WO2021212049A2 - Anticorps monoclonaux anti-sars-cov-2 - Google Patents

Anticorps monoclonaux anti-sars-cov-2 Download PDF

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WO2021212049A2
WO2021212049A2 PCT/US2021/027793 US2021027793W WO2021212049A2 WO 2021212049 A2 WO2021212049 A2 WO 2021212049A2 US 2021027793 W US2021027793 W US 2021027793W WO 2021212049 A2 WO2021212049 A2 WO 2021212049A2
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seq
amino acid
acid sequence
antibody
cdr
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WO2021212049A3 (fr
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Ali ELLEBEDY
Aaron SCHMITZ
Jackson TURNER
Wafaa AL SOUSSI
Michael Diamond
Daved FREMONT
James Brett CASE
Haiyan Zhao
John ERRICO
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Washington University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the present invention relates to antibodies or antigen-binding fragments that are useful for treating infections caused by coronaviruses (e.g., SARS-CoV-2).
  • coronaviruses e.g., SARS-CoV-2
  • the present invention also relates to various pharmaceutical compositions and methods of treating coronavirus infections (e.g., COVID-19) using the antibodies or antigen-binding fragments.
  • SARS-CoV severe acute respiratory syndrome coronavirus
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS-CoV-2 Compared to SARS-CoV and MERS-CoV, SARS-CoV-2 appears to be more readily transmitted from human-to-human, spreading to multiple continents and leading to the WHO declaration of a global pandemic on March 11, 2020.
  • SARS-CoV-2 is a spherical, enveloped virion with a diameter of approximately 90 nm.
  • the virion surface is decorated by the spike protein (S), which binds to human angiotensin-converting enzyme 2 (ACE2) on host cell surfaces, and mediates viral entry.
  • Spike monomers consist of S1 and S2 subunits, which form homotrimers.
  • the S1 subunit is made up of 4 sub-domains, S1 A through S1 D .
  • S1 B encodes the receptor-binding domain (RBD), which directly interacts with ACE2.
  • the S2 subunit contains a fusion peptide, which mediates fusion with host cell membranes after receptor binding by S1.
  • the RBD is observed to have two distinct conformations, termed “up” and “down”, with the "up” position thought to be required for ACE2 binding.
  • anti-RBD antibodies confer protection in animal models of SARS-CoV-2 infection, highlighting their potential use as therapeutic agents.
  • SARS-CoV-2 can rapidly generate mutations in critical epitopes on S, which could render clinical interventions ineffective. Indeed, a database of over 106,000 SARS-CoV-2 sequences isolated from humans reports over 400 amino acid substitutions in the RBD of S alone. Understanding antibody responses to SARS-CoV-2 at the molecular level is critical to predicting vaccine efficacy and designing potent, durable antibody-based therapeutics. Recently, a panel of anti-SARS-CoV-2 antibodies was generated by immunizing mice with recombinant RBD and boosting with S trimers.
  • antibodies or antigen-binding fragments thereof comprise: (a) an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 1–57 and 115–133; or (b) an immunoglobulin light chain variable region comprising an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 58–114 and 134–152.
  • Further aspects of the present invention relate to nucleic acids comprising a nucleotide sequence encoding an immunoglobulin light chain variable region and/or an immunoglobulin heavy chain variable region of any antibody or antigen-binding fragment as described herein.
  • aspects of the present invention relate to expression vectors comprising the nucleic acids, host cells comprising the expression vectors as well as methods of producing the antibodies and antigen-binding fragments thereof as described herein.
  • the vaccines comprise a polypeptide comprising an amino acid sequence comprising at least about 70% identity to an epitope targeted by any antibody or antigen- binding fragment thereof described herein.
  • Further aspects relate to various pharmaceutical compositions comprising any of the antibodies or antigen-binding fragments thereof as described herein.
  • Additional aspects of the present invention relate to methods of preventing or treating a coronavirus infection in a subject in need thereof.
  • the methods comprise administering to the subject any antibody or antigen-binding fragment thereof as described herein, any nucleic acid comprising a nucleotide sequence encoding at least a portion of an antibody or antigen-binding fragment thereof as described herein, any expression vector as described herein, any vaccine as described herein, or any composition comprising at least one of the antibodies or antigen-binding fragments thereof described herein.
