increasing concentrations (1.3e-4 to 50 mg / mL) of either single antibodies (A19-46.1, A19-61.1, and B1-182.1) and antibody combinations (B1-182.1 / A19-46.1 and B1-182.1 / A19-61.1). Wells were assessed for CPE every 3 days, and wells with the highest concentration exhibiting >20% CPE were passaged to medium containing fresh cells and antibodies. For each test condition in each round of selection, the maximum concentration exhibiting >20% CPE is shown. Once 50 mg / mL was reached, the virus was no longer passaged, and dashed lines are used to indicate that the maximum antibody concentration was reached in subsequent rounds. [Figure 21-2] See description of Figure 21-1. [Figure 21-3] See description of Figure 21-1. [Figure 21-4] See description of Figure 21-1. [Figure 22A]Figures 22A-22E. Cryo-EM structure of the SARS-CoV-2 B.1.1.529 (omicron) spike. (A) Cryo-EM map of the SARS-CoV-2 B.1.1.529 spike. Reconstructed density maps at 3.29 Å resolution are shown in side and top views. The contour level of the cryo-EM map is 4.0 arcseconds. (B) Interprotomer interactions introduced by the B.1.1.529 amino acid substitution. Substitutions in one protomer are shown as spheres. Examples of interprotomer interactions introduced by the B.1.1.529 substitution are highlighted in boxes in the enlarged view to the side. Mutations are listed as a percentage of the domain surface (surface) or a percentage of the sequence (sequence). (C) The fragile NTD supersite is shown on a semi-transparent surface with backbone ribbons. Amino acid substitutions, deletions, and insertions are indicated. (D) Fifteen amino acid substitutions clustered at the edge of the RBD altered 16% of the RBD surface area (left) and increased the electropositivity of the ACE2 binding site (right). Mutated residues are shown as sticks. The ACE2 binding site on the electrostatic potential surface is also marked. (E) Mapping of the B.1.1.529 RBD substitutions onto the epitopes of Barnes class I-IV antibodies. The positions of the substitutions are indicated on the surface. Residues that potentially affect the activity of each class of antibody are indicated by their residue numbers. The class I footprint is defined by the CB6 and B1-182.1 epitopes; the class II footprint is defined by the A19-46.1 and LY-CoV555 epitopes; the class III footprint is defined by the A19-61.1, CoV2-2130, LY-CoV1404, and S309 epitopes; and the class IV footprint is defined by the DH1407 and S304 epitopes. Class I and II epitopes overlap with the ACE2 binding site, whereas class III and IV epitopes do not. Class II and III epitopes allow binding to WA-1 when the RBD is in either the up or down conformation. [Figure 22B] See legend to Figure 22A. [Figure 22C] See legend to Figure 22A. [Figure 22D]See legend to Figure 22A. [Figure 22E] See legend to Figure 22A. [Figure 23A] Figures 23A-23C. SARS-CoV-2 monoclonal antibody binding and neutralization. (A) Model of the SARS-CoV-2 WA-1 spike protein (PDB: 6XM3) with red dots indicating the positions of substitutions present in the variants. The total number of mutations and the number and position of receptor-binding domain (RBD) mutations in the spike protein variant in question are also shown. (B) Full-length spike proteins from the indicated SARS-CoV-2 variants were expressed on the surface of transiently transfected 293T cells, and binding to the indicated monoclonal antibodies was assessed by flow cytometry. The mean fluorescence intensity (MFI) of bound antibody on the indicated cells is shown relative to the MFI of the same antibody bound to D614G-expressing cells. Data are expressed as a percentage. A representative experiment (n = 2-3 for each antibody) is shown. (C) Lentivirus pseudotyped with SARS-CoV-2 spike protein from D614G, B.1.1.7, B.1.351, P.1, B.1.617.2, or B.1.1.529 was incubated with serial dilutions of the indicated antibodies, and IC50 and IC80 values ​​were determined. S309 was tested in 293 flpin-TMPRSS2-ACE2 cells, while all other antibodies were tested in 293T-ACE2 cells. *nd = not determined due to incomplete neutralization, which plateaued at <80%. [Figure 23B] See legend to Figure 23A. [Figure 23C] See legend to Figure 23A. [Figure 24A]Figures 24A-24D. Functional and structural basis of class I antibody neutralization and mechanistic basis for retained potency against B.1.1.529 VOCs. (A) Lentivirus pseudotyped with SARS-CoV-2 spike protein of D614G, or D614G plus the indicated point mutations found in the B.1.1.529 spike, was incubated with serial dilutions of the indicated antibodies and transduced into 293T-ACE2 cells to determine IC50 and IC80 values. S375F and G496S viruses were not available and are shown as "untested" (nt). G496R was available and was used in place of G496S. (B) Mapping of B.1.1.529 amino acid substitutions in the epitope of the class I antibody CB6. RBD-bound CB6 was docked into the B.1.1.529 spike structure. B.1.1.529 amino acid substitutions incompatible with CB6 binding were identified and labeled. The K417N mutation caused a clash in the center of the paratope. The B.1.1.529 RBD is shown in a diagram with the amino acid substitutions in stick form. CB6 is shown in a surface representation with the heavy and light chains labeled 153–156, respectively. (C) Docking of the RBD-bound VH1-58-derived class I antibody B1-182.1 into the B.1.1.529 spike structure identified four substitutions with potential steric hindrance. B1-182 is shown in a surface representation with the heavy and light chains labeled. B.1.1.529 amino acid substitutions that may affect VH1-58 antibody binding are labeled. (D) Structural basis for effective neutralization of the B.1.1.529 VOC by VH1-58-derived antibodies. VH1-58 antibodies, such as S2E12, CoV2-2196, A23-58.1, and B1-182.1, share high sequence homology (top right), but their neutralization potency against B.1.1.529 varies. Structural analysis indicated that residue 100C of CDR H3, located in the interface cavity formed by the RBD, heavy chain, and light chain, may determine their potency against B.1.1.529 (left). The size of this residue correlated with potency with a two-tailed p=0.046 (bottom right). Figure 24D shows SEQ ID NOs: 153-156. [Figure 24B] See legend to Figure 24A. [Figure 24C] See legend to Figure 24A. [Figure 24D] See legend to Figure 24A. [Figure 25-1]Figures 25A-25E. Functional and structural basis for class II antibody binding, neutralization, and escape. (A) Lentivirus pseudotyped with SARS-CoV-2 spike protein D614G or D614G + the indicated point mutations found in the B.1.1.529 spike was incubated with serial dilutions of the indicated antibodies and transduced into 293T-ACE2 cells to determine IC50 and IC80 values. S375F and G496S viruses were not available and are shown as "untested" (nt). G496R was available and was used in place of G496S. (B) Cryo-EM structure of the class II antibody A19-46.1 Fab complexed with the B.1.1.529 spike. A global density map is shown on the left, along with the protomers. Two A19-46.1 Fabs are shown bound to the RBD in the up-conformation. The RBD and A19-46.1 structures after localized refinement are shown in a diagrammatic representation on the right. The heavy chain CDRs (CDR H1, CDR H2, and CDR H3) are indicated. The light chain CDRs (CDR L1, CDR L2, and CDR L3, respectively) are also shown. The contour level of the cryo-EM map is 4.0 seconds. (C) Interaction between A19-46.1 and the RBD. CDR H3 and all light chain CDRs were involved in RBD binding (left). The A19-46.1 epitope is shown on the B.1.1.529 RBD surface along with amino acid substitutions. Ser446, A484, and R493 are located at the edges of the epitope of Fab A19-46.1 (right). RBD residues are shown in italic font. (D) Binding of A19-46.1 to the RBD prevents ACE2 receptor binding. Schematic representations of ACE2 and A19-46.1 are shown. (E) Comparison of the binding modes to the RBD for antibodies A19-46.1 and LY-CoV555. Both antibodies target similar regions on the RBD, but different angles of approach result in a clash between LY-CoV555 CDR H3 and the B.1.1.529 mutation Arg493 (left and inset). The B.1.1.529 mutation involved in A19-46.1 binding is only at the edge of its epitope, whereas both Arg493 and A484 are located in the center of the LY-CoV555 epitope (right).The Leu452 to Arg mutation, which knocks out binding of A19-46.1 and LY-CoV555 in other SARS-CoV-2 variants, is colored blue. [Figure 25-2] See description of Figure 25-1. [Figure 25-3] See description of Figure 25-1. [Figure 26-1]Figures 26A-26G. Functional and structural basis for binding, neutralization, and retention of potency of Class III antibodies against the B.1.1.529 VOC. (A) Lentivirus pseudotyped with the SARS-CoV-2 spike protein of D614G or D614G + the indicated point mutations found in the B.1.1.529 spike were incubated with serial dilutions of the indicated antibodies, and IC50 and IC80 values ​​were determined. A19-61.1 and LY-CoV1404 were assayed in 293T-ACE2 cells, while S309 and CoV2-2130 were tested in 293 flpin-TMPRSS2-ACE2 cells. S375F and G496S viruses were not available and are shown as "untested" (nt). G496R was available and was used in place of G496S. (B) Cryo-EM structure of the SARS-CoV-2 WA-1 spike in complex with the class I antibody B1-182.1 and the class III antibody A19-61.1 at 2.83 Å resolution. The full density map is shown along with the protomers. Two RBDs are in the up conformation, each bound to both Fabs, and one RBD is in the down position, bound to A19-61.1. The RBD, B1-182.1, and A19-61.1 are shown (left). A schematic representation of the RBD structure with both Fabs bound, after localized refinement, is shown on the right. The RBD is shown in green, representing the antibody light chain (center). The epitope of A19-61.1 is shown as a surface on the RBD, with the interacting CDRs labeled (right). The contour level of the cryo-EM map was 5.2 arcsec. (C) Structural basis of B.1.1.529 resistance to A19-61.1. Mapping of the A19-61.1 epitope onto the B.1.1.529 RBD showed that G446S clashes with CDR H3 of A19-61.1. The RBD is shown diagrammatically with the mutated residues in sticks and the A19-61.1 epitope on the surface. (D) Structural basis of B.1.1.529 VOC neutralization of CoV2-2130. Docking of CoV2-2130 onto the B.1.1.529 RBD showed that Y50 of CDR L2 creates a minor clash with S446.The RBD is shown in a stick diagram with the mutated residues, and the CoV2-2130 epitope is shown on the surface. (E) Structural basis for S309 neutralization by the B.1.1.529 VOC. The docked complex of S309 and the B.1.1.529 RBD revealed the S371L / S373P / S375F loop. The S371L mutation is adjacent to the S309 epitope, while the G339D mutation is internal to the epitope. The D339 side chain clashes with CDR H3 Y100. The B.1.1.529 RBD is shown in a stick diagram with the mutated residues, and the WA-1 RBD is shown in gray. (F) Structural basis for LY-CoV1404 neutralization by the B.1.1.529 VOC. Docking of LY-CoV1404 onto the B.1.1.529 RBD identified four amino acid substitutions in the epitope, including G446S, which potentially clashes with CDR H2 R60. However, comparison of both the LY-CoV1404-bound and -unbound B.1.1.529 RBDs indicated that the S446 loop is flexible enough to allow LY-CoV1404 binding. The B.1.1.529 residues in the LY-CoV1404 epitope are shown along with the corresponding WA-1 residues. CDR H3 is shown in a diagrammatic representation. (G) Overlay of the epitope footprint of a class III antibody onto the B.1.1.529 RBD. The location of the amino acid substitutions in the B.1.1.529 RBD is indicated. [Figure 26-2] See description of Figure 26-1. [Figure 26-3] See description of Figure 26-1. [Figure 26-4] See description of Figure 26-1. [Figure 27-1]Figures 27A-27C. Potent neutralization of SARS-CoV-2 B.1.1.529 using antibody combinations. (A) Lentivirus pseudotyped with the SARS-CoV-2 spike protein of D614G or D614G + the indicated point mutations found in the B.1.1.529 spike was incubated with serial dilutions of the indicated combinations of antibodies, and IC50 and IC80 values ​​were determined. S375F and G496S viruses were not available and are shown as "untested" (nt). G496R was available and was used in place of G496S. (B) Neutralization IC50 (ng / mL) values ​​for the indicated cocktail (x-axis) or each of its component antibodies. The IC50 for the first antibody is listed as mAb1, and the second antibody is listed as mAb2 or cocktail. (C) Cryo-EM structure of the B.1.1.529 spike in complex with antibodies A19-46.1 and B-182.1 at 3.86 Å resolution. The overall density map, along with the protomers, is shown (left). All RBDs are in the up conformation, with both Fabs bound (center). Binding of one Fab (e.g., B1-182.1) may induce the RBD into the up conformation, facilitating binding of the other Fab (e.g., A19-46.1), which only recognizes the up conformation of the RBD (right). A19-46.1 and B-182.1 are shown, respectively. The contour level of the cryo-EM map is 6.5 s. [Figure 27-2] See description of Figure 27-1. [Figure 28A] Figures 28A-28B. Neutralization of pseudotyped lentivirus with Wuhan 1 (wt) spike (S) or the indicated SARS-CoV-2 variants. Neutralization was determined by incubating virus with various dilutions of antibody before adding to cells. % infection was used to generate values ​​for 50% (IC50) and 80% (IC80) inhibitory concentrations. These values ​​represent the amount of antibody in μg / mL required to reduce infection by 50% and 80%, respectively. (A) Neutralization IC50 and IC80 against variants containing single or compound mutations. (B) Neutralization IC50 and IC80 against variants (VOC / VOI). [Figure 28B] See legend to Figure 28A. [Figure 29-1] Figures 29A-29C. Table for Example 30. (A) Yield and precipitation for B1-182.1 and A23.58.1. (B) Yield, concentration, and precipitation characteristics for B1-182.1_58CDRH3 / B1-182.1light and B1-182heavy / B1-182.1light_5Mut. (C) Neutralization by B1-182.1_58CDRH3 / B1-182.1 (IC50, IC80 in μg / ml) of lentiviruses pseudotyped with the indicated spike proteins from SARS-CoV-2. [Figure 29-2] See description of Figure 29-1. [Figure 30] Serum levels of SARS-CoV-2 antibodies in human FcRn transgenic mice administered at 5 mg / kg via the IV route.

[0013] Sequence Listing Standard letter abbreviations for nucleotide bases and three-letter codes for amino acids are used to depict nucleic acid and amino acid sequences, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood to be included by any reference to the strand shown. The sequence listing is submitted as an ASCII text file [Sequence Listing, February 2, 2022, 67.6 KB], which is incorporated herein by reference.

[0014] SEQ ID NO:1 is B1-182.1 V H SEQ ID NOs: 2, 3, and 4 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0015] SEQ ID NO:5 is B1-182.1 V L SEQ ID NOs: 6, 7, and 8 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0016] SEQ ID NO:9 is A19-61.1 V H SEQ ID NOs: 10, 11, and 12 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0017] SEQ ID NO:13 is A19-61.1 V L SEQ ID NOs: 14, 15, and 16 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0018] SEQ ID NO:17 is A19-46.1 V H SEQ ID NOs: 18, 19, and 20 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0019] SEQ ID NO:21 is A19-46.1 V L SEQ ID NOs: 22, 23, and 24 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0020] SEQ ID NO:25 is A23-58.1 V H SEQ ID NOs:26, 27, and 28 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0021] SEQ ID NO:29 is A23-58.1 V L SEQ ID NOs: 30, 31, and 32 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0022] SEQ ID NO:33 is A20-29.1 V H SEQ ID NOs:34, 35, and 36 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0023] SEQ ID NO:37 is A20-29.1 V L SEQ ID NOs: 38, 39, and 40 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0024] SEQ ID NO:41 is A23-105.1 V H SEQ ID NOs: 42, 43, and 44 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0025] SEQ ID NO:45 is A23-105.1 V L SEQ ID NOs: 46, 47, and 48 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0026] SEQ ID NO:49 is A19-1.1 V H SEQ ID NOs:50, 51, and 52 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0027] SEQ ID NO:53 is A19-1.1 V L SEQ ID NOs:54, 55, and 56 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0028] SEQ ID NO:57 is A19-30.1 V H SEQ ID NOs:58, 59, and 60 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0029] SEQ ID NO:61 is A19-30.1 V L SEQ ID NOs: 62, 63, and 64 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0030] SEQ ID NO:65 is A20-36.1 V H SEQ ID NOs: 66, 67, and 68 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0031] SEQ ID NO:69 is A20-36.1 V L SEQ ID NOs:70, 71, and 72 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0032] SEQ ID NO:73 is A23-97.1 V H SEQ ID NOs:74, 75, and 76 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0033] SEQ ID NO:77 is A23-97.1 V L SEQ ID NOs:78, 79, and 80 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0034] SEQ ID NO:81 is A23-113.1 V H SEQ ID NOs: 82, 83, and 84 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0035] SEQ ID NO:85 is A23-113.1 V L SEQ ID NOs: 86, 87, and 88 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0036] SEQ ID NO:89 is A23-80.1 V HSEQ ID NOs:90, 91, and 92 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0037] SEQ ID NO:93 is A23-80.1 V L SEQ ID NOs: 94, 95, and 96 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0038] SEQ ID NO:97 is A19-82.1 V H SEQ ID NOs:98, 99, and 100 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0039] SEQ ID NO:101 is A19-82.1 V L SEQ ID NOs: 102, 103, and 104 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0040] SEQ ID NO:105 is A20-9.1 V H SEQ ID NOs: 106, 107, and 108 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0041] SEQ ID NO:109 is A20-9.1 V L SEQ ID NOs: 110, 111, and 112 are the amino acid sequences of LCDR1, LCDR2, and LCDR3, respectively.

[0042] SEQ ID NO: 113 to 140 are V H or V L is a nucleic acid sequence encoding

[0043] SEQ ID NO:141 is the RBD sequence of Figure 19A.

[0044] SEQ ID NO:142 is the nucleic acid sequence of a portion of a nucleic acid molecule encoding IgA.

[0045] SEQ ID NO:143 is the amino acid sequence of B1-182.1_58.1CDRH3 heavy / B1-182.1 light. SEQ ID NOs:2, 3, and 28 are the amino acid sequences of HCDR1, HCDR2, and HCDR3, respectively.

[0046] SEQ ID NO:144 is the amino acid sequence of the B1-182.1 heavy / B1-182.1 light_5Mut chain. SEQ ID NOs:6, 145, and 146 are CDR sequences.

[0047] SEQ ID NO:147 is IGHV1-58*01 V H is the amino acid sequence of

[0048] SEQ ID NO:148 is S2E12V H is the amino acid sequence of

[0049] SEQ ID NO:149 is COVOX253 V H is the amino acid sequence of

[0050] SEQ ID NO:150 is IGHV1-58*01 V L is the amino acid sequence of

[0051] SEQ ID NO:151 is S2E12V L is the amino acid sequence of

[0052] SEQ ID NO:152 is COVOX253 V L is the amino acid sequence of

[0053] SEQ ID NO:153 is the amino acid sequence of a portion of A23-58.1.

[0054] SEQ ID NO:154 is the amino acid sequence of a portion of B1-182.1.

[0055] SEQ ID NO:155 is a partial amino acid sequence of CoV2-2196.

[0056] SEQ ID NO:156 is the amino acid sequence of a portion of S2E12. DETAILED DESCRIPTION OF THE INVENTION

[0057] DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS Disclosed herein are monoclonal antibodies that specifically bind to the spike protein of coronaviruses, such as SARS-CoV-2. In some embodiments, these antibodies are useful for inhibiting coronavirus infection and for detecting coronaviruses in biological samples. The antibodies are potently neutralizing and target a unique epitope in the spike glycoprotein of SARS-CoV-2.

[0058] Global genome sequencing has revealed the emergence of SARS-CoV-2 variants that increase transmissibility and reduce the efficacy of vaccine-induced and therapeutic antibodies (see, e.g., Wibmer et al., Nat. Med. 27, 622-625 (2021); Wang et al., Nature. 593, 130-135 (2021); Muik et al., Science. 371, 1152-1153 (2021); Wang et al., Nature. 592, 616-622 (2021)). Recently, there have been serious concerns that antibody responses to natural infection and vaccination with ancestral spike sequences may result in focused responses that lack efficacy against mutations present in more recent variants (e.g., K417N, L452R, T478K, E484K / Q, N501Y in B.1.351, B.1.617.1, and B.1.617.2) (see, e.g., Wibmer et al., Nat. Med. 27, 622-625 (2021); Wang et al., Nature. 593, 130-135 (2021); Muik et al., Science. 371, 1152-1153 (2021); Wang et al., Nature. 592, 616-622 (2021)). Furthermore, neutralization of the P.1 virus could be achieved using serum from subjects infected with B.1.351 (Moyo-Gwete et al., N. Engl. J. Med. 2 (2021), doi:10.1056 / NEJMc2104192), suggesting that shared epitopes in the RBD (i.e., K417N, E484K, and N501Y) mediate cross-reactivity. While the mechanism of cross-reactivity between B.1.351 and P.1 likely focuses on three RBD mutations, the mechanism of broadly neutralizing antibody responses between WA-1 and subsequent variants has not been fully established.Disclosed herein are antibodies isolated and characterized with neutralization breadth that covers emerging SARS-CoV-2 variants, including but not limited to the highly contagious variants B.1.1.7, B.1.351, B.1.617.2, and B.1.1.529. The increased potency and breadth was mediated by binding to the distal region of the RBD, away from E484K / Q, L452R, and other mutational hotspots, which are major determinants of resistance in VOCs.