  • an antibody or antigen-binding fragment that binds to a coronavirus spike protein at an epitope comprising at least one residue selected from the group consisting of R346, Y351, R403, D405, E406, R408, Q409, T415, G416, K417, D420, Y421, K444, V445, G446, G447, N448, Y449, N450, L452, Y453, L455, F456, R457, K458, S459, N460, T470, I472, Y473, Q474, A475, G476, S477, T478, G482, V483, E484, G485, F486, N487, C488, Y489, F490, L492, Q493, S494, Y495, G496, Q498, T500, N501, G502, V503, G504, Y505 and/or at least one residue selected from the group consisting of T333, N334,
  • compositions for preventing or treating a coronavirus infection comprising a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment, wherein the first antibody or antigen-binding fragment binds to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein and/or sterically blocks ACE2 binding, and wherein the second antibody or antigen-binding fragment does not bind to the ACE2 receptor binding domain of the SARS- CoV-2 spike protein, does not sterically block ACE2 binding, and/or binds adjacent to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein.
  • a method for isolating an escape mutant of a SARS coronavirus comprising: a) expressing a SARS-CoV-2 chimeric vesicular stomatitis virus (VSV-SARS-CoV-2) comprising a receptor binding domain of a SARS-CoV- 2 virus; b) applying a neutralizing antibody having an affinity for an epitope on the RBD to the virus; c) incubating in replication competent conditions for a period of time; and d) isolating the escape mutant, wherein the escape mutant comprises at least one mutation relative to the wild-type.
  • An assay is also provided for evaluating an antibody as described herein.
  • a method for generating an antibody selective for an escape mutant isolated according to the methods herein comprising either: a) immunizing an animal with the escape mutant and isolating the antibody, or b) recombinantly expressing the antibody by transfecting a recombinant organism with a nucleic acid encoding an amino acid sequence of the antibody, wherein the amino acid sequence comprises at least one mutation relative to a reference antibody, the mutation increasing the affinity of the antibody to the escape mutant as compared to the reference antibody.
  • an isolated nucleic acid comprising a nucleotide sequence encoding an antibody or antigen binding fragment described herein.
  • FIGS. 1A-1H show the immunization regimen used to generate antibodies, as described herein.
  • FIG.1A Mice were immunized intramuscularly (i.m.) with SARS-2 RBD (10 ⁇ g) in Addavax and then boosted twice with recombinant spike protein (5 ⁇ g) at the indicated time points post-vaccination. Serum and draining LNs were harvested 5 days after the final immunization.
  • FIG.1B IgG serum Ab binding to SARS-2 spike (left panel) and RBD (right panel), measured by enzyme-linked immunosorbent assay (ELISA). Serum from a PBS mouse was used as a negative control. Each curve represents the binding profile from one mouse.
  • FIG.1C Neutralization titers in serum of immunized mice, measured by microneutralization assay against SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020.
  • FIG. 1D Representative gating of total PBs and RBD+ PBs within the PB population in dLN. Cells pregated CD38loCD138+IgDloFas+CD19+CD4- live singlet lymphocytes. Total PBs were bulk-sorted for single-cell RNA sequencing, and RBD+ PBs were single-cell sorted for mAb cloning.
  • FIG.1C Neutralization titers in serum of immunized mice, measured by microneutralization assay against SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020.
  • FIG. 1D Representative gating of total PBs and RBD+ PBs within the PB population in dLN. Cells pregated CD38loCD138+IgDl
  • FIG.1E Bar graph represents binding of the 34 recombinant humanized mAbs derived from the immunized mice RBD+ PBs to mammalian SARS-2 RBD, measure by ELISA.
  • FIG.2 depicts the plasmablast gating strategy. Plasmablasts were defined as live singlet CD19 + CD4- IgDlo Fas + CD38lo CD138+ lymphocytes.