[0059] I. Terminology Overview Unless otherwise specified, terminology is used according to conventional usage. Definitions of many common terms in molecular biology can be found in Krebs et al. (eds.), Lewin's Genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless the context clearly dictates otherwise. For example, the term "an antigen" includes one or more antigens and can be considered equivalent to the phrase "at least one antigen." As used herein, the term "comprises" means "includes." It should be further understood that, unless otherwise indicated, any and all base or amino acid sizes and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and are provided for descriptive purposes. While many methods and materials similar or equivalent to those described herein can be used, certain suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. The following explanations of terms are provided to facilitate review of the various embodiments.

[0060] About: Unless the context indicates otherwise, "about" refers to plus or minus 5% of the reference value. For example, "about" 100 refers to 95 to 105.

[0061] Administration: The introduction of an agent, such as a disclosed antibody, into a subject by a selected route. Administration can be local or systemic. For example, if the selected route is intravascular, the agent (e.g., antibody) is administered by introducing the composition into the subject's blood vessels. Exemplary routes of administration include, but are not limited to, oral, injection (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (e.g., topical), intranasal, vaginal, and inhalation routes.

[0062] Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.

[0063] Antibodies and antigen-binding fragments: Immunoglobulins, antigen-binding fragments, or derivatives thereof that specifically bind to and recognize an analyte (antigen), such as the spike protein of a coronavirus, e.g., the spike protein from SARS-CoV-2. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, so long as they exhibit the desired antigen-binding activity.

[0064] Non-limiting examples of antibodies include, for example, intact immunoglobulins, as well as variants and fragments thereof that retain binding affinity to an antigen. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments either produced by modification of whole antibodies or synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2002). nd ed., Springer-Verlag, 2010).

[0065] Antibodies also include genetically engineered forms such as chimeric antibodies (eg, humanized murine antibodies) and heteroconjugate antibodies (eg, bispecific antibodies).

[0066] An antibody may have one or more binding sites. If more than one binding site is present, the binding sites may be identical to each other or different. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific or bifunctional antibodies have two different binding sites.

[0067] Typically, naturally occurring immunoglobulins have heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as various immunoglobulin variable domain genes. There are two types of light chains: lambda (λ) and kappa (κ). There are five major heavy chain classes (or isotypes): IgM, IgD, IgG, IgA, and IgE, which determine the functional activity of antibody molecules.

[0068] Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). Together, the heavy and light chain variable regions specifically bind to an antigen.

[0069] "V H References to "V" or "VH" refer to the variable region of the heavy chain of an antibody, including that of an antigen-binding fragment such as an Fv, scFv, dsFv, or Fab. L References to "VL" or "VL" refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

[0070] V H and V L comprises a "framework" region separated by three hypervariable regions, also called "complementarity-determining regions" or "CDRs" (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th (See, e.g., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 1991). The sequences of framework regions of different light or heavy chains are relatively conserved within a species. The framework regions of an antibody are the combined framework regions of the constituent light and heavy chains and serve to position and align the CDRs in three-dimensional space.

[0071] CDRs are primarily responsible for binding to an epitope of an antigen. The boundaries of the amino acid sequence of a given CDR are determined by the methods described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5 thed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 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-like domains," Dev. Comp. Immunol., 27(1):55-77, 2003; "IMGT" numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. H CDR3 is the V of the antibody in which it is found H CDR3 from V L CDR1 is the V of the antibody in which it is found L The light chain CDRs may be referred to as LCDR1, LCDR2, and LCDR3. The heavy chain CDRs may be referred to as HCDR1, HCDR2, and HCDR3.

[0072] In some embodiments, the disclosed antibodies comprise a heterologous constant domain, for example, an antibody comprises a constant domain that is different from the native constant domain, for example, a constant domain that comprises one or more modifications to increase half-life (e.g., "LS" mutations).

[0073] A "monoclonal antibody" is an antibody obtained from a population of substantially homogeneous antibodies; i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for variant antibodies that may contain, for example, naturally occurring mutations or arise during the production of the monoclonal antibody preparation and are generally present in minor amounts. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies can be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin locus; such methods, as well as other exemplary methods for producing monoclonal antibodies, are described herein. In some examples, the monoclonal antibody is isolated from a subject. Monoclonal antibodies may have conservative amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin functions (see, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014.

[0074] A "humanized" antibody or antigen-binding fragment comprises a human framework region and one or more CDRs from a non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragment. The non-human antibody or antigen-binding fragment providing the CDRs is referred to as the "donor," and the human antibody or antigen-binding fragment providing the framework is referred to as the "acceptor." In one embodiment, all CDRs are from the donor immunoglobulin of the humanized immunoglobulin. Constant regions need not be present, but if present, can be substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, e.g., about 95% or more identical. Thus, all parts of a humanized antibody or antigen-binding fragment, except possibly for the CDRs, are substantially identical to the corresponding parts of a natural human antibody sequence.

[0075] A "chimeric antibody" is an antibody that contains sequences derived from two different antibodies, typically from different species. In some instances, a chimeric antibody contains one or more CDRs and / or framework regions from one human antibody and CDRs and / or framework regions from another human antibody.

[0076] A "fully human antibody" or "human antibody" is an antibody that contains sequences from (or derived from) the human genome and no sequences from another species. In some embodiments, a human antibody contains CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques to generate antibodies based on sequences from the human genome, for example, by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1 stEd. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

[0077] SARS-CoV-2-neutralizing antibody or antigen-binding fragment: An antibody or antigen-binding fragment that specifically binds to a SARS-CoV-2 antigen (e.g., spike protein) in a manner that inhibits a biological function associated with SARS-CoV-2, thereby inhibiting infection. The antibody can neutralize the activity of SARS-CoV-2. For example, a SARS-CoV-2-neutralizing antibody or antigen-binding fragment can interfere with the virus by directly binding to the virus and limiting 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 examples, antibodies specific for the coronavirus spike protein neutralize the infectious titer of SARS-CoV-2.

[0078] In some embodiments, the antibody or antigen-binding fragment that specifically binds to and neutralizes SARS-CoV-2 inhibits infection of cells by at least 50% compared to a control antibody or antigen-binding fragment.

[0079] A "broadly neutralizing antibody" is an antibody that binds to and inhibits the function of a related antigen, e.g., an antigen that shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical antigenic surface. With respect to antigens from pathogens, e.g., viruses, the antibody can bind to antigens from more than one class and / or subclass of pathogen and inhibit the function of the antigen. For example, with respect to coronaviruses, the antibody can bind to antigens such as spike proteins from coronaviruses, including SARS-CoV-2, and inhibit the function of the antigen.

[0080] Biological sample: A sample obtained from a subject. Biological samples include, but are not limited to, cells, tissues, and bodily fluids, such as blood, blood derivatives and fractions (e.g., serum), cerebrospinal fluid; and all clinical samples useful for detecting disease or infection in a subject, including biopsy tissue or surgically removed tissue, such as unfixed, frozen, or fixed in formalin or paraffin. In a specific example, the biological sample is obtained from a subject with or suspected of having a SARS-CoV-2 infection.

[0081] Bispecific antibodies: Recombinant molecules consisting of two different antigen-binding domains that ultimately bind to two different antigenic epitopes. Bispecific antibodies include molecules in which two antigen-binding domains are chemically or genetically linked. The antigen-binding domains can be linked using a linker. The antigen-binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. Bispecific antibodies can include one or more constant domains, but do not necessarily include constant domains.

[0082] Conditions sufficient to form an immune complex: Conditions that allow an antibody or antigen-binding fragment to bind to its cognate epitope to a detectably greater extent than binding to substantially all other epitopes and / or to substantially eliminate binding to substantially all other epitopes. Conditions sufficient to form an immune complex depend on the type of binding reaction and are typically those utilized in immunoassay protocols or conditions encountered in vivo. For a description of immunoassay formats and conditions, see Greenfield (Ed.), Antibodies: A Laboratory Manual, 2004, pp. 111-114, 1997. nd ed. New York: Cold Spring Harbor Laboratory Press, 2014. The conditions used in this method are "physiological conditions," which includes reference to conditions (e.g., temperature, osmolality, pH) typical inside a living mammal or mammalian cell. While it is recognized that some organs are subjected to extreme conditions, the environment within an organism and within a cell is typically at approximately 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. The osmolality is within a range that supports cell viability and growth.

[0083] Immune complex formation can be detected by conventional methods, such as immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblot (e.g., Western blot), magnetic resonance imaging (MRI), computed tomography (CT) scan, radiography, and affinity chromatography.

[0084] Conjugate: A complex of two molecules that are linked to each other, e.g., covalently linked. In one embodiment, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to SARS-CoV-2 that is covalently linked to an effector molecule, e.g., a detectable label. Linkage can be by chemical or recombinant means. In one embodiment, linkage is chemical, where reaction between the antibody moiety and the effector molecule generates a covalent bond formed between the two molecules to form a single molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because a conjugate can be prepared from two molecules with separate functionalities, e.g., an antibody and an effector molecule, it may also be referred to as a "chimeric molecule."

[0085] Conservative variant: A "conservative" amino acid substitution is one that does not substantially affect or impair the function of a protein, such as its ability to interact with a target protein. For example, a SARS-CoV-2-specific antibody can contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or even up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity to spike protein binding and / or SARS-CoV-2 neutralization activity. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid.

[0086] Individual substitutions, deletions, or additions that change, add, or remove a single amino acid or a small number of amino acids (e.g., less than 5%, in some embodiments, less than 1%) in the encoded sequence are conservative mutations in which the change results in the substitution of one amino acid with a chemically similar amino acid.

[0087] The following six groups are examples of amino acids that are considered conservative substitutions for one another: 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).

[0088] Non-conservative substitutions reduce the activity or function of an antibody, for example, its ability to specifically bind to the spike protein of a coronavirus. For example, if an amino acid residue is essential for the function of a protein, even an otherwise conservative substitution may disrupt its activity. Therefore, conservative substitutions do not change the basic function of the protein of interest.

[0089] Contacting: To bring into direct physical association; this includes both solid and liquid forms and can occur either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, e.g., amino acids on the surface of a polypeptide, such as an antigen, contacting another polypeptide, such as an antibody. Contacting can also include contacting cells, for example, by bringing an antibody into direct physical association with the cell.

[0090] Control: Reference Standard. In some embodiments, the control is a negative control, e.g., a sample obtained from a healthy patient not infected with coronavirus. In other embodiments, the control is a positive control, e.g., a tissue sample obtained from a patient diagnosed with coronavirus infection. In still other embodiments, the control is a historical control or a standard reference value or range of values ​​(e.g., previously tested control samples, e.g., a group of patients with known prognosis or outcome, or a group of samples representing baseline or normal values).

[0091] The difference between the test sample and the control can be an increase or a decrease.The difference can be a qualitative difference or a quantitative difference, for example, a statistically significant difference.In some examples, the difference is at least about 5%, 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% increase or decrease compared to the control.

[0092] Coronavirus: A family of positive-sense, single-stranded RNA viruses known to cause severe respiratory illness. Viruses from the Coronaviridae family currently known to infect humans are from the alphacoronavirus and betacoronavirus genera. Additionally, the gammacoronavirus and deltacoronavirus genera are thought to have the potential to infect humans in the future.

[0093] Non-limiting examples of betacoronaviruses 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), Murine Hepatitis Virus (MHV-CoV), Bat SARS-like coronavirus WIV1 (WIV1-CoV), and Human coronavirus HKU9 (HKU9-CoV). Non-limiting examples of alphacoronaviruses 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 a deltacoronavirus is swine deltacoronavirus (SDCV).

[0094] The viral genome is capped, polyadenylated, and surrounded by nucleocapsid proteins. Coronavirus virions contain a viral envelope containing a type I fusion glycoprotein called the spike (S) protein. Most coronaviruses share a common genome organization, including a replicase gene.

[0095] Degenerate variant: In this disclosure, "degenerate variant" refers to a polynucleotide encoding a polypeptide (e.g., an antibody heavy or light chain) that contains a degenerate sequence as a result of the genetic code. There are 20 naturally occurring amino acids, many of which are specified by more than one codon. Thus, all degenerate nucleotide sequences that encode a peptide are included, as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

[0096] Detectable marker: A detectable molecule (also known as a label) that is directly or indirectly conjugated to a second molecule, such as an antibody, to facilitate detection of the second molecule. For example, a detectable marker may be detectable by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging techniques (e.g., CT scan, MRI, ultrasound, fiberoptic examination, and laparoscopy). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance on selecting detectable markers suitable for various purposes 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, 2017, including supplements).

[0097] Detect: To identify the existence, presence, or fact of something.

[0098] Dual variable domain immunoglobulin: A bispecific antibody containing two heavy chain variable domains and two light chain variable domains. However, unlike IgG, both the heavy and light chains of a DVD-immunoglobulin molecule contain an additional variable domain (VD) connected to the N-terminus of the VH and VL of an existing monoclonal antibody (mAb) via a linker sequence. Thus, 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. For a schematic and space-filling diagram, see Figure 1 of Jakob et al. (商標) The molecule functions to simultaneously bind two different antigens on each DFab.

[0099] Effective amount: The quantity of a particular substance that is sufficient to achieve a desired effect in a subject to which it is administered. For example, this may be the amount necessary to inhibit a coronavirus infection, e.g., a SARS-CoV-2 infection, or to measurably alter the outward symptoms of such an infection.

[0100] In one example, the desired response is to inhibit, reduce, or prevent SARS-CoV-2 infection. It is not necessary to completely eliminate, reduce, or prevent SARS-CoV-2 infection for the method to be effective.

[0101] In some embodiments, administration of an effective amount of the disclosed antibodies or antigen-binding fragments that bind to the coronavirus spike protein can reduce or inhibit SAR-CoV-2 infection (eliminating or preventing detectable infection) by a desired amount, e.g., 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%, relative to a suitable control (as measured by infection of cells, or by the number or percentage of subjects infected with the coronavirus, or by increased survival of infected subjects, or by a reduction in symptoms associated with the infection).

[0102] The effective amount of an antibody or antigen-binding fragment that specifically binds to a coronavirus spike protein to be administered to a subject to inhibit infection will vary depending on several factors related to the subject, such as the subject's overall health and / or weight. An effective amount can be determined by varying the dosage and measuring the resulting response, such as a reduction in pathogen titer. An effective amount can also be determined by various in vitro, in vivo, or in situ immunoassays.

[0103] The effective amount includes divided doses that contribute to achieving effective response in combination with previous or subsequent administration.For example, the effective amount of an agent can be administered in a single dose or several doses, for example, daily, during a course of treatment lasting several days or weeks.However, the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the mode of administration.The unit dosage form of the agent can be packaged in a certain amount or in multiples of an effective amount, for example, in a vial (for example, with a pierceable cap) or syringe containing sterile components.

[0104] Effector molecule: A molecule or detectable marker that is intended to have or produce a desired effect, for example, a desired effect on the cell that the effector molecule targets. Effector molecules can include, for example, polypeptides and small molecules. Some effector molecules can have or produce more than one desired effect.

[0105] Epitope: Antigenic determinant. These are specific chemical groups or peptide sequences on an antigenic molecule that elicit a specific immune response. For example, an epitope is a region of an antigen to which B cells and / or T cells respond. An antibody can bind to a specific antigenic epitope, such as an epitope on the spike protein of a coronavirus.

[0106] Expression: Transcription or translation of a nucleic acid sequence. For example, a coding nucleic acid sequence (e.g., a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some instances is processed into mRNA. A coding nucleic acid sequence (e.g., a gene) can also be expressed when its mRNA is translated into an amino acid sequence, e.g., a protein or protein fragment. In certain instances, a heterologous gene is expressed when it is transcribed into RNA. In other instances, a 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 such as mRNA, or through the activation, inactivation, compartmentalization, or degradation of a particular protein molecule after it has been produced.

[0107] Expression control sequence: A nucleic acid sequence that is functionally linked to a heterologous nucleic acid sequence and regulates the expression of the heterologous nucleic acid sequence. An expression control sequence is functionally linked to a nucleic acid sequence when it controls and regulates the transcription and, if appropriate, translation of the nucleic acid sequence. Thus, an expression control sequence can include an appropriate promoter, enhancer, transcription terminator, a start codon (ATG) in front of a protein-encoding gene, splice signals for introns, maintaining the correct reading frame of the gene to enable proper translation of mRNA, and a stop codon. The term "control sequence" is intended to include at least components whose presence can affect expression, and can also include additional components whose presence is advantageous, such as leader sequences and fusion partner sequences. An expression control sequence can include a promoter.

[0108] Expression vector: A vector containing a recombinant polynucleotide comprising an expression control sequence operably linked to a nucleotide sequence to be expressed. An expression vector contains sufficient cis-acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

[0109] Polynucleotides can be inserted into expression vectors containing promoter sequences that facilitate efficient transcription of the inserted gene sequence in 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.

[0110] Fc region: The constant region of an antibody excluding the first heavy chain constant domain. The Fc region generally 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 can also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, the Fc region may or may not include the tailpiece and may or may not include the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains Cγ2 and Cγ3, and optionally the lower part of the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region can vary, the human IgG heavy chain Fc region is usually defined as including residues from C226 or P230 to the Fc carboxyl terminus, with numbering according to the EU numbering system. Residues can also be identified by Kabat positions. For IgA, the Fc region includes immunoglobulin domains Cα2 and Cα3, and optionally the lower hinge region between Cα1 and Cα2.

[0111] Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originates from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein, such as an scFv, is expressed in a cell, such as a mammalian cell. Methods for introducing heterologous nucleic acid molecules into cells or organisms, including, for example, electroporation, lipofection, particle gun acceleration, and homologous recombination, are well known in the art for transformation with nucleic acids.

[0112] Host cell: A cell in which a vector can be propagated and its DNA expressed. The cell can be prokaryotic or eukaryotic. The term also includes any progeny of the subject host cell. It will be understood that all progeny may not be identical to the parent cell since there may be mutations that occur during replication. However, when the term "host cell" is used, such progeny are included.

[0113] IgA: A polypeptide belonging to a class of antibodies substantially encoded by recognized immunoglobulin alpha genes. In humans, this class or isotype includes IgA1 and IgA2. IgA antibodies can exist as monomers, polymers primarily in dimeric form (termed pIgA), and secretory IgA. The constant chain of wild-type IgA contains an 18-amino acid extension at its C-terminus called the tailpiece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain that links two IgA monomers via a conserved cysteine ​​residue in the tailpiece.

[0114] IgG: A polypeptide belonging to a class or isotype of antibody substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.

[0115] Immune complex: The binding of an antibody or antigen-binding fragment (e.g., scFv) to a soluble antigen results in the formation of an immune complex. The formation of an immune complex can be detected by conventional methods, such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blot), magnetic resonance imaging, CT scan, radiography, and affinity chromatography.

[0116] Inhibiting or treating a disease: For example, inhibiting the full development of a disease or condition in a subject at risk for a disease, such as SARS-CoV-2 infection. "Treatment" refers to a therapeutic intervention that ameliorates signs or symptoms of a disease or pathological condition after they have begun to develop. In the context of a disease or pathological condition, the term "ameliorating" refers to any observable beneficial effect of treatment. Inhibiting a disease can include preventing or reducing the risk of disease, for example, preventing or reducing the risk of viral infection. A beneficial effect can be evidenced by a delay in the onset of clinical symptoms of the disease in a subject susceptible to the disease, a reduction in the severity of some or all clinical symptoms of the disease, a slowing of disease progression, a reduction in viral load, an improvement in overall health, or the subject's well-being, or other parameters specific to a particular disease. "Prophylactic" treatment is treatment administered to a subject who does not show signs of the disease or who shows only early signs, with the intent of reducing the risk of developing the condition.

[0117] The term "reduce" is a relative term, and therefore, if a disease or condition is quantitatively reduced after administration of an agent, or is reduced compared to a reference agent after administration of an agent, the agent reduces the disease or condition. Similarly, the term "prevent" does not necessarily mean that an agent completely eliminates the disease or condition, as long as at least one characteristic of the disease or condition is eliminated. Thus, a composition that reduces or prevents infection can eliminate such infection, but does not necessarily completely eliminate it, as long as the infection is reduced to a measurable extent, for example, by at least about 50%, for example, by at least about 70%, or about 80%, or even about 90%, in the absence of the agent or compared to a reference agent.

[0118] Isolated: A biological component (e.g., a nucleic acid, peptide, protein, or protein complex, e.g., an antibody) that has been substantially separated from, produced free of, or purified from other biological components, i.e., other chromosomal and extrachromosomal DNA and RNA and proteins, in the cells of an organism in which it naturally occurs. Thus, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. The term also encompasses nucleic acids, peptides, and proteins prepared by recombinant expression in a host cell, as well as chemically synthesized nucleic acids. An isolated nucleic acid, peptide, or protein, e.g., an antibody, can 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.

[0119] Kabat location: Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th The position of a residue in an amino acid sequence according to the numbering convention detailed in the National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991).

[0120] Linker: A bifunctional molecule that can be used to link two molecules into one continuous molecule, for example, to link a detectable marker to an antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0121] The terms "conjugate," "connect," "bind," or "link" can refer to bringing two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently linking a radionuclide or other molecule that is an effector molecule or detectable marker to a polypeptide, e.g., an scFv. Linking can be by either chemical or recombinant means. "Chemical means" refers to a reaction between an antibody moiety and an effector molecule such that a covalent bond is formed between the two molecules to form one molecule.

[0122] Nucleic acid (molecule or sequence): A deoxyribonucleotide or ribonucleotide polymer, 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). If single-stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can include natural nucleotides (e.g., A, T / U, C, and G) and can include analogs of natural nucleotides, e.g., labeled nucleotides.

[0123] "cDNA" refers to a DNA that is complementary to or identical to an mRNA, in either single- or double-stranded form.

[0124] "Encode" refers to the inherent property of a particular sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined sequence of amino acids, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and usually provided in a sequence listing, and the non-coding strand used as a template for transcription of a gene or cDNA can be referred to as encoding the protein or other product of the gene or cDNA. Unless otherwise specified, a "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. Protein- and RNA-encoding nucleotide sequences can contain introns.

[0125] Functionally linked: A first nucleic acid sequence is functionally linked to a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For example, a promoter, such as a CMV promoter, is functionally linked to a coding sequence when the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to connect two protein coding regions, are in the same reading frame.

[0126] Pharmaceutically acceptable carriers: Useful pharmaceutically acceptable carriers are conventionally used. Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

[0127] Generally, the nature of the carrier will depend on the particular mode of administration used. For example, parenteral formulations usually comprise injectable fluids containing pharmaceutically and physiologically acceptable liquids, such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol, or the like, as a vehicle. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the administered pharmaceutical composition can also contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (e.g., non-natural preservatives), and pH buffering agents, such as sodium acetate or sorbitan monolaurate. In certain instances, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject, for example, by injection. In some embodiments, the active agent and the pharmaceutically acceptable carrier are provided in a unit dosage form, such as a pill, or in a selected amount contained in a vial. The unit dosage form can comprise a single dosage or multiple dosages (e.g., contained in a vial from which a metered dosage of the agent can be selectively dispensed).

[0128] Polypeptide: A polymer in which the monomers are amino acid residues and are connected together by amide bonds. When the amino acids are alpha amino acids, either the L-optical isomer or the D-optical isomer can be used, with the L-isomer being preferred. The term "polypeptide" or "protein" as used herein encompasses any amino acid sequence and is intended to include modified sequences such as glycoproteins. Polypeptides include both naturally occurring proteins and recombinantly or synthetically produced proteins. Polypeptides have an amino terminus (N-terminus) and a carboxy terminus. In some embodiments, the polypeptide is a disclosed antibody or fragment thereof.

[0129] Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (e.g., an antibody) is more enriched than the peptide or protein in its natural environment within a cell. In one embodiment, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

[0130] Recombinant: A recombinant nucleic acid is one that has a sequence that does not occur in nature or that is created by the artificial combination of two otherwise separate sequence segments. This artificial combination can be achieved by chemical synthesis or, more commonly, by the artificial manipulation of isolated nucleic acid segments, e.g., by genetic engineering techniques. A recombinant protein is one that has a sequence that does not occur in nature or that is created by the artificial combination of two otherwise separate sequence segments. In some embodiments, a recombinant protein is encoded by a heterologous (e.g., recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, into an expression vector having signals that allow for the expression of the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into a chromosome of the host cell.

[0131] SARS-CoV-2: SARS-CoV-2, also known as the Wuhan coronavirus or 2019 novel coronavirus, is a positive-sense, single-stranded RNA virus of the Betacoronavirus genus that has emerged as a highly lethal cause of severe acute respiratory infection. The viral genome is capped, polyadenylated, and surrounded by nucleocapsid protein. SARS-CoV-2 virions contain a viral envelope with a large spike glycoprotein. Like most coronaviruses, the SARS-CoV-2 genome shares a common genome organization, with the replicase gene located in the 5' two-thirds of the genome and the structural genes located in the 3' one-third of the genome. The SARS-CoV-2 genome encodes a standard set of structural protein genes in the following order: 5'-spike (S)-envelope (E)-membrane (M) and nucleocapsid (N)-3'. Symptoms of SARS-CoV-2 infection include fever and respiratory illness, such as a dry cough and shortness of breath. Severe infection can progress to severe pneumonia, multiple organ failure, and death. The time from exposure to the onset of symptoms is approximately 2 to 14 days.

[0132] Standard methods for detecting viral infections can be used to detect SARS-CoV-2 infection, including, but not limited to, evaluation of patient symptoms and background and genetic testing, such as reverse transcription polymerase chain reaction (rRT-PCR). Tests can be performed on patient samples, such as respiratory or blood samples.

[0133] B.1.1.529, also known as the Omicron variant, is the original SARS-CoV-2 variant first reported to the World Health Organization on November 21, 2021. This variant has a total of 60 mutations compared to the original lineage of SARS-CoV-2, 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), approximately half of which are located in the receptor-binding domain (319–541).

[0134] SARS spike (S) protein: A class I fusion glycoprotein that is initially synthesized as a precursor protein approximately 1256 amino acids in size for SARS-CoV and approximately 1273 amino acids in size for SARS-CoV-2. Individual precursor S polypeptides form homotrimers and undergo glycosylation in the Golgi apparatus, further processing to remove the signal peptide, and cleavage by cellular proteases between approximately positions 679 / 680 for SARS-CoV and approximately positions 685 / 686 for SARS-CoV-2, resulting in the generation of separate S1 and S2 polypeptide chains that remain associated as S1 / S2 protomers within the homotrimer, thus representing a trimer of heterodimers. The S1 subunit is distal to the viral membrane and contains an N-terminal domain (NTD) and a receptor-binding domain (RBD), which is thought to mediate viral attachment to its host receptor. The S2 subunit is thought to contain the fusion protein machinery, including the fusion peptide, two heptad repeats (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and a cytosolic tail domain.

[0135] The numbering used in the disclosed SARS-CoV-2 S protein and fragments thereof is relative to the S protein of SARS-CoV-2, the sequence of which has been deposited under NCBI reference number YP_009724390.1, which is incorporated herein by reference in its entirety.

[0136] Sequence identity: The identity between two or more nucleic acid sequences or two or more amino acid sequences is expressed in terms of the percentage identity between the sequences. Sequence identity can be measured in terms of the percentage identity; the higher the percentage, the more identical the sequences. The V of an antibody that specifically binds to a target antigen L or V HHomologues and variants of are typically characterized by possessing at least about 75% sequence identity, e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity, counted over the full length alignment with the amino acid sequence of interest.

[0137] Any suitable method can be used to align sequences for comparison. Non-limiting examples of programs and alignment algorithms include those described in Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, 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, and Altschul et al., J. Mol. Biol. 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information, and on 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. Further information can be found on the NCBI website.

[0138] Generally, once two sequences are aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue occurs in both sequences. The percent sequence identity between two sequences is determined by dividing the number of matches by either the length of the sequence shown in the specified sequence or the concatenated length (e.g., 100 consecutive nucleotides or amino acid residues from the sequence shown in the specified sequence), and then multiplying the resulting value by 100.

[0139] Specific binding: When referring to an antibody or antigen-binding fragment, this refers to a binding reaction that determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biological substances. Thus, under specified conditions, the antibody preferentially binds to a particular target protein, peptide, or polysaccharide (e.g., an antigen present on the surface of a pathogen, e.g., the spike protein of a coronavirus) and does not bind in significant amounts to other proteins present in the sample or subject. With respect to spike proteins, an epitope may be present on the SARS-CoV-2 spike protein, such that the antibody binds to the spike protein on both types of viruses but not to other proteins. Specific binding can be determined by standard methods. For a description of immunoassay formats and conditions that can be used to determine specific immune reactivity, see Harlow & Lane, Antibodies, A Laboratory Manual, 2014. nd ed., Cold Spring Harbor Publications, New York (2013).

[0140] In the context of antibody-antigen complexes, the specific binding of antigen to antibody is approximately 10 -7 Less than 10 molar, e.g., about 10 -8 Less than 10 -9 Less than, or even about 10 -10 K less than Moller D K Drefers to the dissociation constant for a given interaction, e.g., a polypeptide-ligand interaction or an antibody-antigen interaction. For example, for a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

[0141] An antibody that specifically binds to an epitope on a coronavirus spike protein is an antibody that substantially binds to the coronavirus spike protein, e.g., the NTD or RBD of the spike protein from SARS-CoV-2, including proteins in the virus, the substrate to which the spike protein is attached, or a biological sample. It is recognized, of course, that a certain degree of non-specific interaction may occur between the antibody and a non-target. Typically, specific binding results in much stronger binding between the antibody and the spike protein than between the antibody and another different coronavirus protein (e.g., MERS) or between the antibody and a non-coronavirus protein. Specific binding typically results in a greater than 2-fold, e.g., greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in the amount of antibody bound (per unit time) to a protein containing the epitope or to cells or tissues expressing the target epitope compared to a protein, cell, or tissue lacking the epitope. Specific binding of a protein under such conditions requires an antibody that has been selected for its specificity for a particular protein. A variety of immunoassay formats are suitable for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

[0142] Subject: Living multi-cellular vertebrate organisms, a category that includes humans and non-human mammals, such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, etc. In one example, the subject is a human. In a particular example, the subject is a human. In a further example, a subject in need of inhibiting SARS-CoV-2 infection is selected. For example, the subject is either uninfected and at risk of SARS-CoV-2 infection, or infected and in need of treatment.

[0143] Transformed: Transformed cells are cells that have been introduced with nucleic acid molecules by molecular biological techniques.As used herein, the terms transformed and the like (for example, transformation, transfection, transduction, etc.) encompass all the techniques that can introduce nucleic acid molecules into such cells, including transduction with viral vectors, transformation with plasmid vectors, and the introduction of DNA by electroporation, lipofection and particle gun acceleration methods.

[0144] Vector: An entity comprising a nucleic acid molecule (e.g., a DNA or RNA molecule) operably linked to a coding sequence for a protein of interest and having a promoter capable of expressing the coding sequence. Non-limiting examples include naked or packaged (lipid and / or protein) DNA, naked or packaged RNA, a virus or a bacterium or other microorganism subcomponent that may be replication-incompetent, or a virus or a bacterium or other microorganism subcomponent that may be replication-competent. A vector is sometimes referred to as a construct. A recombinant DNA vector is a vector containing recombinant DNA. A vector can contain a nucleic acid sequence, such as an origin of replication, that enables the vector to replicate in a host cell. A vector can also contain one or more selectable marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector that contains at least some nucleic acid sequences derived from one or more viruses. In some embodiments, the viral vector comprises a nucleic acid molecule encoding the disclosed antibody or antigen-binding fragment that specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the viral vector can be an adeno-associated virus (AAV) vector.

[0145] Under conditions sufficient for: A phrase used to describe any environment that allows for a desired activity.

[0146] II. Description of Certain Embodiments Isolated monoclonal antibodies and antigen-binding fragments that specifically bind to coronavirus spike proteins are provided. The antibodies and antigen-binding fragments may be fully human antibodies and antigen-binding fragments. The antibodies and antigen-binding fragments can neutralize coronaviruses, such as SARS-CoV-2. In some embodiments, the disclosed antibodies can inhibit coronavirus infection in vivo and can be administered before or after infection with a coronavirus, such as SARS-CoV-2. Bispecific antibodies comprising the variable domains of these antibodies are also provided. Additionally, compositions comprising the antibodies and antigen-binding fragments and a pharmaceutically acceptable carrier are disclosed herein. Nucleic acids encoding the antibodies, antigen-binding fragments, and variable domains, as well as expression vectors (e.g., adeno-associated virus (AAV) virus vectors) comprising these nucleic acids are also provided. The antibodies, antigen-binding fragments, nucleic acid molecules, host cells, and compositions can be used for research, diagnostic, therapeutic, and prophylactic purposes. For example, the disclosed antibodies and antigen-binding fragments can be used to diagnose a subject with a coronavirus infection or can be administered to inhibit coronavirus infection in a subject. The binding characteristics of each of the antibodies listed below are also provided in the Examples section.

[0147] A. Monoclonal antibodies that specifically bind to the coronavirus spike protein, and antigen-binding fragments thereof The following description of monoclonal antibodies refers to isolated monoclonal antibodies comprising heavy and / or light chain variable domains (or antigen-binding fragments thereof) comprising CDR1, CDR2, and / or CDR3 according to the IMGT numbering system (unless the context indicates otherwise). Various CDR numbering systems (e.g., Kabat, Chothia, or IMGT numbering systems) can be used to determine the location of the CDRs. The amino acid sequences of the heavy and light chains and CDRs of the disclosed monoclonal antibodies according to the IMGT numbering system are provided in the sequence listings, but are merely exemplary.

[0148] In some embodiments, a monoclonal antibody is provided that comprises the heavy chain CDRs and light chain CDRs of any one of the antibodies described herein. In some embodiments, a monoclonal antibody is provided that comprises the heavy chain variable region and light chain variable region of any one of the antibodies described herein.

[0149] Table A: IMGT CDRs and SEQ ID NOs of antibodies TIFF2024506315000002.tif135151TIFF2024506315000003.tif237151TIFF2024506315 000004.tif236151TIFF2024506315000005.tif235151TIFF2024506315000006.tif27151

[0150] Binding to SARS-CoV-1 and / or SARS-CoV-2 is shown in Figure 1D.

[0151] Monoclonal antibody B1-182.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the B1-182.1 antibody and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus.

[0152] The monoclonal antibody B1-182.1 binds to an epitope in the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. As disclosed below, the B1-182.1 antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor, which has a unique competitive profile compared to other antibodies. The antibody neutralizes SARS-CoV-2 pseudotyped lentiviral particles and Nanoluc live viral particles. This is three to four times more potent than the lead clinical candidate, LY-CoV555, and is one of the most potent antibodies reported for SARS-CoV-2. Information regarding the LY-CoV55 antibody is provided in Jones det al., bioRxiv.2020 Oct 1;2020.09.30.318972 (rev. October 9, 2020), preprint, PMID 33024963, available electronically at pubmed.ncbi.nlm.nih.gov / 33024963 / , which is incorporated herein by reference; clinical data for this antibody are available in Chen et al., New Engl. J. Med. 384(3): 229-237, 2021, which is incorporated herein by reference; see also pubmed.ncbi.nlm.nih.gov / 33113295 / .

[0153] In some embodiments, B1-182.1 maintains high potency against the following variants: D614G, N439K / D614G, Y543F / D614G, A222V / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, as well as B.1.1.7 (VOC 202012 / 01) containing amino acid changes at H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H; and has increased potency against N501Y / D614G. The D614G variant is the predominant variant in circulation. The E484K variant is not neutralized by leading antibodies (e.g., LY-CoV555, REGN-10989) or shows a significant loss of efficacy (REGN-10933). The Y453F variant is not neutralized by REGN-10933. In some embodiments, B1-182.1 binds to the B.1.1.529 variant.

[0154] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the B1-182.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0155] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:1. Hand specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:5. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment comprises V independently comprising an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 1 and 5, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0156] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 2, 3, and 4, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 6, 7, and 8, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0157] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 2, 3, and 4, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 6, 7, and 8, respectively; L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:5, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0158] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:1. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:5. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 1 and 5, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0159] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0160] b. Monoclonal antibody A19-61.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A19-61.1 antibody and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus.

[0161] The monoclonal antibody A19-61.1 binds to an epitope in the RBD of the SARS-CoV-2 spike protein. This antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor, with a unique competitive profile compared to other antibodies. This antibody neutralizes pseudotyped lentivirus and Nanoluc live virus particles. Neutralization of Nanoluc live virus is one of the most potent reported for an antibody targeting SARS-CoV-2.

[0162] In some embodiments, monoclonal antibody A19-61.1 has increased potency against the D614G variant and maintains its potency against variants: N439K / D614G, Y543F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, as well as B.1.1.7 (VOC 202012 / 01) containing amino acid changes at the following variants: N439K / D614G, Y543F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, and H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H.

[0163] In some examples, the antibody or antigen-binding fragment comprises a V sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A19-61.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0164] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:9. Hand specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 13. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment comprises V independently comprising an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs:9 and 13, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0165] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 10, 11, and 12, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 14, 15, and 16, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0166] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 10, 11, and 12, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 14, 15, and 16, respectively; L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:9, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:9, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:13, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:13, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0167] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:9. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO: 13. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0168] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0169] c. Monoclonal antibody A19-46.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A19-46.1 antibody and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus.

[0170] The monoclonal antibody A19-46.1 binds to an epitope in the S1 domain of the SARS-CoV-2 spike protein. The antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor, with a unique competitive profile compared to other antibodies. The antibody neutralizes SARS-CoV-2 pseudotyped lentivirus and Nanoluc live virus particles. Neutralization of SARS-CoV-2 Nanoluc live virus is one of the most potent reported for an antibody targeting SARS-CoV-2.

[0171] In some embodiments, monoclonal antibody A19-46.1 has increased potency against the D614G variant and maintains its potency against variants: N439K / D614G, Y543F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, as well as B.1.1.7 (VOC 202012 / 01) containing amino acid changes at the following variants: N439K / D614G, Y543F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, and H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H.

[0172] In some examples, the antibody or antigen-binding fragment comprises a V sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A19-46.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0173] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:17. Hand specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:21. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 17 and 21, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0174] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 18, 19, and 20, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 22, 23, and 24, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0175] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 18, 19, and 20, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 22, 23, and 24, respectively; L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:17, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:17, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:21, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:21, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2 and / or SARS-CoV-1.

[0176] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:17. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:21. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 17 and 21, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0177] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0178] d. Monoclonal antibody A23-58.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A784-58.1 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0179] Monoclonal antibody 789-58.1 has an epitope in the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. This antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor, and has a unique competitive profile compared to other antibodies. This antibody exhibits strong IC10 activity against SARS-CoV-2 pseudotyped lentiviral particles. 50 and IC 80 This antibody is 3-4 times more potent than the monoclonal antibody LY-CoV555. The neutralization of live SARS-CoV-2 Nanoluc virus is one of the most potent reported for an antibody targeting SARS-CoV-2.

[0180] In some embodiments, monoclonal antibody A23-58.1 has a slight increase in high potency against E484K / D614G and maintains high potency against D614G, N439K / D614G, Y543F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, as well as B.1.1.7 (VOC 202012 / 01) containing the amino acid changes H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H. In some embodiments, A23-58.1 binds to the B.1.1.529 variant.

[0181] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A23-58.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0182] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:25. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:29. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 25 and 29, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0183] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 26, 27, and 28, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 30, 31, and 32, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0184] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 26, 27, and 28, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 30, 31, and 32, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:25, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:25, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:29, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:29, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0185] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:25. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:29. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 25 and 29, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0186] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0187] e. Monoclonal antibody A20-29.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A20-29.1 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0188] Monoclonal antibody A20-29.1 binds to an epitope in the RBD domain of the SARS-CoV-2 spike protein. A20-29.1 can also bind to the original SARS-CoV-1 spike protein. This antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor and has a unique competitive profile compared to other antibodies. It does not compete with antibodies from the LY-CoV555 competitor group, making it potentially useful in therapeutic cocktails containing antibodies from that class. As disclosed herein, this antibody neutralizes SARS-CoV-2 pseudotyped lentiviral particles.

[0189] In some embodiments, monoclonal antibody A20-29.1 maintains similar potency against the D614G, N439K / D614G, E484K / D61G, Y543F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G variants.

[0190] In some examples, the antibody or antigen-binding fragment comprises a V sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A20-29.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0191] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:33. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:37. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 33 and 37, respectively. H and V L and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 and / or SARS-CoV-1.