  • FIGS. 3A-3D depict data associated with experiments to test cross-reactivity and neutralization of the anti-RBD mAbs generated in Example 2. Bar graphs represent the minimum positive concentrations of anti-RBD mAbs to either SARS-2 RBD (FIG.3A), SARS-1 RBD (FIG.3B), or MERS RBD (FIG.3C), measured by ELISA.
  • FIG.4A SARS- CoV- 2
  • FIG. 4B SARS-CoV
  • FIG.4C MERS-CoV
  • FIG.4D Microneutralization assay of clonally distinct anti- RBD mAbs against SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020. Representative of 2 (1B10) or 3 (all other mAbs) independent experiments.
  • FIG.4E Bilayer interferometry traces of mAb competition for ACE2 binding to SARS- CoV-2 RBD.
  • FIGS. 5A-5D depict data associated with sorted plasmablasts.
  • FIG.5A Gene expression-based clustering visualized via t-distributed stochastic neighbor embedding.
  • FIG. 5B Plasmablasts found in clones containing RBD + and RBD- mAbs.
  • FIG.5C Isotypes of plasmablasts found in clones containing RBD + mAbs.
  • FIGS. 6A-6D depict clonal and transcriptional characterization of plasmablasts.
  • FIG.6A Distance-to-nearest-neighbor plots for choosing a distance threshold for inferring clones via hierarchical clustering.
  • FIG.6B Dot plot showing the average log-normalized expression of genes used for defining the t-SNE clusters (FIG.5A) and the fraction of cells expressing each gene in each cluster.
  • FIG.6C Distribution of plasmablasts found in clones containing RBD + mAbs on the tSNE plot.
  • FIG.6D IGHV mutation frequency of plasmablasts of the indicated isotype found in clones containing RBD+ and RBD- mAbs. P-values from two-sided Mann-Whitney.
  • FIGS. 7A-7B depict in vivo protection by mAb 2B04.
  • FIG.7A SARS-CoV- 2 challenge model.
  • BALB/c mice received ⁇ IFNAR1 mAb i.p.24 hours prior to i.n. administration of AdV-hACE2.
  • Mice received mAb 2B04 or isotype i.p.4 days later, followed by i.n.
  • FIGS. 8A-8D depict anti-SARS-CoV-2 mAbs 2B04 and 2H04 binding to the RBD of the trimeric spike protein.
  • FIG.8A 2B04 up/down/down (U/D/D) density map (top panel).
  • the S1/S2 portion of the spike excluding the RBD is shown, with the 'Up' RBD subunit shown as well.2B04 heavy chain is shown, with the light chain shown as well.
  • Pie charts in the bottom panels of FIG.8A and FIG.8B represent distribution of particles belonging to the two 2B04 or two 2H04 maps, respectively.
  • FIG.8C 2H04 down/down/down (D/D) density map.
  • the Fab and RBD portions of the maps are contoured at a higher level than the core S1/S2 portions of the map for FIG.8A, FIG.8B, and FIG.8C.
  • FIGS. 9A-9H depict cryo-EM validation for 2B04 and 2H04 maps.
  • FIG.9A Example micrographs and CTF estimates for 2B04-spike datasets imaged on holey lacey carbon grids or lacey carbon grids with ultra-thin carbon film.
  • FIG.9B Example micrographs and CTF estimates for 2H04-spike datasets imaged with holey lacey carbon grids or lacey carbon grids with ultra-thin carbon film.
  • FIG.9C Particle orientation distribution for 2B04- spike up/down/down reconstruction.
  • FIG.9D Particle orientation distribution for 2H04-spike down/down/down reconstruction.
  • FIG.9E GSFSC curve for 2B04-spike up/down/down refinement.
  • FIG.9F GSFSC curve for 2H04-spike down/down/down refinement.
  • FIG.9G Local resolution map for 2B04-spike up/down/down map.
  • FIG.9H Local resolution map for 2H04-spike down/down/down map.
  • FIGS. 10A-10B depict the cryo-EM processing strategy.
  • FIG.10A Flowchart depicting cryo-EM data processing steps for the 2B04-spike up/down/down and 2B04/RBD locally refined maps.
  • FIG.10B Flowchart depicting cryo-EM data processing steps for the 2H04-spike down/down/down and 2H04/RBD locally refined maps.