[0192] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 34, 35, and 36, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 38, 39, and 40, respectively. L and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 and / or SARS-CoV-1.

[0193] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 34, 35, and 36, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 38, 39, and 40, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:33, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:33, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:37, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:37, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2 and / or SARS-CoV-1.

[0194] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:33. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:37. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 33 and 37, respectively. H and V L and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 and / or SARS-CoV-1.

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

[0196] f. Monoclonal antibody A23-105.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A23-105.1 antibody and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some examples, the antibody or antigen-binding fragment comprises a V-seq sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A23-105.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0197] Monoclonal antibody A23-105.1 binds to an epitope in the RBD of the SARS-CoV-2 spike protein. This antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor and has a competitive profile similar to that of LY-CoV555. Monoclonal antibody A23-105.1 is neutralizing. Neutralization of SARS-CoV-2 Nanoluc live virus by A23-105.1 demonstrates its potency.

[0198] In some embodiments, A23-105.1 has increased potency against the D614G variant and maintains similar potency against the D614G, N439K / D614G, Y543F / D614G, A222V / D614G, N501Y / D614G, and del69-70 / D614G variants. Like LY-CoV555, it loses activity against the variants E484K / D61G and N501 / E484K / K417N / D614G.

[0199] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:41. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:45. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 41 and 45, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0200] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 42, 43, and 44, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 46, 47, and 48, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0201] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 42, 43, and 44, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 46, 47, and 48, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:41, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:41, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:45, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:45, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0202] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:41. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:45. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 41 and 45, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

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

[0204] g. Monoclonal antibody A19-1.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A19-1.1 antibody and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus.

[0205] The monoclonal antibody A19-1.1 binds to the original SARS CoV-1 spike protein and prevents infection by directly blocking viral binding to the ACE2 viral receptor, with a competitive profile similar to that of LY-CoV555.

[0206] In some embodiments, monoclonal antibody A19-1.1 has increased potency against the D614G variant and maintains similar potency against the D614G, N439K / D614G, Y543F / D614G, A222V / D614G, N501Y / D614G, and del69-70 / D614G variants. Like LY-CoV555, it loses activity against the variants E484K / D61G and N501 / E484K / K417N / D614G.

[0207] In some examples, the antibody or antigen-binding fragment comprises a V sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A19-1.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-1.

[0208] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:49. Hand specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:53. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 49 and 53, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0209] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 50, 51, and 52, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 54, 55, and 56, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0210] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 50, 51, and 52, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 54, 55, and 56, respectively; L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:49, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:49, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:53, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:53, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0211] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:49. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:53. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 49 and 53, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0212] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0213] h. Monoclonal antibody A19-30.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A19-30.1 antibody and specifically binds to the spike protein of a coronavirus.

[0214] Monoclonal antibody A19-30.1 binds to an epitope in the RBD domain of the SARS-CoV-2 spike protein. This antibody does not prevent infection by directly blocking viral binding to the ACE2 viral receptor. It has a unique competitive profile compared to other antibodies. Because this antibody does not compete with antibodies in the LY-CoV555 competitor group, it is useful in therapeutic cocktails that include LY-CoV555 class antibodies. It does not act by neutralizing SARS-CoV-2 pseudotyped lentiviral particles.

[0215] In some examples, the antibody or antigen-binding fragment comprises a V sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A19-30.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and acts using a non-neutralizing mechanism against coronavirus infection, such as antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, or antibody-dependent complement killing of cells or virus particles. The coronavirus may be SARS-CoV-2.

[0216] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:57. H and specifically binds to the coronavirus spike and inactivates the coronavirus or kills infected cells. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:61. Land specifically binds to the coronavirus spike protein and inactivates the coronavirus or kills infected cells. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 57 and 61, respectively. H and V L The coronavirus may be SARS-CoV-2, and the antibody specifically binds to the spike protein of the coronavirus and inactivates the coronavirus or kills infected cells.

[0217] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 58, 59, and 60, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 62, 63, and 64, respectively. L The coronavirus may be SARS-CoV-2, and the antibody specifically binds to the spike protein of the coronavirus and inactivates the coronavirus or kills infected cells.

[0218] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 58, 59, and 60, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 62, 63, and 64, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:57, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:57, and V Lcomprises an amino acid sequence at least 90% identical to SEQ ID NO:61, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:61, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and inactivates the coronavirus or kills infected cells. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0219] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:57. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:61. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 57 and 61, respectively. H and V L The coronavirus may be SARS-CoV-2, and the antibody specifically binds to the spike protein of the coronavirus and inactivates the coronavirus or kills infected cells.

[0220] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0221] i. Monoclonal antibody A20-36.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A20-36.1 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0222] Monoclonal antibody A20-36.1 binds to an epitope in the SD2 region of the S1 domain of the SARS-CoV-2 spike protein. Monoclonal antibody A20-36.1 can also bind to the original SARS-CoV-1 spike protein. This antibody does not prevent infection by directly blocking viral binding to the ACE2 viral receptor. It has a unique competitive profile. Because this antibody does not compete with antibodies from the LY-CoV555 competitor group, it is useful in therapeutic cocktails containing antibodies from that class. This monoclonal antibody neutralizes SARS-CoV-2 pseudotyped lentiviral particles.

[0223] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A20-36.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0224] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:65. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:69. Land specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 65 and 69, respectively. H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0225] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 66, 67, and 68, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 70, 71, and 72, respectively. L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0226] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 66, 67, and 68, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 70, 71, and 72, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:65, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:65, and V Lcomprises an amino acid sequence at least 90% identical to SEQ ID NO:69, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:69, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2 or SARS-CoV-1.

[0227] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:65. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:69. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 65 and 69, respectively. H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0228] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0229] j. Monoclonal antibody A23-97.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A23-97.1 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0230] Monoclonal antibody A23-97.1 binds to an epitope in the RBD of the SARS CoV-2 spike protein. Monoclonal antibody A23-97.1 can also bind to the original SARS CoV-1 spike protein. This antibody does not prevent infection by directly blocking viral binding to the ACE2 viral receptor. It has a unique competitive profile compared to other antibodies. This antibody does not compete with antibodies in the LY-CoV555 competitor group, making it useful in therapeutic cocktails containing antibodies from that class.

[0231] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A23-97.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0232] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:73. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:77. Land specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 73 and 77, respectively. H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0233] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 74, 75, and 76, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 78, 79, and 80, respectively. L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0234] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 74, 75, and 76, respectively. H , comprising a VL comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 78, 79, and 80, respectively; H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:73, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:73, and V Lcomprises an amino acid sequence at least 90% identical to SEQ ID NO:77, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:77, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2 or SARS-CoV-1.

[0235] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:73. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:77. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 73 and 77, respectively. H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0236] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0237] k. Monoclonal antibody A23-113.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A23-113.1 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0238] Monoclonal antibody A23-113.1 binds to an epitope in the RBD of the SARS-CoV-2 spike protein. It can also bind to the original SARS-CoV-1 spike protein. This monoclonal antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor and has a similar competitive profile to A23-97.1.

[0239] In some examples, the antibody or antigen-binding fragment comprises a V sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A23-113.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0240] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:81. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:85. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 81 and 85, respectively.H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0241] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 82, 83, and 84, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 86, 87, and 88, respectively. L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0242] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 82, 83, and 84, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 86, 87, and 88, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:81, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:81, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:85, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:85, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2 or SARS-CoV-1.

[0243] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:81. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:85. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 81 and 85, respectively. H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0244] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0245] l. Monoclonal antibody A23-80.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A23-80.1 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0246] Monoclonal antibody A23-80.1 binds to an epitope in the RBD of the SARS-CoV-2 spike protein. This antibody does not prevent infection by directly blocking viral binding to the ACE2 viral receptor. This antibody has a unique competitive profile compared to other antibodies. It neutralizes SARS-CoV-2 pseudotyped lentiviral particles.

[0247] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A23-80.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0248] In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:89. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:93. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 89 and 93, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0249] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 90, 91, and 92, respectively. Hand / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 94, 95, and 96, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0250] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 90, 91, and 92, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 94, 95, and 96, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:89, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:89, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:93, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:93, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0251] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:89. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:93. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 89 and 93, respectively. H and VL The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0252] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0253] m. Monoclonal antibody A19-82.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A19-82.1 antibody and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus.

[0254] The monoclonal antibody A19-82.1 binds to an epitope in the RBD of the SARS-CoV-2 spike protein. This antibody can also bind to the original SARS-CoV-1 spike protein. This antibody does not prevent infection by directly blocking viral binding to the ACE2 viral receptor. It has a unique competitive profile compared to other antibodies. Because this antibody does not compete with antibodies in the LY-CoV555 competitor group, it is useful in therapeutic cocktails containing antibodies from that class.

[0255] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A19-82.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0256] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:97. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 101. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs:97 and 101, respectively. H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0257] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 98, 99, 100, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 102, 103, and 104, respectively. L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0258] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 98, 99, and 100, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 102, 103, and 104, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:97, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:97, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:101, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:101, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2 or SARS-CoV-1.

[0259] In some embodiments, the antibody or antigen-binding fragment comprises a VV comprising the amino acid sequence set forth as SEQ ID NO:97. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO: 101. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 97 and 101, respectively. H and V L and specifically binds to the spike protein of the coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2 or SARS-CoV-1.

[0260] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0261] Monoclonal antibody A20-9.1 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the A20-9.1 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0262] Monoclonal antibody A20-9.1 binds to an epitope in the SD2 region of the S1 domain of the SARS-CoV-2 spike protein. This antibody does not prevent infection by directly blocking viral binding to the ACE2 viral receptor. It has a unique competitive profile compared to other antibodies. This antibody does not compete with antibodies in the LY-CoV555 competitor group and is therefore useful in therapeutic cocktails containing antibodies from that class.

[0263] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the A20-9.1 antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0264] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:105. Hand specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 109. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 105 and 109, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0265] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 106, 107, and 108, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 110, 111, and 112, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0266] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 106, 107, and 108, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 110, 111, and 112, respectively; L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:105, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:105, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 109, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 109, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0267] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:105. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO: 109. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 105 and 109, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0268] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0269] Monoclonal antibody B1-182.1_58CDRH3 In some embodiments, the antibody or antigen-binding fragment is based on or derived from the B1-182.1_58CDRH3 antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0270] The monoclonal antibody B1-182.1_58CDRH3 binds to the same epitope in the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein as B1-182.1. The B1-182.1_58CDRH3 antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor, with a competitive profile similar to that of B1-182.1 and A23-58.1. This antibody neutralizes SARS-CoV-2 pseudotyped lentiviral particles. This is 3-4 times more potent than the lead clinical candidate, LY-CoV555, and is one of the most potent antibodies reported for SARS-CoV-2. Neutralization data are shown in Figure 29C.

[0271] In some embodiments, B1-182.1_58CDRH3 maintains high potency against the following variants: D614G, N439K / D614G, Y543F / D614G, A222V / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, as well as B.1.1.7 (VOC 202012 / 01) containing amino acid changes at H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H; and has increased potency against N501Y / D614G. The D614G variant is the predominant variant in circulation. The E484K variant is not neutralized by leading antibodies (e.g., LY-CoV555, REGN-10989) or shows a significant loss of efficacy (REGN-10933). The Y453F variant is not neutralized by REGN-10933. In some embodiments, B1-182.1_58CDRH3 binds to the B.1.1.529 variant.

[0272] In some examples, the antibody or antigen-binding fragment comprises a V sequence comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the B1-182.1_58CDRH3 antibody (e.g., according to IMGT, Kabat, or Chothia).H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0273] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:143. H and specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence that is at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:5. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 143 and 5, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0274] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 2, 3, and 58, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 6, 7, and 8, respectively. LThe antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0275] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 2, 3, and 58, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 6, 7, and 8, respectively; L Including V H comprises an amino acid sequence that is at least 90% identical to SEQ ID NO:143, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:143, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:5, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:5, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, the sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0276] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:143. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:5. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 143 and 5, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0277] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0278] p. Monoclonal antibody B1-182.1 heavy / B1-182.1 light_5Mut In some embodiments, the antibody or antigen-binding fragment is based on or derived from the B1-182.1 heavy / B1-182.1 light_5Mut antibody and specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus.

[0279] The monoclonal antibody B1-182.1 heavy / B1-182.1 light_5Mut binds to an epitope in the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein. As disclosed below, the B1-182.1 heavy / B1-182.1 light_5Mut antibody prevents infection by directly blocking viral binding to the ACE2 viral receptor, with a competitive profile similar to that of B1-182.1 and A23-58.1. The antibody neutralizes SARS-CoV-2 pseudotyped lentiviral particles. It is 3-4 times more potent than the lead clinical candidate, LY-CoV555, and is one of the most potent antibodies reported for SARS-CoV-2.

[0280] In some embodiments, B1-182.1heavy / B1-182.1light_5Mut maintains high potency against the following variants: D614G, N439K / D614G, Y543F / D614G, A222V / D614G, del69-70 / D614G, and N501 / E484K / K417N / D614G, as well as B.1.1.7 (VOC 202012 / 01) containing amino acid changes at H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H; and has increased potency against N501Y / D614G. The D614G variant is the predominant variant in circulation. The E484K variant is not neutralized by leading antibodies (e.g., LY-CoV555, REGN-10989) or shows a significant loss of efficacy (REGN-10933). The Y453F variant is not neutralized by REGN-10933. In some embodiments, it binds to the B1-182.1 heavy / B1-182.1 light_5Mut B.1.1.529 variant.

[0281] In some examples, the antibody or antigen-binding fragment comprises a V molecule comprising HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, respectively, of the B1-182.1 heavy / B1-182.1 light_5Mut antibody (e.g., according to IMGT, Kabat, or Chothia). H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0282] In some embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO:1. Hand specifically binds to coronavirus spikes and neutralizes coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V that comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 144. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In additional embodiments, the antibody or antigen-binding fragment independently comprises an amino acid sequence at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NOs: 1 and 144, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0283] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 2, 3, and 4, respectively. H and / or V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 6, 145, and 146, respectively. L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0284] In some embodiments, the antibody or antigen-binding fragment comprises a VHQ domain comprising HCDR1, HCDR2, and HCDR3 set forth as SEQ ID NOs: 2, 3, and 4, respectively. H , V comprising LCDR1, LCDR2, and LCDR3 set forth as SEQ ID NOs: 6, 145, and 146, respectively; L Including V Hcomprises an amino acid sequence that is at least 90% identical to SEQ ID NO:1, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:1, and V L comprises an amino acid sequence at least 90% identical to SEQ ID NO:144, e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:144, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this embodiment, sequence-specific variations fall outside the CDRs. The coronavirus can be SARS-CoV-2.

[0285] In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO:1. H and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In more embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NO: 144. L and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In some embodiments, the antibody or antigen-binding fragment comprises a V comprising the amino acid sequence set forth as SEQ ID NOs: 1 and 144, respectively. H and V L The antibody specifically binds to the spike protein of a coronavirus and neutralizes the coronavirus, which may be SARS-CoV-2.

[0286] In some embodiments, the disclosed antibodies inhibit viral entry and / or replication.

[0287] 1. Further Antibodies In some examples, an antibody that binds to an epitope of interest can be identified based on its ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with an antibody provided herein in a binding assay. In other examples, an antibody that binds to an epitope of interest can be identified based on its ability to cross-compete (e.g., competitively inhibit binding in a statistically significant manner) with one or more of the antibodies provided herein in a binding assay.

[0288] Human antibodies that bind to the same epitope on a coronavirus protein spike, e.g., the NTD or RBD of the spike protein, as bound by the disclosed antibodies, can be generated using any suitable method. Such antibodies can be prepared, for example, by administering an immunogen to a transgenic animal that has been engineered to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci, which have replaced endogenous immunoglobulin loci or are extrachromosomally present or randomly integrated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are generally inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, e.g., XENOMOUSE. (商標) See also U.S. Patent Nos. 6,075,181 and 6,150,584, which describe HUMAB® technology; U.S. Patent No. 5,770,429, which describes HUMAB® technology; U.S. Patent No. 7,041,870, which describes KM MOUSE® technology; and U.S. Patent Application Publication No. US2007 / 0061900, which describes VELOCIMOUSE® technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0289] Other human antibodies that bind to the same epitope can also be produced by hybridoma-based method.Human myeloma cell lines and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, for example, Kozbor J. Immunol., 133: 3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987);and Oerner et al., J. Immunol., 147: 86 (1991)).The human antibody produced by human B cell hybridoma technology is also described in Li et al., Proc. Natl. Acad. Sci. USA, 103: 3557-3562 (2006). Additional methods include those described, for example, in U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005). Human antibodies can also be produced by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains.

[0290] Antibodies and antigen-binding fragments that specifically bind to the same epitope can also be isolated by screening combinatorial libraries for antibodies with the desired binding properties, for example, by generating phage display libraries and screening such libraries for antibodies with the desired binding properties. Such methods are reviewed, for example, by Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and are also described, for example, in McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0291] In one particular phage display method, V is used as a vector for the detection of virions, as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). H Genes and V LGene repertoires can be cloned separately by polymerase chain reaction (PCR), randomly combined into phage libraries, and then screened for antigen-binding phage. Phage typically display antibody fragments either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need to construct hybridomas. Alternatively, as described by Griffiths et al., EMBO J, 12: 725-734 (1993), naive repertoires (e.g., from humans) can be cloned to provide a single source of antibodies against a wide range of non-self antigens and even self antigens without any immunization. Finally, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), naive libraries can also be generated synthetically by cloning unrearranged V gene segments from stem cells, encoding highly variable CDR3 regions, and using PCR primers containing random sequences to achieve in vitro rearrangement. Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Application Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Competitive binding assays similar to those disclosed in the Examples section below can be used to select antibodies with desirable binding properties.

[0292] 2. Further Description of Antibodies and Antigen-Binding Fragments The antibody or antigen-binding fragment of the antibody disclosed herein may be a human antibody or a fragment thereof. Chimeric antibodies are also provided. The antibody or antigen-binding fragment may include any suitable framework region, for example (but not limited to) a human framework region from another source, or an optimized framework region. Alternatively, a heterologous framework region, for example (but not limited to) a mouse or monkey framework region, may be included in the heavy or light chain of the antibody.

[0293] The antibody can be of any isotype, for example, an IgA, IgM, or IgG antibody, e.g., IgG1, IgG 2、 The class of antibody that specifically binds to the coronavirus spike protein can be converted to another class. L or V H The nucleic acid molecules encoding V are then isolated so as to be free of any nucleic acid sequences encoding the light or heavy chain constant regions, respectively. L or V H Nucleic acid molecules encoding C from different classes of immunoglobulin molecules L or C H This is operably linked to a nucleic acid sequence encoding, for example, C L or C H This can be achieved using vectors or nucleic acid molecules containing the chains. For example, an originally IgG antibody that specifically binds to the spike protein can be class switched to IgA. Class switching can be used to switch the IgG subclass to another, e.g., from IgG1 to IgG 2、 IgG 3、 Or it can be converted to IgG4.

[0294] In some examples, the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomers, and the like.

[0295] The antibody or antigen-binding fragment can be derivatized or linked to another molecule (e.g., another peptide or protein). Generally, the antibody or antigen-binding fragment is derivatized such that binding to the spike protein is not adversely affected by the derivatization or labeling. For example, the antibody or antigen-binding fragment can be functionally linked (by chemical bonding, genetic fusion, non-covalent bonding, or other methods) to one or more other branched entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate binding of the antibody or antibody portion to another molecule (e.g., a streptavidin core region or a polyhistidine tag).

[0296] (a) Binding affinity In some embodiments, the antibody or antigen-binding fragment is 1.0 x 10 -8 M or less, 5.0×10 -8 M or less, 1.0×10 -9 M or less, 5.0×10 -9 M or less, 1.0×10 -10 M or less, 5.0×10 -10 M or less, or 1.0 x 10 -11 M or less (e.g., K D It specifically binds to the coronavirus spike protein with an affinity measured by K D can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed using a Fab version of the antibody of interest and its antigen. In one assay, the solution binding affinity of the Fab for the antigen is measured in the presence of a titration series of unlabeled antigen at a minimum concentration of ( 125I) Fab is equilibrated with labeled antigen, followed by capturing the bound antigen on an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293(4):865-881, 1999). To establish the conditions for the assay, MICROTITER® multiwell plates (Thermo Scientific) are coated overnight with 5 μg / ml of 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 2-5 hours at room temperature (approximately 23°C). Non-adsorbent plates (NUNC (商標) Catalog No. 269620) at 100 μM or 26 pM [ 125 The [I]-antigen is mixed with serial dilutions of the Fab of interest (e.g., consistent with the evaluation of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57(20):4593-4599, 1997). The Fab of interest is then incubated overnight; however, incubation may be continued for a longer period (e.g., about 65 hours) to ensure equilibrium is reached. The mixture is then transferred to a capture plate and incubated at room temperature (e.g., 1 hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. Once the plate has dried, 150 μl / well of scintillant (MICROSCINT (商標) -20; PerkinElmer) was added, and TOPCOUNT (商標) Plates are counted for 10 minutes in a gamma counter (PerkinElmer). Concentrations of each Fab that give 20% or less of maximum binding are selected for use in competitive binding assays.

[0297] In another assay, K DK can be measured using a surface plasmon resonance assay using biolayer interferometry (BLI); see the Examples section. In other embodiments, K can be measured using a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ) at 25°C with an immobilized antigen CM5 chip of approximately 10 response units (RU). Briefly, a 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) in 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 bound protein. After injection of the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are added to the wells of the Fab in 0.05% polysorbate 20 (TWEEN-20). (商標) ) in PBS containing surfactant (PBST) at a flow rate of approximately 25 l / min at 25° C. The binding rate (k on ) and dissociation rate (k off ) is calculated. off / k on The equilibrium dissociation constant (K D ) is calculated. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). The surface plasmon resonance assay described above determines whether the on-rate is 10 6 M -1 s -1 If the concentration exceeds 100 ppm, a spectrometer, e.g., a spectrophotometer with stop flow (Aviv Instruments) or an 8000 series SLM-AMINCO with a stirred cuvette, may be used. (商標)On-rates can be determined by using a fluorescence quenching technique to measure 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, in the presence of increasing concentrations of antigen, as measured by a spectrophotometer (ThermoSpectronic), at 25°C. Affinity can also be measured by high-throughput SPR using a Carterra LSA.

[0298] (b) Multispecific antibody In some embodiments, multispecific antibodies, e.g., bispecific antibodies, are provided that include the antibodies or antigen-binding fragments provided herein that specifically bind to coronavirus spike proteins. Any suitable method can be used to design and generate multispecific antibodies, such as crosslinking two or more antibodies or antigen-binding fragments (e.g., scFvs) of the same or different types. Exemplary methods for generating multispecific antibodies include those described in PCT Patent Application Publication No. WO 2013 / 163427, incorporated herein by reference in its entirety. Non-limiting examples of suitable crosslinkers include heterobifunctional crosslinkers, which have two distinct reactive groups separated by a suitable spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or homobifunctional crosslinkers (such as disuccinimidyl suberate).

[0299] Multispecific antibodies may have any suitable format that allows the antibodies or antigen-binding fragments provided herein to bind to coronavirus spike proteins. Bispecific single-chain antibodies may 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. Further 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; Loffler et al., Blood, 95(6):2098-2103, 2000; and Bruhl et al., J. Immunol., 166(4):2420-2426, 2001. The generation of bispecific Fab-scFv (bibody) molecules has been 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 bibodies, scFv molecules can be fused to one of the VL-CL(L) or VH-CH1 chains, e.g., to generate a bibody in which one scFv is fused to the C-terminus of a Fab chain.

[0300] Bispecific tetravalent immunoglobulins, known as dual variable domain immunoglobulins or DVD-immunoglobulin molecules, are disclosed in Wu et al., MAbs. 2009;1:339-47, doi: 10.4161 / mabs.1.4.8755, incorporated herein by reference. See also Nat Biotechnol. 2007 Nov;25(11):1290-7, doi: 10.1038 / nbt1345. Epub 2007 Oct 14, also incorporated herein by reference. DVD-immunoglobulin molecules contain two heavy chains and two light chains. However, unlike IgG, both the heavy and light chains of DVD-immunoglobulin molecules contain an additional variable domain (VD) connected via a linker sequence to the N-terminus of the VH and VL of an existing monoclonal antibody (mAb). Thus, when the heavy and light chains combine, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites. See Jakob et al., Mabs 5: 358-363, 2013, incorporated herein by reference. For a schematic and space-filling diagram, see Figure 1 of Jakob et al. The DVD-immunoglobulin molecule functions to simultaneously bind two different antigens on each DFab.

[0301] The outermost or N-terminal variable domain is designated VD1, and the innermost variable domain is designated VD2; VD2 is proximal to the C-terminal CH1 or CL. As disclosed in Jakob et al., supra, DVD-immunoglobulin molecules can be produced and purified to homogeneity in large quantities, can have pharmacological properties similar to those of conventional IgG1, and can exhibit in vivo efficacy. Any of the disclosed monoclonal antibodies can be included in the DVD-immunoglobulin format.

[0302] (c) antigen-binding fragment Heavy chain and V LAntigen-binding fragments, such as Fab, F(ab'), and Fv, comprising the following, which specifically bind to the coronavirus spike protein, are encompassed by the present disclosure. 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, the fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digesting whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) The fragment of an antibody molecule, Fab', can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; (3) (Fab')2, the fragment of an antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) V expressed as two chains L、 and V L Fv, which is a genetically engineered fragment comprising: (5) V linked by a suitable polypeptide linker as a genetically fused single chain molecule. H and V L Single-chain antibodies (e.g., scFv) are defined as genetically engineered molecules containing the V (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012, doi:10.1155 / 2012 / 980250; Marbry and Snavely, IDrugs, 13(8):543-549, 2010). H Domains and V L The intramolecular orientation of the domains is not critical for the provided antibodies (e.g., the provided multispecific antibodies). Therefore, both possible configurations (V H - Domain - Linker Domain - V L -Domain;V L - Domain - Linker Domain - VH -domain) can be used. (6) Single-chain antibody dimers (scFV2), defined as dimers of scFVs, are also called "miniantibodies."

[0303] Any suitable method for generating the antigen-binding fragments described above can be used. Non-limiting examples include those described in Harlow and Lane, Antibodies: A Laboratory Manual, 2002. nd , Cold Spring Harbor Laboratory, New York, 2013.

[0304] Antigen-binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression of DNA encoding the fragment in host cells (e.g., E. coli cells). Antigen-binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen-binding fragments can be generated by enzymatic cleavage of antibodies with pepsin to produce a 5S fragment designated F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide bonds, to produce a 3.5S Fab' monovalent fragment.

[0305] Other methods of cleaving antibodies can also be used, such as separation of the heavy chain to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymatic, chemical, or genetic techniques, so long as the fragments bind to the antigen recognized by the intact antibody.

[0306] (d) Variant In some embodiments, amino acid sequence variants of the antibodies and bispecific antibodies provided herein are provided. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody or bispecific antibody. Amino acid sequence variants of antibodies include those that have the V H Domain and / or V L The domains can be prepared by introducing appropriate modifications into the nucleotide sequence encoding them or by peptide synthesis. Such modifications include, for example, deletions from and / or insertions into and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired properties, e.g., antigen binding.

[0307] In some embodiments, variants with one or more amino acid substitutions are provided.The sites of interest for substitutional mutagenesis include CDR and framework regions.Amino acid substitutions can be introduced into the antibody of interest, and the product can be screened for desired activity, such as maintaining / improving antigen binding, reducing immunogenicity, or improving ADCC or CDC.

[0308] The variants are typically V H Area and V L It is believed that amino acid substitutions are made to V to retain the amino acid residues necessary for correct folding and stabilization between the regions, and to preserve the charge characteristics of the residues in order to preserve the low pI and low toxicity of the molecule. H Area and V L It can be done in the area.

[0309] In some embodiments, the V of the antibody Hcontains 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 1. In some embodiments, the V of the antibody L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:5.

[0310] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:9. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:13.

[0311] In a further aspect, the V H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 17. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:21.

[0312] In yet other embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO:25. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:29.

[0313] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:33. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:37.

[0314] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO:41. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:45.

[0315] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO:49. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:53.

[0316] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:57. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:61.

[0317] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO:65. Lcontains 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:69.

[0318] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:73. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:77.

[0319] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO:81. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:85.

[0320] In more embodiments, the V of the antibody Hcontains 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO:89. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:93.

[0321] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:97. L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:101.

[0322] In more embodiments, the V of the antibody H 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs: 105. In some embodiments, the V of the antibody L 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., conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NOs:109.

[0323] In some embodiments, the antibody or antigen-binding fragment can include 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., conservative amino acid substitutions) in the framework regions of the antibody / diabody heavy chain or the antibody / diabody light chain or the antibody / diabody heavy and light chains compared to a known framework region or compared to the framework regions of the antibody, and can maintain specific binding activity for an epitope of the spike protein.

[0324] In some embodiments, substitutions, insertions, or deletions can occur in one or more CDRs, as long as such changes do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative changes (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity can be made in the CDRs. Variant V provided above H and V L In some embodiments of the sequences, each CDR is either unchanged or contains no more than one, two, or three amino acid substitutions. H and V L In some embodiments of the sequences, only framework residues are modified, so that the CDRs are unchanged.

[0325] To increase the binding affinity of antibodies, V is engineered to bind to a specific target protein, a process similar to the in vivo somatic mutation process involved in affinity maturation of antibodies during the innate immune response. L and V H Segments can be randomly mutated, such as within the HCDR3 or LCDR3 regions. Thus, in vitro affinity maturation can be performed by using PCR primers complementary to the HCDR3 or LCDR3, respectively, to generate V H and V L This can be achieved by amplifying a region of interest. In this process, primers are "spiked" at certain positions with a random mixture of the four nucleotide bases, so that the resulting PCR product is VH and / or V L V with random mutations introduced into the CDR3 region of H and V L These randomly mutated V H and V L The V segments can be tested to determine their binding affinity to the spike protein. H is one of SEQ ID NOs: 1, 9, 17, 25, 33, 41, 49, 57, 65, 73, 81, 89, 97, or 105. In other examples, V L is one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, 77, 85, 93, 101, or 109, respectively.

[0326] In some embodiments, the antibody or antigen-binding fragment is altered to increase or decrease the extent to which the antibody or antigen-binding fragment is glycosylated. Addition or deletion of glycosylation sites can conveniently be accomplished by altering the amino acid sequence so that one or more glycosylation sites are created or removed.

[0327] If the antibody contains an Fc region, the carbohydrate attached thereto can be altered. Native antibodies produced by mammalian cells typically contain branched, biantennary oligosaccharides, generally N-linked to Asn297 in the CH2 domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol. 15(1):26-32, 1997. Oligosaccharides can include 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 embodiments, oligosaccharide modifications in antibodies can be performed to generate antibody variants with improved specific properties.

[0328] In one embodiment, variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan of Asn297 relative to the sum of all glycans (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the asparagine residue located at approximately position 297 in the Fc region; however, due to minor sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylation variants may have improved ADCC function. See, for example, U.S. Patent Application Publications US 2003 / 0157108 (Presta, L.); US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating to "defucosylated" or "fucose-deficient" antibody variants include: US 2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US 2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO 2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; 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 defucosylated antibodies include Lec 13 CHO cells, which are deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249(2):533-545, 1986; U.S. Patent Application Publications US 2003 / 0157108 and WO 2004 / 056312, especially Example 11), as well as knockout cell lines, such as knockout CHO cells for the alpha-1,6-fucosyltransferase gene, FUT8 (see, e.g., Yamane-Ohnuki et al., Biotechnol. Bioeng., 87(5):614-622, 2004; Kanda et al., Biotechnol. Bioeng., 94(4):680-688, 2006; and WO2003 / 085107).

[0329] Further provided are antibody variants having bisected oligosaccharides, for example, 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.

[0330] In some embodiments, the constant region of the antibody or bispecific antibody contains one or more amino acid substitutions to optimize the in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in some embodiments, the antibody contains amino acid substitutions that increase binding to FcRn. Non-limiting examples of such substitutions include IgG constant region T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176(1):346-356, 2006); M428L and N434S ("LS" mutations, see, e.g., Zalevsky, et al., Nature Biotechnol., 28(2):157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006). 2006); and M252Y, S254T, and T256E (see, e.g., Dall'Acqua et al., J. Biol. Chem., 281(33):23514-23524, 2006). The disclosed antibodies and antigen-binding fragments can be linked to or can include an Fc polypeptide comprising any of the substitutions listed above, for example, an Fc polypeptide can comprise an M428L and N434S substitution according to the EU index numbering system.

[0331] In some embodiments, the antibodies or bispecific antibodies provided herein can be further modified to include additional nonprotein moieties. Moieties suitable for derivatization of antibodies 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, these can be the same or different molecules. Generally, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc.

[0332] B. Conjugates The antibodies, antigen-binding fragments, and bispecific antibodies disclosed herein that specifically bind to coronavirus spike proteins can be conjugated to agents such as effector molecules or detectable markers. Both covalent and non-covalent attachment means can be used. These include (but are not limited to) toxins and radioactive agents, such as 125 I, 32 P,14 C. 3 H, and 35 A variety of effector molecules and detectable markers can be used, including S, as well as other labels, targeting moieties, enzymes, ligands, etc. The choice of a particular effector molecule or detectable marker will depend on the particular target molecule or target cell and the desired biological effect.

[0333] The procedure for attaching a detectable marker to an antibody, antigen-binding fragment, or bispecific antibody varies depending on the chemical structure of the effector. Polypeptides typically contain various functional groups, such as carboxyl (-COOH) groups, free amine (-NH) groups, or sulfhydryl (-SH) groups, which are available for reaction with appropriate functional groups on the polypeptide to result in attachment of an effector molecule or detectable marker. Alternatively, the antibody, antigen-binding fragment, or bispecific antibody can be derivatized to expose or attach additional reactive functional groups. Derivatization can include attachment of any suitable linker molecule. The linker can form covalent bonds with both the antibody or antigen-binding fragment and the effector molecule or detectable marker. Suitable linkers include, but are not limited to, linear or branched carbon linkers, heterocyclic carbon linkers, or peptide linkers. When the antibody, antigen-binding fragment, or bispecific antibody, and the effector molecule or detectable marker are polypeptides, the linkers can be attached to the constituent amino acids through their side chains (e.g., via a disulfide bond to cysteine) or alpha carbon, or through the amino and / or carboxyl groups of the terminal amino acids.

[0334] Considering the numerous methods reported for attaching various radiodiagnostic compounds, radiotherapeutic compounds, labels (e.g., enzymes or fluorescent molecules), toxins, and other agents to antibodies, one can determine an appropriate method for attaching a given agent to an antibody or antigen-binding fragment or bispecific antibody.

[0335] The antibody, antigen-binding fragment, or bispecific antibody can be conjugated with a detectable marker; for example, a detectable marker that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or imaging diagnostic techniques (e.g., CT, computerized axial tomography (CAT), MRI, magnetic resonance imaging (MTR), ultrasound, fiberoptic examination, and laparoscopy). Specific non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzyme linkages, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, useful detectable markers include fluorescent compounds such as fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, etc. Bioluminescent markers such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP) are also useful. Antibodies, antigen-binding fragments, or bispecific antibodies can also be conjugated with enzymes useful for detection, such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc. When antibodies or antigen-binding fragments are conjugated with a detectable enzyme, they can be detected by adding an additional reagent that the enzyme uses to generate a distinguishable reaction product. For example, in the presence of the agent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a colored reaction product that is visually detectable. Antibodies, antigen-binding fragments, or bispecific antibodies can also be conjugated with biotin, and can be detected by indirect measurement of avidin or streptavidin binding. Note that avidin itself can be conjugated with an enzyme or fluorescent label.

[0336] Antibodies, antigen-binding fragments, or bispecific antibodies can be conjugated with paramagnetic agents such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also useful as labels. Antibodies can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. Antibodies, antigen-binding fragments, or bispecific antibodies can also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (e.g., a leucine zipper pair sequence, a binding site for a second antibody, a metal binding domain, an epitope tag).

[0337] For example, for diagnostic purposes, antibodies, antigen-binding fragments, or bispecific antibodies can also be conjugated to radiolabeled amino acids. For example, radiolabels can be used to detect coronaviruses by radiography, emission spectroscopy, or other diagnostic techniques. Examples of labels for polypeptides 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. Radiolabels can be detected, for example, using photographic film or a scintillation counter, fluorescent markers can be detected using a photodetector to detect emitted illumination, enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

[0338] The average detectable marker moiety per antibody, antigen-binding fragment, or bispecific antibody in the conjugate can range, for example, from 1 to 20 moieties per antibody or antigen-binding fragment. In some embodiments, the average number of effector molecules or detectable marker moieties per antibody or antigen-binding fragment in the 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 / antibody ratio) can be controlled in various ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partial or limited reduction conditions for cysteine ​​thiol modifications; or (iv) manipulating the amino acid sequence of the antibody by recombinant techniques to alter the number and position of cysteine ​​residues to control the number or position of linker-effector molecule attachments.

[0339] C. Polynucleotides and Expression Nucleic acid molecules (e.g., cDNA or RNA molecules, e.g., mRNA) encoding the amino acid sequences of the antibodies, antigen-binding fragments, bispecific antibodies, and conjugates disclosed herein that specifically bind to coronavirus spike proteins are provided. Nucleic acids encoding these molecules can be prepared by cloning the amino acid sequences (e.g., CDR sequences and V sequences) provided herein. H Array and V L In some embodiments, nucleic acid molecules can be readily generated using sequences (e.g., framework or constant region sequences) available in the art, as well as the genetic code. In some embodiments, nucleic acid molecules can be ... H , V L , or V H and V Land (e.g., in a bicistronic expression vector). In some embodiments, the nucleic acid molecule encodes an scFv. In some embodiments, the nucleic acid molecule can be expressed in a host cell (e.g., a mammalian cell) to produce the disclosed antibody or antigen-binding fragment. Nucleic acid molecules encoding scFvs are provided.

[0340] Using the genetic code, various functionally equivalent nucleic acid sequences, e.g., sequences that differ but encode the same antibody sequence, or V L and / or V H A nucleic acid encoding a conjugate or fusion protein comprising the nucleic acid sequence of can be constructed.

[0341] In a non-limiting example, the isolated nucleic acid molecule is selected from the group consisting of the V of the A23-58.1, A19-61.1, A19-46.1, A23-105.1, A23-97.1, A19-82.1, A19-1.1, A23-113.1, A20-29.1, A19-30.1, A20-36.1, A20-9.1, A23-80.1, or B1-182.1 antibody. H Exemplary nucleic acid sequences are provided herein. In another non-limiting example, the nucleic acid molecule encodes the V of the A23-58.1, A19-61.1, A19-46.1, A23-105.1, A23-97.1, A19-82.1, A19-1.1, A23-113.1, A20-29.1, A19-30.1, A20-36.1, A20-9.1, A23-80.1, or B1-182.1 monoclonal antibody. L In a further non-limiting example, the nucleic acid molecule can encode a bispecific antibody, such as a DVD-immunoglobulin format. The nucleic acid can also encode an scFv. The nucleic acid molecule can also encode a conjugate.

[0342] Nucleic acid molecules encoding antibodies, antigen-binding fragments, bispecific antibodies, and conjugates that specifically bind to coronavirus spike proteins can be prepared by any suitable method, including, for example, cloning appropriate sequences, or by direct chemical synthesis using standard methods. Chemical synthesis produces single-stranded oligonucleotides, which can be converted into double-stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

[0343] Exemplary nucleic acids can be prepared by 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).

[0344] Nucleic acids can also be prepared by amplification methods, including polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and self-sustained sequence replication systems (3SR).

[0345] Nucleic acid molecules can be expressed in recombinantly engineered cells, such as bacterial cells, plant cells, yeast cells, insect cells, and mammalian cells. Antibodies, antigen-binding fragments, and conjugates can be expressed in the V (optionally linked to an effector molecule or detectable marker). H and / or V LThe antibodies and antigen-binding fragments can be expressed as individual proteins comprising V, or as a fusion protein. Any suitable method for expressing and purifying antibodies and antigen-binding fragments can be used; non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011). Immunoadhesins can also be expressed. Thus, in some examples, V H and V L As well as immunoadhesin-encoding nucleic acids are provided. The nucleic acid sequence can optionally encode a leader sequence.

[0346] To generate scFv, V L Domains and V H V as a continuous single-chain protein with domains connected by flexible linkers H Array and V L The V fragment encodes a flexible linker, e.g., the V fragment encodes the amino acid sequence (Gly4-Ser)3, so that the V fragment can be expressed. H DNA fragments encoding V L DNA fragments encoding the nucleotides can be operably linked (e.g., 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 Dubel (Eds.), Antibody Engineering, Vols. 1-2, 2002). nd ed., Springer-Verlag, 2010;Greenfield (Ed.), Antibodies: A Laboratory Manual, 2 nd(See, e.g., J. Am. Chem. Soc. 1999, 14:101-104, 1999, ed. New York: Cold Spring Harbor Laboratory Press, 2014.) Optionally, a cleavage site, such as a furin cleavage site, can be included in the linker.

[0347] Single-chain antibodies contain a single V H and V L If only V is used, it can be monovalent, and two V H and V L When used, it can be bivalent, or have more than two V H and V L When the encoded V is used, it can be multivalent. Bispecific or multivalent antibodies can be generated that specifically bind to the coronavirus spike protein and another antigen. H and V L optionally, a furin cleavage site is H Domains and V L For example, the nucleic acid molecule may contain a DVD-Ig domain. (商標) When encoding a bispecific antibody of this type, a linker may also be encoded.

[0348] 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. The cells can be prokaryotic or eukaryotic. Numerous expression systems available for protein expression can be used to express the disclosed antibodies and antigen-binding fragments, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells, such as COS, CHO, HeLa, and myeloma cell lines. Stable transfer methods, meaning that the foreign DNA is continuously maintained in the host, can be used. Hybridomas expressing the antibody of interest are also encompassed by the present disclosure.

[0349] Expression of nucleic acids encoding the antibodies, antigen-binding fragments, and bispecific antibodies (e.g., DVD-immunoglobulin antibodies) described herein can be achieved by operably linking the DNA or cDNA to a promoter (either constitutive or inducible) and subsequently incorporating it into an expression cassette. The promoter may be any promoter of interest, including a cytomegalovirus promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassette may be suitable for replication and integration in either prokaryotes or eukaryotes. A typical expression cassette contains specific sequences useful for regulating the expression of DNA encoding a protein. For example, an expression cassette can include an appropriate promoter, enhancer, transcription and translation terminator, initiation sequence, a start codon (i.e., ATG) in front of the protein-encoding gene, splicing signals for introns, sequences for maintaining the correct reading frame of the gene to enable proper translation of the mRNA, and a stop codon. The vector can encode a selectable marker, such as a marker that encodes drug resistance (eg, ampicillin or tetracycline resistance).

[0350] To obtain high-level expression of the cloned gene, it is desirable to construct an expression cassette containing, for example, a strong promoter to direct transcription, a ribosome binding site for translation initiation (e.g., an internal ribosome binding sequence), and a transcription / translation terminator. For E. coli, this can include a promoter, such as the T7, trp, lac, or lambda promoter, a ribosome binding site, and preferably, a transcription termination signal. For eukaryotic cells, the control sequences can include, for example, promoters and / or enhancers derived from immunoglobulin genes, HTLV, SV40, or cytomegalovirus, as well as polyadenylation sequences, and can further include splice donor and / or acceptor sequences (e.g., splice acceptor and donor sequences of CMV and / or HTLV). The cassette can be transferred into the selected host cell by any suitable method, such as transformation or electroporation for E. coli, and calcium phosphate treatment, electroporation, or lipofection for mammalian cells. Cells transformed with the cassette can be selected by resistance to antibiotics conferred by genes contained in the cassette, such as the amp, gpt, neo, and hyg genes.

[0351] The nucleic acid encoding the polypeptide described herein can be modified without reducing its biological activity.Some modifications can be made to facilitate cloning, expression, or incorporation into a fusion protein of the target molecule.Such modifications include, for example, a stop codon, a sequence for creating a conveniently located restriction site, and a sequence for adding methionine to the amino terminus to provide an initiation site, or additional amino acids (e.g., polyHis) that are useful in the purification process.

[0352] Once expressed, antibodies, antigen-binding fragments, bispecific antibodies, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (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 need not be 100% pure. Once purified, partially or to homogeneity as desired, if used prophylactically, the polypeptides should be substantially free of endotoxin.

[0353] Methods for the expression of antibodies, antigen-binding fragments, bispecific antibodies, and conjugates from mammalian cells and bacteria such as E. coli, and / or their refolding into suitable active forms have been described and are applicable to the antibodies disclosed herein. See, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual, 2004, pp. 211-214, 1997. nd ed. New York: Cold Spring 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.

[0354] D. Methods and Compositions 1. Inhibiting coronavirus infection Disclosed herein is a method for inhibiting coronavirus infection, such as SARS-CoV-2 infection, in a subject. The method includes administering to a subject at risk of or with coronavirus infection an effective amount (i.e., an amount effective to inhibit infection in the subject) of the disclosed antibody, antigen-binding fragment, or bispecific antibody, or a nucleic acid encoding such an antibody, antigen-binding fragment, or bispecific antibody. The method can be used before or after exposure. In some embodiments, the antibody or antigen-binding fragment can be used in the form of a bispecific antibody, e.g., a DVD-immunoglobulin. The antigen-binding fragment can be an scFv.

[0355] The method does not need to completely eliminate or inhibit infection to be effective. For example, the method can reduce infection by a desired amount, e.g., 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%, compared to coronavirus infection in the absence of treatment (eliminating or preventing detectable coronavirus infection). In some embodiments, the subject can also be treated with an effective amount of an additional agent, e.g., an antiviral agent.

[0356] In some embodiments, administration of an effective amount of the disclosed antibodies, antigen-binding fragments, bispecific antibodies, or nucleic acid molecules inhibits the establishment of infection and / or subsequent disease progression in a subject, which can include any statistically significant reduction in the activity (e.g., growth or invasion) or symptoms of coronavirus infection in a subject.

[0357] Disclosed herein is a method for inhibiting coronavirus replication in a subject. The method comprises administering to a subject at risk of or with a coronavirus infection an effective amount (i.e., an amount effective to inhibit 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. The method can be used before or after exposure.

[0358] Methods for treating a coronavirus infection in a subject are disclosed. Methods for preventing a coronavirus infection in a subject are also disclosed. These methods include administering one or more of the disclosed antibodies, antigen-binding fragments, bispecific antibodies, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, as disclosed herein.

[0359] Antibodies, antigen-binding fragments thereof, and bispecific antibodies can be administered by intravenous infusion. The dosage of the antibody, antigen-binding fragment, or bispecific antibody varies, but is generally in the range of 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 embodiments, the dosage of the antibody, antigen-binding fragment, or bispecific antibody can be 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. The antibody, antigen-binding fragment, or bispecific antibody is administered according to a dosing schedule determined by a physician. In some examples, the antibody, antigen-binding fragment, or bispecific antibody is administered weekly, every two weeks, every three weeks, or every four weeks.

[0360] In some embodiments, the method of inhibiting infection in a subject further comprises administering one or more additional agents to the subject. 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.

[0361] In some embodiments, the method includes administering a first antibody that specifically binds to a coronavirus spike protein disclosed herein and a second antibody that also specifically binds to a coronavirus protein, e.g., a different epitope of a coronavirus protein. In some embodiments, the first antibody is one of A23-58.1, A19-61.1, A19-46.1, A23-105.1, A23-97.1, A19-82.1, A19-1.1, A23-113.1, A20-29.1, A19-30.1, A20-36.1, A20-9.1, A23-80.1, or B1-182.1. In more embodiments, the first antibody is selected from the group consisting of A23-58.1, A19-61.1, A19-46.1, A23-105.1, A23-97.1, A19-82.1, A19-1.1, A23-113.1, A20-29.1, A19-30.1, A20-36.1, A20-9.1, A23-80.1, B1-182.1, or B1-182.1_58CDRH3. and the second antibody is another of A23-58.1, A19-61.1, A19-46.1, A23-105.1, A23-97.1, A19-82.1, A19-1.1, A23-113.1, A20-29.1, A19-30.1, A20-36.1, A20-9.1, A23-80.1, B1-182.1, or B1-182.1_58CDRH3. In some embodiments, one antibody binds to one epitope on the spike protein and the other antibody binds to a different epitope on the spike protein. An effective amount of one, two, three, four, five, or six of A23-58.1, A19-61.1, A19-46.1, A23-105.1, A23-97.1, A19-82.1, A19-1.1, A23-113.1, A20-29.1, A19-30.1, A20-36.1, A20-9.1, A23-80.1, B1-182.1, or B1-182.1_58CDRH3 can be administered to a subject. An effective amount of LY-CoV555 or an antibody of this class can be administered to a subject. In more embodiments, the first antibody is B1-182.1 and the second antibody is A19-46.1.In further embodiments, the first antibody is B1-182.1 and the second antibody is A19-61.1. In more embodiments, the first antibody is B1-182.1_58CDRH3 and the second antibody is A19-46.1. In more embodiments, the first antibody is B1-182.1_58CDRH3 and the second antibody is A19-61.1. In further embodiments, more than two antibodies are administered to the subject. Thus, in some examples, three, four, or five antibodies are administered to the subject.

[0362] In some embodiments, a subject is administered DNA or RNA encoding the disclosed antibodies, antigen-binding fragments, or bispecific antibodies to induce in vivo antibody production, e.g., using the subject's cellular machinery. Any suitable method of nucleic acid administration can be used; non-limiting examples are provided in U.S. Pat. Nos. 5,643,578, 5,593,972, and 5,817,637. U.S. Pat. No. 5,880,103 describes several methods for delivering protein-encoding nucleic acids to organisms. One approach for nucleic acid administration is direct administration using plasmid DNA, e.g., a mammalian expression plasmid. To increase expression, the nucleotide sequence encoding the disclosed antibodies, antigen-binding fragments, or bispecific antibodies can be placed under the control of a promoter. Methods include liposomal delivery of nucleic acids. Such methods can be applied to the production of antibodies or antigen-binding fragments thereof. In some embodiments, the pVRC8400 vector (described in Barouch et al., J. Virol., 79(14), 8828-8834, 2005, incorporated herein by reference) is used to express the disclosed antibodies or antigen-binding fragments in a subject.

[0363] In some embodiments, a subject (e.g., a human subject at risk for or with a coronavirus infection) can be administered an effective amount of an AAV viral vector containing one or more nucleic acid molecules encoding the disclosed antibodies, antigen-binding fragments, or bispecific antibodies. The AAV viral vector is designed for expression of the nucleic acid molecules encoding the disclosed antibodies, antigen-binding fragments, or bispecific antibodies, and administration of an effective amount of the AAV viral vector to a subject results in expression of an effective amount of the 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 antibodies, antigen-binding fragments, or bispecific antibodies in a subject include those provided in Johnson et al., Nat. Med., 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541):87-91, 2015, each of which is incorporated herein by reference in its entirety.

[0364] In one embodiment, nucleic acids encoding the disclosed antibodies, antigen-binding fragments, or bispecific antibodies are introduced directly into tissue. For example, nucleic acids can be loaded onto gold microparticles by standard methods, such as Bio-Rad's HELIOS (商標) It can be introduced into the skin by a device such as a gene gun. The nucleic acid can be "naked", consisting of a plasmid under the control of a strong promoter.

[0365] Typically, DNA is injected into the muscle, but can also be injected directly into other sites. The dosage for injection is usually about 0.5 μg / kg to about 50 mg / kg, typically about 0.005 mg / kg to about 5 mg / kg (see, e.g., U.S. Patent No. 5,589,466).

[0366] Single or multiple administrations of the disclosed antibodies, antigen-binding fragments, or bispecific antibodies, compositions containing conjugates, or nucleic acid molecules encoding such molecules can be administered at a dosage and frequency as needed and tolerated by the patient. Dosages can be administered once, or periodically until the desired results are achieved or side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit coronavirus infection without causing unacceptable toxicity to the patient.

[0367] The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. Dosages are usually administered at or above the ED 50 The circulating concentration range includes 0.1 to 1.0 mg / kg of steroids and 0.1 to 1.0 mg / kg of steroids, with little or minimal toxicity. The dosage may vary within this range depending on the dosage form used and the route of administration utilized. The effective amount can be determined from cell culture assays and animal studies.

[0368] Antibodies, antigen-binding fragments, or bispecific antibodies specific for coronavirus spike proteins, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, can be administered to a subject in a variety of ways, including local administration and systemic administration, such as by subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. In one embodiment, antibodies, antigen-binding fragments, bispecific antibodies, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, are administered once daily by a single subcutaneous, intravenous, intraarterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. Antibodies, antigen-binding fragments, bispecific antibodies, conjugates, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, can also be administered by direct injection at or near the site of disease. A further method of administration is via an osmotic pump (e.g., an Alzet pump) or minipump (e.g., an Alzet miniosmotic pump) that allows for controlled, sustained, and / or sustained release delivery of an antibody, antigen-binding fragment, conjugate, or nucleic acid molecule encoding such a molecule, or a composition comprising such a molecule, over a predetermined period of time. The osmotic pump or minipump can be implanted subcutaneously or near the target site.

[0369] 2. Composition Compositions are provided comprising one or more of the coronavirus spike protein-specific antibodies, antigen-binding fragments, conjugates, or nucleic acid molecules encoding such molecules in a pharmaceutically acceptable carrier, as disclosed herein. In some embodiments, the compositions comprise the A23-58.1, A19-61.1, A19-46.1, A23-105.1, A23-97.1, A19-82.1, A19-1.1, A23-113.1, A20-29.1, A19-30.1, A20-36.1, A20-9.1, A23-80.1, B1-182.1, or B1-182.1_58CDRH3 antibodies, or antigen-binding fragments thereof, as disclosed herein. In some embodiments, the compositions comprise two, three, four, or more antibodies that specifically bind to the coronavirus spike protein. For example, the compositions are useful, e.g., for inhibiting or detecting a coronavirus infection, e.g., a SARS-CoV-2 infection. For example, the compositions are useful, e.g., for inhibiting or detecting a coronavirus infection, e.g., a SARS-CoV-1 infection.

[0370] The composition can be prepared in a unit dosage form, such as a kit, for example, a kit, for administration to a subject.The amount and timing of administration are at the discretion of the administering physician to achieve the desired purpose.Antibody, antigen-binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecule can be formulated for systemic or local administration.In one example, antigen-binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecule is formulated for parenteral administration, for example, intravenous administration.

[0371] In some embodiments, the antibody, antigen-binding fragment, bispecific antibody, or conjugate thereof in the composition is 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%) pure. In some embodiments, 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) macromolecular contaminants, such as other mammalian (e.g., human) proteins.

[0372] Compositions for administration can comprise a solution of an antibody, antigen-binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such a molecule dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers can be used, such as buffered saline. These solutions are sterile and generally free of undesirable material. These compositions can be sterilized by any suitable technique. The compositions can contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, and the like, e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of the antibody in these formulations can vary widely and will be selected primarily based on fluid volume, viscosity, body weight, and the like, in accordance with the particular mode of administration selected and the needs of the subject.

[0373] A typical composition for intravenous administration contains about 0.01 to about 30 mg / kg of antibody, antigen-binding fragment, bispecific antibody, or conjugate (or a corresponding dose of a conjugate comprising the antibody or antigen-binding fragment) per subject per day. Any suitable method can be used to prepare an administrable composition; non-limiting examples are described in Remington: The Science and Practice of Pharmacy, 22 nded., London, UK: Pharmaceutical Press, 2013. In some embodiments, the composition can be a liquid formulation comprising one or more antibodies, antigen-binding fragments, or bispecific antibodies in a concentration range of 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.

[0374] Antibodies, antigen-binding fragments thereof, bispecific antibodies, or nucleic acids encoding such molecules can be provided in lyophilized form and rehydrated with sterile water before administration, but they can also be provided in sterile solutions of known concentrations. Solutions containing antibodies, antigen-binding fragments, bispecific antibodies, or nucleic acids encoding such molecules can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of 0.5 to 15 mg / kg body weight. Considerable experience is available in the field of antibody drug administration, and antibody drugs have been commercially available in the United States since the approval of rituximab in 1997. Antibodies, antigen-binding fragments, conjugates, or nucleic acids encoding such molecules can be administered by slow infusion rather than intravenous push or bolus. In one example, a high loading dose is administered, followed by lower maintenance doses. For example, an initial loading dose of 4 mg / kg can be infused over approximately 90 minutes, followed by weekly maintenance doses of 2 mg / kg infused over 30 minutes for 4 to 8 weeks if the previous dose is well tolerated.

[0375] Controlled-release parenteral formulations can be prepared as implants, oily injections, or particulate systems. For a general 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 microparticles, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain an active protein agent, such as a cytotoxin or drug, as a central core. In microparticles, the active protein agent is dispersed throughout the particle. Particles, microparticles, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 μm, so only nanoparticles are administered intravenously. Microparticles are typically approximately 100 μm in diameter and are administered subcutaneously or intramuscularly. See, e.g., 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.

[0376] Polymer can be used for the ion-controlled release of the composition disclosed herein.Any suitable polymer can be used, such as degradable or non-degradable polymer matrix designed for use in controlled drug delivery.Alternatively, hydroxyapatite is used as the microcarrier for the controlled release of protein.In yet another aspect, liposome is used for the controlled release and drug targeting of lipid-encapsulated drugs.

[0377] 3. Methods of detection and diagnosis Methods for detecting the presence of coronavirus spike protein in vitro or in vivo are also provided. In one example, the presence of coronavirus spike protein is detected in a biological sample from a subject and can be used to identify infected subjects. The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies, and pathology specimens. Biological samples also include tissue sections, e.g., frozen sections obtained for histological purposes. Biological samples further include bodily fluids, e.g., blood, serum, plasma, sputum, spinal fluid, or urine. The detection method can include contacting a cell or sample with an antibody, antigen-binding fragment, or bispecific antibody, or a conjugate thereof (e.g., a conjugate comprising a detectable marker), that specifically binds to the coronavirus spike protein under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting the detectable marker conjugated to the antibody or antigen-binding fragment).

[0378] In one embodiment, the antibody, antigen-binding fragment, or bispecific antibody is directly labeled with a detectable marker. In another embodiment, the antibody (or antigen-binding fragment, or bispecific antibody) that binds to the coronavirus spike protein (first antibody) is unlabeled, and a second antibody or other molecule capable of binding to the first antibody is used for detection. A second antibody capable of specifically binding to a particular species and class of first antibody is selected. For example, if the first antibody is human IgG, the second antibody can be anti-human IgG. Other molecules capable of binding to antibodies include, but are not limited to, Protein A and Protein G, both of which are commercially available. Suitable labels for antibodies, antigen-binding fragments, bispecific antibodies, or second antibodies are known and described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic materials, and radioactive materials.

[0379] In some embodiments, the disclosed antibodies, antigen-binding fragments thereof, or bispecific antibodies are used to test vaccines. For example, to test whether a vaccine composition containing a coronavirus spike protein or a fragment thereof exhibits a conformation containing the epitope of the disclosed antibodies. Accordingly, provided herein is a method for testing a vaccine, comprising contacting a sample containing a vaccine, e.g., a coronavirus spike protein immunogen, with the disclosed antibodies, antigen-binding fragments, or bispecific antibodies under conditions sufficient for immune complex formation, and detecting the immune complex to detect a vaccine containing the epitope of interest in the sample. In one example, detection of an immune complex in the sample indicates that the vaccine component, e.g., the immunogen, exhibits a conformation capable of binding to the antibody or antigen-binding fragment.

[0380] Because some isolated mAbs bind only to SARS-CoV-2 and not to SARS-CoV-1, the methods can also include using an assay to distinguish between SARS-CoV-2. In some embodiments, a comparison is made between binding of a sample to antibodies that bind to SARS-CoV-1 and binding of a sample to antibodies that bind only to SARS-CoV-2. Thus, the disclosed methods can be used to distinguish between SARS-CoV-1 and SARS-CoV-2 in a sample. [Example]

[0381] Monoclonal antibodies A23-58.1 (A23-58.1), A19-61.1 (A19-61.1), A19-46.1 (A19-46.1), A23-105.1 (A23-105.1), A23-97.1 (A23-97.1), A19-82.1 (A19-82.1), and A19-1.1 (A19-1.1) , A23-113.1 (A23-113.1), A20-29.1 (A20-29.1), A19-30.1 (A19-30.1), A20-36.1 (A20-36.1), A20-9.1 (A20-9.1), A23-80.1 (A23-80.1), and B1-182.1 (B1-182.1) were isolated from single memory B cells from peripheral mononuclear blood cells of survivors of SARS-CoV-2 infection and screened for SARS-CoV-2 spike protein binding.

[0382] To obtain these antibodies, blood was obtained between days 25 and 55 after symptom onset from 22 convalescent subjects who experienced mild to moderate symptoms after WA-1 infection. Four subjects, A19, A20, A23, and B1, had both high neutralizing and binding activity against WA-1 variants (Figure 16A) and were selected for antibody isolation. CD19+ / CD20+ / IgM- / IgA+ or IgG+ B cells were sorted for binding to the stabilized version of S (S-2P), the complete S1 subunit, or the receptor binding domain plus subdomain-1 region of S1 (RBD-SD1) (Figure 16B). In total, 889 B cells were sorted, recovering 709 (80%) paired heavy and light chain antibody sequences, and 200 antibodies were selected for expression. We measured the binding of these 200 antibodies to the stabilized spike, the intact S1 subunit, the RBD, or the NTD using the MSD binding assay. There was a broad range of responses across all spike domains in the MSD binding assay: 77 bound to the RBD, 46 bound to the NTD, 58 were inferred to bind to the S2 subunit based on binding to S but not S1, and 19 bound to unspecified epitopes or failed to recognize the spike (Figure 16C).

[0383] Using SMRTseq, we identified a single B cell receptor sequence. The heavy and light chain variable sequences of the B cell receptor sequence were synthesized, cloned into a human vector, expressed, and characterized for their binding, structural, and functional capabilities. The antibody is a potent neutralizing antibody that targets a unique epitope in the spike glycoprotein of SARS-CoV-2.

[0384] Example 1 In vitro and in vivo evaluation of antibodies against SARS CoV2 Functional Properties: We aimed to determine the in vitro functionality of the mAbs, including their reactivity with the coronavirus surface spike protein (S). mAbs were tested for binding to S2P, HexaPro, S1, RBD, and / or NTD by ELISA, MSD, and biolayer interferometry (BLI).

[0385] Epitope Mapping: Comprehensive mapping was performed to determine the binding properties of the mAb and epitopes on S2P, HexaPro, S1, RBD, and / or NTD by evaluation by ELISA, MSD, and using BLI competition with other mAbs and ACE2 with known epitopes.

[0386] Affinity measurements: BLI was used to determine the affinity of the mAbs for S2P.

[0387] Neutralization: Pseudotyped lentiviral particles bearing the coronavirus spike protein were used to determine neutralization of viral infection by mAbs. Virus-induced infection was determined by measuring the expression of a luciferase reporter gene encoded in the viral genome.

[0388] Structural assessment: Use class averages of single particle negative stain electron microscopy and / or 2D and 3D reconstructions from CyroEM to determine the mode of recognition and molecular interactions required for the antibody to function and bind to the target antigen protein.

[0389] Example 2 Selection of coronavirus-specific monoclonal antibodies by single-cell sorting and SMRTseq Monoclonal antibodies whose variable domains are discussed below were isolated by single-cell sorting of SARS-CoV-2-specific memory B cells. Briefly, peripheral blood mononuclear cells were stained with a flow cytometry panel to identify memory B cell populations. Cells were co-stained with fluorescently labeled SARS-CoV-2 antigens. Probes included the RBD, NTD, S1, and S2P. Each probe was labeled with a unique fluorescent color to allow phenotyping of memory B cells by their ability to bind to each probe and the relative potency of binding to each probe. Memory B cells that showed binding to the RBD or S2P probe were sorted as individual cells into wells of a 96-well plate. These single cells were then subjected to immunoglobulin heavy and light chain sequencing using the SMRTseq method.

[0390] Example 3 A23-58.1(A23-58.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A23-58.1 is a monoclonal antibody (mAb) whose variable domains were isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 48 days after symptom onset. The nucleotide and amino acid sequences of the heavy and light chains of the expression version of A23-58.1 are as follows: The heavy and light chain variable immunoglobulin sequences encoding the antigen-binding regions of A23-58.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and kappa light chain. These vectors were used to express the antibody, which was purified using standard methods.

[0391] Table 1. Gene family of A23-58.1 and nucleotide and amino acid sequences of heavy and light chains. CDR1, CDR2, and CDR3 are It is bold and underlined. TIFF2024506315000007.tif95156

[0392] Binding to SARS-CoV-2 spike protein: The purified monoclonal antibody A23-58.1 was evaluated for its ability to bind to four SARS CoV2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS CoV1 spike protein antigen. Binding was confirmed using an ELISA immunoassay, which showed that A23-58.1 could bind to SARS CoV2 S2P, S1, and RBD, but not to SARS CoV2 NTD or SARS CoV1 S2P (Figure 1). With the exception of D614G / N439K, which had slightly reduced binding, A23-58.1 maintained or increased binding to all variants tested, including D614G, D614G / Y453F, D614G / 501Y, D614G / del69-70, D614G / del69-70 / N501Y, B.1.1.7, D614G / K471N, D614G / E484K, D614G / K417N / E484K / N501Y, and B.1.351 variants. See Figure 1.

[0393] Epitope mapping: By evaluating how A23-58.1 competes with previously published antibodies and the antibodies discovered in this report, the approximate epitope can be determined. Biolayer interferometry (BLI) was used to determine the competitive classification. Briefly, purified SARS CoV2 S2P was loaded onto a biosensor. A competitor mAb (a mAb that determines the class or approximate epitope) was then allowed to bind to the antigen, and the degree of binding was recorded. Next, an analyte mAb was then allowed to bind, and the degree of binding was recorded. The percent inhibition of analyte binding was calculated as follows: Calculate as TIFF2024506315000008.tif10128.

[0394] The competition profile of A23-58.1 is shown in Figure 2. This shows that A23-58.1 competes similarly with LY-CoV555, but not with A19-46.1, A19-61.1, and A23-80.1, which LY-CoV555 competes with. Similarly, A19-46.1, A19-61.1, and A23-80.1 each block the binding of LY-CoV555, but not A23-58.1. Taken together, this indicates that A23-58.1 has a unique binding mode and epitope within the RBD. Figure 2.

[0395] Kinetics of binding to SARS CoV2 S2P protein: Using BLI, the Fab protein generated from A23-58.1 was evaluated for binding to SARS CoV2 S2P. The Fab of A23-58.1 had an affinity constant (KD) of 7.3 nM, 7.13 x 10 5 / sec kon, and 5.2 x 10 -3 koff in / molar*sec, indicating binding to SARS CoV2 S2P protein.

[0396] Neutralization: A23-58.1 was tested for neutralizing activity in a pseudotyped virus entry assay (Figure 1). A23-58.1 had a neutralizing IC 50 (0.0025 μg / mL) and IC 80 (0.0107 μg / mL), which is the IC 50 :0.0071, IC 80 The neutralization IC of SARS-CoV-2 Nanoluc live virus is 3-4 times more potent than that of 0.0357 μg / mL. 50 (0.0021 μg / mL) and IC 80 (0.0045 μg / mL) is one of the most potent reported for an antibody targeting SARS CoV-2. See Figure 1.

[0397] Further testing against naturally occurring SARS CoV-2 spike variants in pseudotyping neutralization assays demonstrated that A23-58.1 exhibited high potency (IC) against the following variants: D614G, N439K / D614G, Y453F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, N501Y / del69-70 / D614G, N501Y / E484K / K417N / D614G, and B.1.1.7 (VOC 202012 / 01) containing the following amino acid changes: H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H. 50 :<0.0006~0.0058ug / mL; IC 80 It was shown that E484K / D614G (IC 50 :0.0102, IC 80 : 0.0251 μg / mL) and B.1.351 (IC 50 :0.0130, IC 80 : 0.1196 μg / mL), but still highly potent. This is in contrast to LY-CoV555 and REGN-10989, which lose neutralizing activity against the E484K / D614G, N501Y / E484K / K417N / D614G, and B.1.351 variants (>10 μg / mL). REGN-10933 loses neutralizing activity against the Y453F / E614G, N501Y / E484K / K417N / D614G, and B.1.351 variants (>10 μg / mL). CB6 loses neutralizing activity against the N501Y / E484K / K417N / D614G and B.1.351 variants (>10 μg / mL). See Figure 1.

[0398] Blocking ACE2 receptors: The spike proteins of SARS CoV1 and SARS CoV2 target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody binding of spike glycoproteins can be performed using a modified version of the BLI competition assay used for epitope mapping. Briefly, purified SARS CoV2 S2P is loaded onto a biosensor. A competitor mAb (the mAb being evaluated to determine whether it blocks ACE2 binding) is then allowed to bind to the antigen, and the degree of binding is recorded. Soluble ACE2 protein is then allowed to bind, and the degree of binding is recorded. The percent inhibition of analyte binding is calculated as follows: The calculation was as follows: TIFF2024506315000009.tif10128.

[0399] The competition profile of A23-58.1 ACE2 is shown in Figure 2 and shows that A23-58.1 prevents ACE2 from binding to S2P.

[0400] Electron microscopy studies: Fab proteins were generated from A23-58.1 IgG by enzymatic digestion. Excess Fab was incubated in the presence of a stabilized 6-proline (S-6P) spike protein ectodomain (described in Hseih et al., "Structure-based design of prefusion-stabilized SARS-CoV-2 Spikes," Science, 369(6510):1501-1505, 2020, incorporated herein by reference). The complexed material was then analyzed by cryo-electron microscopy single particle analysis (Figure 3). This data shows that A23-58.1 binds to spikes with three Fabs per S-6P trimer when the RBD domain is in the up position. See Figure 3.

[0401] Example 4 A19-61.1(A19-61.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A19-61.1 is a monoclonal antibody whose variable domain was isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 41 days after symptom onset. Flow cytometry data suggested this antibody. The nucleotide and amino acid sequences of the heavy and light chains of the expressed version of A19-61.1 can be found in Table 2. The heavy and light chain variable immunoglobulin sequences in Table 2 encoding the antigen-binding region of A19-61.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and kappa light chain. These vectors were used to express the antibody, which was purified using standard methods.

[0402] Table 2: Gene family of A19-61.1 and nucleotide and amino acid sequences of the heavy and light chains. CDR1, CDR2, and CDR3 are It is bold and underlined. TIFF2024506315000010.tif100156

[0403] Binding to SARS-CoV-2 spike protein: Purified monoclonal antibody A19-61.1 was evaluated for its ability to bind to four SARS CoV2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS CoV1 spike protein antigen (Figure 1). Binding was confirmed using an ELISA immunoassay, which showed that A19-61.1 binds to SARS CoV2 S2P, S1, and RBD, but not to SARS CoV2 NTD or SARS CoV1 S2P. With the exception of D614G / N439K, which has slightly reduced binding, A19-61.1 maintains or slightly increases binding to all variants tested, including D614G, D614G / Y453F, D614G / 501Y, D614G / del69-70, D614G / del69-70 / N501Y, B.1.1.7, D614G / K471N, D614G / E484K, D614G / K417N / E484K / N501Y, and B.1.351 variants. See Figure 1.

[0404] Epitope mapping: The approximate epitope of A19-61.1 can be determined by assessing how it competes with previously published antibodies and the antibodies disclosed herein. The competitive classification was determined using the biolayer interferometry (BLI) assay described above. The competition profile of A19-61.1 is shown in Figure 4. A19-61.1 binding blocks the binding of S309 and LY-CoV555, and both S309 and LY-CoV555 block A19-61.1 binding. However, the remaining competition profiles differ from these two antibodies. For example, A23-113.1, A19-46.1, and A23-58.1 competed differentially (see below). Negative EM showed that this antibody binds to the RBD on the spike in an RBD-down position, with an epitope close to the spike's triad axis (Figure 4B). Taken together, this indicates that A19-61.1 has a unique binding mode and epitope within the RBD.

[0405] Kinetics of binding to SARS CoV2 S2P protein Using BLI, the Fab protein generated from A19-61.1 was evaluated for binding to SARS CoV2 S2P. The A19-61.1 Fab had an affinity constant (KD) of 2.33 nM, a binding affinity of 3.04 x 10 5 / sec kon, and 7.06 x 10 -4 demonstrated binding to SARS CoV2 S2P protein with a koff of / molar*sec.

[0406] Neutralization A19-61.1 was tested for neutralizing activity in a pseudotyped virus entry assay (see Figure 2). A19-61.1 showed a neutralizing IC 50 (0.0709 μg / mL) and IC 80 (0.1633 μg / mL). Further testing against naturally occurring SARS CoV-2 spike variants in pseudotyping neutralization assays revealed that A19-61.1 is a variant of B.1.1.7 (VOC) containing the following amino acid changes: D614G, N439K / D614G, Y453F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, N501Y / del69-70 / D614G, E484K / D614G, and N501Y / E484K / K417N / D614G, H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H. High efficacy (IC 50 : 0.0020~0.0237ug / mL; IC 80The antibodies maintained neutralization activity (>10 μg / mL) against the E484K / D614G, N501Y / E484K / K417N / D614G, and B.1.351 variants. This contrasts with LY-CoV555 and REGN-10989, which lost neutralization activity against the E484K / D614G, N501Y / E484K / K417N / D614G, and B.1.351 variants (>10 μg / mL). REGN-10933 lost neutralization activity against the Y453F / E614G, N501Y / E484K / K417N / D614G, and B.1.351 variants (>10 μg / mL). CB6 lost neutralization activity against the N501Y / E484K / K417N / D614G and B.1.351 variants (>10 μg / mL). See Figure 1. SARS CoV-2 Nanoluc live virus neutralization IC 50 (0.0022 μg / mL) and IC 80 (0.0134 μg / mL), one of the most potent reported for an antibody targeting SARS CoV-2.

[0407] ACE2 receptor blockade The spike proteins of SARS-CoV1 and SARS-CoV2 target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody binding of spike glycoproteins can be performed using a modified version of the BLI competition assay described above. The competition profile of A19-61.1 ACE2 is shown in Figure 4, which demonstrates that A19-61.1 prevents ACE2 from binding to S2P. Figure 4.

[0408] Example 5 A19-46.1(A19-46.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A19-46.1 is a mAb whose variable domains were isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 41 days after symptom onset. The nucleotide and amino acid sequences of the heavy and light chains of the expressed version of A19-46.1 can be found in Table 3. The heavy and light chain variable immunoglobulin sequences encoding the antigen-binding regions of A19-46.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and lambda light chain. These vectors were used to express antibodies, which were purified using standard methods.

[0409] Table 3. Gene family and nucleotide and amino acid sequences of the heavy and light chains of SARS2.A789-d41-46.1. CDR1, CDR2, and CDR3 are It is bold and underlined. TIFF2024506315000011.tif101156

[0410] Binding to SARS-CoV-2 spike protein The purified monoclonal antibody A19-46.1 was evaluated for its ability to bind to four SARS CoV2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS CoV1 spike protein antigen (Figure 1). Binding was confirmed using an ELISA immunoassay, which showed that A19-46.1 can strongly bind to SARS CoV2 S2P, S1, and RBD, but cannot bind to SARS CoV1 S2P. It also weakly binds to SARS CoV2 NTD. See Figure 1. With the exception of D614G / N439K and D614G / E484K, which had slightly reduced binding, A19-46.1 maintained or slightly increased binding to all variants tested, including D614G, D614G / Y453F, D614G / 501Y, D614G / del69-70, D614G / del69-70 / N501Y, B.1.1.7, D614G / K471N, D614G / K417N / E484K / N501Y, and B.1.351 variants. See Figure 1.

[0411] Epitope mapping The approximate epitope of A19-46.1 can be determined by evaluating its competition with previously published antibodies and the antibodies discovered in this study. The competitive classification was determined using the biolayer interferometry (BLI) assay described above. The competitive profile of A19-46.1 is shown in Figure 5.

[0412] Similarly, A19-46.1, A19-61.1, and A23-80.1 each block the binding of LY-CoV555 but not A23-58.1.

[0413] Negative EM showed that this antibody binds to the RBD on the spike in the RBD-down position, but at a different angle than that of A19-61.1 (Figure 4B). Collectively, this indicates that A19-46.1 has a unique binding mode and epitope within the RBD.

[0414] Kinetics of binding to SARS CoV2 S2P protein Using BLI, the Fab protein generated from A19-46.1 was evaluated for binding to SARS CoV2 S2P. The Fab from A23-58.1 had an affinity constant (KD) of 3.58 nM, 3.79 x 10 5 / sec kon, and 1.35 x 10 -3 koff in / molar*sec, indicating binding to SARS CoV2 S2P protein.

[0415] Neutralization A19-46.1 was tested for neutralizing activity in a pseudotyped virus entry assay (Figure 1). A19-46.1 was found to have a neutralization IC50 (0.0398 μg / mL) and IC80 (0.1287 μg / mL) with SARS-CoV-2 pseudotyped lentiviral particles. Further testing against naturally occurring SARS CoV-2 spike variants in pseudotyping neutralization assays revealed that A19-46.1 is a virulent variant of B.1.1.7 (VOC) containing the following variants: D614G, N439K / D614G, Y453F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, N501Y / del69-70 / D614G, E484K / D614G, and N501Y / E484K / K417N / D614G, as well as B.1.1.7 (VOC) containing amino acid changes H69del-V70del-Y144del-N501Y-A570D-D614G-P681H-T716I-S982A-D1118H. 202012 / 01) and has high efficacy (IC 50 : 0.0149~0.1265ug / mL; IC 80The antibodies maintained neutralization activity (>10 μg / mL) against the E484K / D614G, N501Y / E484K / K417N / D614G, and B.1.351 variants. This contrasts with LY-CoV555 and REGN-10989, which lost neutralization activity against the E484K / D614G, N501Y / E484K / K417N / D614G, and B.1.351 variants (>10 μg / mL). REGN-10933 lost neutralization activity against the Y453F / E614G, N501Y / E484K / K417N / D614G, and B.1.351 variants (>10 μg / mL). CB6 lost neutralization activity against the N501Y / E484K / K417N / D614G and B.1.351 variants (>10 μg / mL). See Figure 1. SARS CoV-2 Nanoluc live virus neutralization IC 50 (0.0048 μg / mL) and IC 80 (0.0535 μg / mL) is one of the most potent reported for an antibody targeting SARS CoV-2.

[0416] ACE2 receptor blockade The SARS-CoV-1 and SARS-CoV-2 spike proteins target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody binding of the spike glycoprotein can be performed using a modified version of the BLI competition assay described above. The competition profile of A19-46.1 ACE2 is shown in Figure 5, which demonstrates that A19-46.1 prevents ACE2 from binding to S2P.

[0417] Example 6 4.5 A23-105.1(A23-105.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A23-105.1 is a mAb whose variable domains were isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 48 days after symptom onset. The nucleotide and amino acid sequences of the heavy and light chains of the expressed version of A23-105.1 can be found in Table 4. The heavy and light chain variable immunoglobulin sequences in Table 4 encoding the antigen-binding regions of A23-105.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and kappa light chain. These vectors were used to express antibodies, which were purified using standard methods.

[0418] Table 4: Gene family of A23-105.1 and nucleotide and amino acid sequences of the heavy and light chains. CDR1, CDR2, and CDR3 are It is bold and underlined. TIFF2024506315000012.tif103156

[0419] Binding to SARS-CoV-2 spike protein The purified monoclonal antibody A23-105.1 was evaluated for its ability to bind to four SARS CoV-2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS CoV-1 spike protein antigen (Figure 1). Binding was confirmed using an ELISA immunoassay, which showed that A23-105.1 could bind to SARS CoV2 S2P, S1, and RBD, but not to SARS CoV2 NTD or SARS CoV1 S2P. This is consistent with flow cytometry probe staining, indicating that the epitope resides in the RBD domain.

[0420] Epitope mapping The approximate epitope of A23-105.1 can be determined by evaluating its competition with previously published antibodies and the antibodies discovered in this study. The competitive classification was determined using the biolayer interferometry (BLI) assay described above.

[0421] The competition profile of A23-105.1 is shown in Figure 6. A23-105.1 competes similarly with LY-CoV555, but does not compete with A19-46.1, A19-61.1, and A23-80.1. Taken together, this indicates that A23-105.1 has a unique binding mode and epitope within the RBD.

[0422] Kinetics of binding to SARS CoV2 S2P protein Using BLI, the Fab protein generated from A23-105.1 was evaluated for binding to SARS CoV2 S2P. The Fab of A23-105.1 had an affinity constant (K) of 2.59 nM. D ), 1.68×10 5 / sec kon, and 4.36 x 10 -4 koff in / molar*sec, indicating binding to SARS CoV2 S2P protein.

[0423] Neutralization A23-105.1 was tested for neutralizing activity in a pseudotyped virus entry assay (Figure 1). A23-105.1 had a neutralizing IC 50 (0.0889 μg / mL) and IC 80 (0.2277 μg / mL) (Figure 1). Neutralization of SARS CoV-2 Nanoluc live virus by A23-105.1 was found to have an IC 50 (0 . 0186μg / mL) and IC 80 (0.06 μg / mL), indicating that this is a highly potent mAb.

[0424] ACE2 receptor blockade The SARS-CoV-1 and SARS-CoV-2 spike proteins target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody binding of the spike glycoprotein can be performed using a modified version of the BLI competition assay described above. The competition profile of A23-105.1 ACE2 is shown in Figure 6, demonstrating that A23-105.1 indeed prevents ACE2 from binding to S2P.

[0425] Example 7 4.6 A19-1.1(A19-1.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A19-1.1 is a mAb whose variable domains were isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 41 days after symptom onset. The nucleotide and amino acid sequences of the heavy and light chains of the expressed version of A19-1.1 can be found in Table 5. The heavy and light chain variable immunoglobulin sequences in Table 5 encoding the antigen-binding regions of A19-1.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and lambda light chain. These vectors were used to express antibodies, which were purified using standard methods.

[0426] Table 5. Gene family of A19-1.1 and nucleotide and amino acid sequences of the heavy and light chains. CDR1, CDR2, and CDR3 are It is bold and underlined. TIFF2024506315000013.tif95156

[0427] Binding to SARS-CoV-2 spike protein Purified monoclonal antibody A19-1.1 was evaluated for its ability to bind to four SARS CoV2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS CoV1 spike protein antigen (Figure 1). Binding was confirmed using an ELISA immunoassay, which showed that A19-1.1 could bind to SARS CoV2 S2P, S1, and RBD, but not to SARS CoV-2 NTD or SARS CoV-1 S2P. This is consistent with flow cytometry probe staining, indicating that the epitope resides in the S1 domain.

[0428] Epitope mapping By assessing how A19-1.1 competes with previously published antibodies and the antibodies discovered in this study, we can determine the approximate epitope. The competitive classification was determined using the biolayer interferometry (BLI) assay described above. The competitive profile of A19-1.1 is shown in Figure 7. Collectively, this indicates that A19-1.1 has a unique binding mode and epitope within the RBD, yet competes with LY-CoV555.

[0429] Neutralization A19-1.1 was tested for neutralizing activity in a pseudotyped virus entry assay (Figure 1). A19-1.1 had a neutralizing IC of 0.01 for SARS-CoV-2 pseudotyped lentiviral particles. 50 (0.1259 μg / mL) and IC 80 (0.5305 μg / mL).

[0430] ACE2 receptor blockade The spike proteins of SARS-CoV1 and SARS-CoV2 target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody binding of spike glycoproteins can be performed using a modified version of the BLI competition assay described above. The competition profile of A19-1.1 ACE2 is shown in Figure 7, demonstrating that A19-1.1 prevents ACE2 from binding to S2P.

[0431] Example 8 4.7 A20-29.1(A20-29.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A20-29.1 is a mAb whose variable domains were isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 50 days after symptom onset. Flow cytometry data suggested this antibody. The nucleotide and amino acid sequences of the heavy and light chains of the expressed version of A20-29.1 can be found in Table 6. The heavy and light chain variable immunoglobulin sequences in Table 6 encoding the antigen-binding region of A20-29.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and kappa light chain. These vectors were used to express the antibody, which was purified using standard methods.

[0432] Table 6: Gene family of A20-29.1 and nucleotide and amino acid sequences of heavy and light chains. CDR1, CDR2, and CDR3 are It is bolded and underlined in red. TIFF2024506315000014.tif104156

[0433] Binding to SARS-CoV-2 spike protein The purified monoclonal antibody A20-29.1 was evaluated for its ability to bind to four SARS-CoV-2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS-CoV1 spike protein antigen (Figure 1). Binding was confirmed using an ELISA immunoassay, demonstrating that A20-29.1 can bind to SARS-CoV2 S2P, S1, and RBD, but not to SARS-CoV2 NTD. This is consistent with flow cytometry probe staining, indicating that the epitope resides in the RBD domain. Furthermore, A20-29.1 binds to SARS-CoV-1 S2P, demonstrating cross-coronavirus binding activity.

[0434] Epitope mapping The approximate epitope of A20-29.1 can be determined by evaluating its competition with previously published antibodies and the antibodies discovered in this study. The competitive classification was determined using the biolayer interferometry (BLI) assay described above.

[0435] The competition profile of A20-29.1 is shown in Figure 8. Negative-stain EM of A20-29.1, A19-46.1, and A19-61.1 confirmed that these antibodies bind to the RBD of the spike in the RBD-down position (Figure 4B). A20-29.1 has a different angle of access to the RBD domain compared to A19-46.1 and A19-61.1. The epitope of A20-29.1 is located at the base of the RBD, while the epitopes of A19-46.1 and A19-61.1 are located on the RBD region closer to the three-fold axis of the spike. Taken together, this indicates that A20-29.1 has a unique binding mode and epitope within the RBD.

[0436] Kinetics of binding to SARS CoV2 S2P protein The Fab protein generated from A20-29.1 was evaluated for binding to SARS CoV2 S2P using the Fab. The Fab of A20-29.1 had an affinity constant (K) of 0.263 nM. D ), 4.8×10 5 / sec kon, and 1.26 x 10 -4 koff in / molar*sec, indicating binding to SARS CoV2 S2P protein.

[0437] Neutralization A20-29.1 was tested for neutralizing activity in a pseudotyped virus entry assay (Figure 1). A20-29.1 had a neutralizing IC 50 (0.3483 μg / mL) and IC 80 Further testing against naturally occurring SARS CoV-2 spike variants in a pseudotyping neutralization assay showed that A20-29.1 had similar potency (IC) against the following variants: D614G, N439K / D614G, Y453F / D614G, A222V / D614G, N501Y / D614G, del69-70 / D614G, N501Y / del69-70 / D614G, E484K / D614G, and N501Y / E484K / K417N / D614G. 50 : 0.1732~0.5792ug / mL; IC 80 This contrasts with LY-CoV555 and REGN-10989, which lost neutralizing activity against the E484K / D614G and N501Y / E484K / K417N / D614G variants (>10 μg / mL), and REGN-10933, which lost neutralizing activity against the Y453F / E614G variant (>10 μg / mL), and with LY-CoV555, CB6, REGN-10989, and REGN-10933, which lost neutralizing activity against the N501Y / E484K / K417N / D614G variant.

[0438] ACE2 receptor blockade The SARS-CoV-1 and SARS-CoV-2 spike proteins target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody binding of the spike glycoprotein can be performed using a modified version of the BLI competition assay described above. The A20-29.1 ACE2 competition profile is shown in Figure 8, which indicates that A20-29.1 does not prevent ACE2 from binding to S2P. Combined with the lack of competition with LY-CoV555 class antibodies, this indicates that A20-29.1 is a highly neutralizing antibody that can be used as a non-competitive partner for antibodies within the LY-CoV555 class.

[0439] Example 9 4.8 A19-30.1(A19-30.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A19-30.1 is a mAb whose variable domain was isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 41 days after symptom onset. Flow cytometry data suggested this antibody. The nucleotide and amino acid sequences of the heavy and light chains of the expressed version of A19-30.1 can be found in Table 7. The heavy and light chain variable immunoglobulin sequences in Table 7 encoding the antigen-binding region of A19-30.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and lambda light chain. These vectors were used to express the antibody, which was purified using standard methods.

[0440] Table 7. Gene family of A19-30.1 and nucleotide and amino acid sequences of heavy and light chains. CDR1, CDR2, and CDR3 are It is bolded and underlined in red. TIFF2024506315000015.tif104156

[0441] Binding to SARS-CoV-2 spike protein The purified monoclonal antibody A19-30.1 was evaluated for its ability to bind to four SARS CoV2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS CoV1 spike protein antigen (Figure 1). Binding was confirmed using an ELISA immunoassay, which showed that A19-30.1 could bind to SARS CoV2 S2P, S1, and RBD, but not to SARS CoV2 NTD or SARS CoV1 S2P, indicating that the epitope resides in the RBD domain.

[0442] Epitope mapping By assessing how A19-30.1 competes with previously published antibodies and the antibodies discovered in this study, we were able to determine its approximate epitope. Using the biolayer interferometry (BLI) assay described above, we determined the competitive classification. The competitive profile of A19-30.1 is shown in Figure 9. Collectively, this indicates that A19-30.1 possesses a unique binding mode and epitope within the RBD.

[0443] Kinetics of binding to SARS CoV2 S2P protein Using BLI, the Fab protein generated from A19-30.1 was evaluated for binding to SARS CoV-2 S2P. The Fab of A19-30.1 had an affinity constant (K) of 0.916 nM. D ), 7.62 x 10 4 / sec kon, and 6.95 x 10 -5 koff in / molar*sec, indicating binding to SARS CoV2 S2P protein.

[0444] Neutralization A19-30.1 was tested for neutralizing activity in a pseudotyped virus entry assay (Figure 1). A19-30.1 was found to be non-neutralizing against SARS CoV2 pseudotyped lentiviral particles. This suggests that A19-30.1 acts via antibody Fc-dependent mechanisms, such as antibody-dependent cellular cytotoxicity, antibody-dependent phagocytosis, and / or antibody-dependent complement-mediated killing, to kill infected cells or directly lyse viral particles.

[0445] ACE2 receptor blockade The spike proteins of SARS-CoV1 and SARS-CoV2 target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody-bound spike glycoproteins can be performed using a modified version of the BLI competition assay described above. The A19-30.1 ACE2 competition profile is shown in Figure 9, which indicates that A19-30.1 does not prevent ACE2 from binding to S2P. Combined with the lack of competition with LY-CoV555 class antibodies, this indicates that A19-30.1 is a potent neutralizing antibody that can be used as a non-competitive partner for antibodies within the LY-CoV555 class.

[0446] Example 10 4.9 A20-36.1(A20-36.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A20-36.1 is a mAb whose variable domains were isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV-2 survivor 50 days after symptom onset. The nucleotide and amino acid sequences of the heavy and light chains of the expression version of A20-36.1 can be found in Table 8. The heavy and light chain variable immunoglobulin sequences in Table 8 encoding the antigen-binding regions of A20-36.1 were synthesized and cloned into immunoglobulin expression vectors containing human constant regions for the IgG1 heavy chain and lambda light chain. These vectors were used to express antibodies, which were purified using standard methods.

[0447] Table 8: Gene family of A20-36.1 and nucleotide and amino acid sequences of heavy and light chains. CDR1, CDR2, and CDR3 are It is bold and underlined. TIFF2024506315000016.tif115156

[0448] Binding to SARS-CoV-2 spike protein The purified monoclonal antibody A20-36.1 was evaluated for its ability to bind to four SARS-CoV-2 spike-derived protein antigens (S2P, S1, RBD, and NTD) and the S2P SARS-CoV-1 spike protein antigen (Figure 1). Binding was confirmed using an ELISA immunoassay, demonstrating that A20-36.1 can bind to SARS-CoV-2 S2P and S1 but not to SARS-CoV-2 RBD or NTD. A follow-up mapping ELISA examining the SD1 and SD2 domains of S1 suggested that the antibody targets a region within SD2 of S1. This data is consistent with the flow cytometry probe staining. Furthermore, the antibody was shown to bind to SARS-CoV-1 S2P.

[0449] Epitope mapping By evaluating how A20-36.1 competes with previously published antibodies and the antibodies disclosed herein, the approximate epitope can be determined. The above-mentioned biolayer interferometry (BLI) assay was used to determine the competitive classification. The competitive profile of A20-36.1 is shown in Figure 10. Taken together, this indicates that A20-36.1 has a unique binding mode and epitope within the SD2 region of S1.

[0450] Kinetics of binding to SARS CoV2 S2P protein Using BLI, the Fab protein generated from A20-36.1 was evaluated for binding to SARS CoV2 S2P. The Fab from A23-58.1 had an affinity constant (K) of 1.82 nM. D ), 5.99 x 10 5 / sec kon, and 1.09 x 10 -3 koff in / molar*sec, indicating binding to SARS CoV2 S2P protein.

[0451] Neutralization A20-36.1 was tested for neutralizing activity in a pseudotyped virus entry assay (see Table 4.9.2-1). A20-36.1 had a neutralizing IC of 1.000 for SARS CoV2 pseudotyped lentiviral particles. 50 (1.3851 μg / mL) and IC 80 (3.7620 μg / mL).

[0452] ACE2 receptor blockade The spike proteins of SARS-CoV1 and SARS-CoV2 target expressing cells by binding to ACE2 via the receptor-binding domain on the spike protein. Given the critical requirement for ACE2 binding in the viral life cycle, this vulnerability is exploited by several classes of antibodies to neutralize infection. Inhibition of ACE2 binding by antibody binding of the spike glycoprotein can be performed using a modified version of the BLI competition assay described above. The A20-36.1 ACE2 competition profile is shown in Figure 10, which indicates that A20-36.1 does not prevent ACE2 from binding to S2P. Combined with the lack of competition with LY-CoV555 class antibodies, this indicates that A20-36.1 is a neutralizing antibody that can be used as a non-competitive partner for antibodies within the LY-CoV555 class.

[0453] Example 11 4.10 A23-97.1(A23-97.1) Identifying coronavirus antibodies using FACS probe sorting and SMRTseq sequencing A23-97.1 is a mAb whose variable domains were isolated through pairing of heavy and light chain immunoglobulin genes from single memory B cells obtained from a SARS CoV2 survivor 48 days after symptom onset. Flow cytometry data suggested this antibody. The nucleotide and amino acid sequences of the heavy and light chains of the expressed version of A23-97.1 can be found in Table 9. The heavy and light chain variable immunoglobulin sequences in T...

Claims

1. a) V, as set forth as SEQ ID NOs: 1 and 5, respectively H and V L heavy chain variable (VV) including heavy chain complementarity determining regions (HCDR) 1, HCDR2, and HCDR3, and light chain complementarity determining regions (LCDR) 1, LCDR2, and LCDR3 H ) region and the light chain variable region (V L ). b) V, as set forth as SEQ ID NOs: 9 and 13, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; c) V, as set forth as SEQ ID NOs: 17 and 21, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; d) V, as set forth as SEQ ID NOs: 25 and 29, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; e) V, as set forth as SEQ ID NOs: 33 and 37, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; f) V, as set forth as SEQ ID NOs: 41 and 45, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; g) V, as set forth as SEQ ID NOs: 49 and 53, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; h) V, as set forth as SEQ ID NOs: 57 and 61, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; i) V, as set forth as SEQ ID NOs: 65 and 69, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; j) V, as set forth in SEQ ID NOs: 73 and 77, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L , k) V as set forth in SEQ ID NO: 81 and 85 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; l) V as set forth in SEQ ID NO: 89 and 93 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; m) V as set forth in SEQ ID NO: 97 and 101 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ; n) V as set forth in SEQ ID NO: 105 and 109 H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L ;or o) V, as set forth as SEQ ID NOs: 143 and 5, respectively H and V L V, including HCDR1, HCDR2, and HCDR3, and LCDR1, LCDR2, and LCDR3 H and V L 1. An isolated monoclonal antibody or antigen-binding fragment thereof comprising: An isolated monoclonal antibody or an antigen-binding fragment thereof, wherein the monoclonal antibody specifically binds to the coronavirus spike protein and neutralizes SARS-CoV-2.

2. a) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 2, 3, 4, 6, 7, and 8, respectively; b) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 10, 11, 12, 14, 15, and 16, respectively; c) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 18, 19, 20, 22, 23, and 24, respectively; d) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 26, 27, 28, 30, 31, and 32, respectively; e) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 34, 35, 36, 38, 39, and 40, respectively; f) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 42, 43, 44, 46, 47, and 48, respectively; g) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 50, 51, 52, 54, 55, and 56, respectively; h) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 58, 59, 60, 62, 63, and 64, respectively; i) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 66, 67, 68, 70, 71, and 72, respectively; j) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 74, 75, 76, 78, 79, and 80, respectively; k) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 82, 83, 84, 86, 87, and 88, respectively; l) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs:90, 91, 92, 94, 95, and 96, respectively; m) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 98, 99, 100, 102, 103, and 104, respectively; n) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 106, 107, 108, 110, 111, and 112, respectively; or o) the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3 comprise the amino acid sequences set forth as SEQ ID NOs: 2, 3, 58, 6, 7, and 8, respectively; 2. The antibody or antigen-binding fragment of claim 1.

3. a) The above V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively; b) The above V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs:9 and 13, respectively; c) Said V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 17 and 21, respectively; d) The above V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 25 and 29, respectively; e) The above V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 33 and 37, respectively; f) Said V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 41 and 45, respectively; g) The V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 49 and 53, respectively; h) the V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs:57 and 61, respectively; i) The above V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 65 and 69, respectively; j) The above V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs:73 and 77, respectively; k) The above V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 81 and 85, respectively; l) Said V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 89 and 93, respectively; m) Said V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs:97 and 101, respectively; n) the V H and the above V L comprises an amino acid sequence that is at least 90% identical to the amino acid sequence set forth as SEQ ID NOs: 105 and 109, respectively; or o) The above V H and the above V L comprising an amino acid sequence that is at least 90% identical to the amino acid sequences set forth as SEQ ID NOs: 143 and 5, respectively; 3. An antibody or antigen-binding fragment according to claim 1 or claim 2.

4. 4. The antibody or antigen-binding fragment of any one of claims 1 to 3, comprising a human framework region.

5. a) The above V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively; b) The above V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:9 and 13, respectively; c) Said V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively; d) The above V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:25 and 29, respectively; e) The above V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:33 and 37, respectively; f) Said V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs: 41 and 45, respectively; g) The V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs: 49 and 53, respectively; h) the V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:57 and 61, respectively; i) The above V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:65 and 69, respectively; j) The above V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:73 and 77, respectively; k) The above V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:81 and 85, respectively; l) Said V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs: 89 and 93, respectively; m) Said V H and the above V L comprises the amino acid sequences set forth as SEQ ID NOs:97 and 101, respectively; n) the V H and the above V L comprises the amino acid sequence set forth as SEQ ID NOs: 105 and 109, respectively; or o) The above V H and the above V L comprising the amino acid sequences set forth as SEQ ID NOs: 143 and 5, respectively; An antibody or antigen-binding fragment according to any one of claims 1 to 4.

6. The antibody of any one of claims 1 to 5, comprising a human constant domain.

7. The antibody of any one of claims 1 to 6, which is a human antibody.

8. The antibody of any one of claims 1 to 7, which is IgA.

9. The antibody of any one of claims 6 to 8, comprising a recombinant constant domain comprising a modification that increases the half-life of the antibody.

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

11. The antibody or antigen-binding fragment of any one of claims 1 to 10, wherein the antibody specifically binds to the N-terminal domain of coronavirus spike protein.

12. The antibody or antigen-binding fragment of any one of claims 1 to 11, wherein the antibody specifically binds to the receptor binding domain (RBD) of coronavirus spike protein.

13. The antibody or antigen-binding fragment of any one of claims 1 to 12, wherein the antibody neutralizes SARS-CoV-1.

14. An antigen-binding fragment according to any one of claims 1 to 5 or 11 to 13.

15. Fv, Fab, F(ab') 2 , scFV, or scFV 2 15. The antigen-binding fragment of claim 14, which is a fragment.

16. The antibody or antigen-binding fragment of any one of claims 1 to 15, conjugated to a detectable marker.

17. A bispecific antibody comprising an antibody or antigen-binding fragment according to any one of claims 1 to 16.

18. 18. The bispecific antibody of claim 17, which is a dual variable domain immunoglobulin.

19. The antibody or antigen-binding fragment according to any one of claims 1 to 15, H Or V L 19. An isolated nucleic acid molecule encoding an antigen-binding fragment or a dual variable domain immunoglobulin according to claim 18.

20. The above V H or V L 20. The nucleic acid molecule of claim 19, which is a cDNA sequence encoding

21. 21. The nucleic acid molecule of claim 19 or claim 20, operably linked to a promoter.

22. A vector comprising the nucleic acid molecule of any one of claims 19 to 21.

23. A host cell comprising the nucleic acid molecule or vector of any one of claims 19 to 22.

24. an effective amount of the antibody, antigen-binding fragment, bispecific antibody, nucleic acid molecule, or vector of any one of claims 1 to 22; A pharma- ceutically acceptable carrier; 23. A pharmaceutical composition for use in inhibiting SARS-CoV-1 or SARS-CoV-2 infection comprising:

25. 1. A method for producing an antibody or antigen-binding fragment that specifically binds to a SARS-CoV-2 spike protein, comprising the steps of: Expressing in a host cell one or more nucleic acid molecules encoding the antibody or antigen-binding fragment of any one of claims 1 to 16; and Purifying the antibody or antigen-binding fragment.

26. 1. A method for detecting the presence of coronavirus in a biological sample from a subject, comprising the steps of: contacting the biological sample with an effective amount of the antibody or antigen-binding fragment of any one of claims 1 to 16 under conditions sufficient to form an immune complex; and Detecting the presence of said immune complex in said biological sample, wherein the presence of said immune complex in said biological sample indicates the presence of said coronavirus in said sample.

27. 27. The method of claim 26, wherein detecting the presence of an immune complex in the biological sample indicates that the subject has a SARS-CoV-2 infection.

28. A pharmaceutical composition comprising the antibody, antigen-binding fragment, nucleic acid molecule, or vector of any one of claims 1 to 22 for inhibiting a coronavirus infection in a subject, wherein the subject has or is at risk of a coronavirus infection.

29. 29. The pharmaceutical composition of claim 28, wherein the coronavirus is SARS-CoV-2.

30. 28. The method of claim 27, wherein the SARS-CoV-2 is the B.1.1.529 variant.

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