  • FIGS. 11A-11D depict 2B04 and 2H04 binding to the RBD of the trimeric spike protein.
  • FIG.11A Density map for 2B04 U/D/D spike reconstruction shown as an outline, with the model shown internally as a cartoon. S1/S2 with the RBD portion and 2B04 are shown.
  • FIG.11B Density map for 2B04 U/U/D spike reconstruction shown as an outline, with the model shown internally as a cartoon and the same components as FIG. 11A.
  • FIG. 11C Density map for 2H04 D/D/D spike reconstruction shown as an outline, with the model shown internally as a cartoon.
  • S1/S2 with the RBD portion and 2H04 are shown.
  • FIG. 11D Density map for 2H04 U/D/D spike reconstruction shown as an outline, with the model shown internally as a cartoon and the same components as in FIG.11C.
  • FIGS. 12A-12D depict validation for locally refined maps.
  • FIG.12C Example density and model fits for an RBD beta-strand (left) and at the 2B04/RBD interface (right).
  • FIG.12D Example density and model fits for an RBD beta-strand (left) and at the 2H04/RBD interface (right).
  • FIGS. 13A-13F depict how 2B04 and 2H04 target distinct epitopes on the RBD.
  • Complementarity-determining regions (CDRs) of 2B04 are shown as cartoon ribbons, with the heavy chain and the light chain included.
  • the RBM is shown as a transparent surface with underlying ribbon diagram.
  • FIG.13C 2H04 targets an epitope adjacent to the RBM on the SARS-CoV-2 RBD, distal from the RBM loop.
  • CDR regions of 2H04 are shown as cartoon ribbons, with the heavy chain and the light chain included.
  • FIG.13E Alignment of 2B04 and 2H04 models to the 2B04 RBD.2B04 contacts are included as in FIG.13A, and 2H04 contacts are included as in FIG. 13C.
  • N450 the only shared contact between 2B04 and 2H04, is shown on the RBD.
  • FIG.13F BLI analysis of RBD mutants to 2B04, 2H04, and ACE2. Results represent mean ( ⁇ standard deviation, SD) from two independent experiments.
  • FIG.14 depicts how 2H04 contacts the core fucose of the N343 glycan on SARS-CoV-2 RBD.2H04 is depicted as a surface, with the heavy chain and the light chain included.
  • FIGS. 15A-15C depict how 2B04 and 2H04 share epitopes with human antibodies.
  • FIG.15A Comparison of human antibodies that target the RBM of the SARS- CoV-2 RBD and 2B04. mAb Groups were formed based on overlap between their interfacial residues as determined by PISA analysis.
  • FIG.15B Comparison of human antibodies that target non-RBM epitopes of the SARS-CoV-2 RBD with 2H04.
  • FIG.15C Multiple sequence alignment of the SARS-CoV-2 RBD (residues 333-527) with binding footprints highlighted for human antibodies that are predicted to compete with 2B04 or 2H04 based on structural analysis.
  • SARS- CoV RBD is shown at the bottom, with substitutions relative to SARS-CoV-2 RBD highlighted.
  • SARS-CoV-2 RBD contacts with ACE2 are identified by stars at the bottom of the alignment.
  • FIGS. 16A-16D depict binding affinity of 2B04 and 2H04 to mammalian cell- derived SARS-CoV-2 RBD.
  • FIG.16A Binding of 2B04 to mammalian cell-derived SARS- CoV-2 RBD.
  • FIG.16B Binding of 2H04 to mammalian cell-derived SARS-CoV-2 RBD.
  • FIG.16C Binding of 2H04 to bacterially derived SARS-CoV RBD.
  • FIG.16D Binding of 2B04 to bacterially derived SARS-CoV RBD. Kinetic values were fitted to a 1:1 Langmuir binding model (K D , kinetic). Steady-state analysis is shown below kinetic plots (K D , equilibrium) with inset Scatchard plots. Data were analyzed using Biaevaluation 3.1. One representative trace of two or three independent experiments is shown. [0041] FIGS. 17A-17F depict how SARS-CoV-2 can escape neutralization by 2B04 and 2H04. FIG.17A Logo plot depicting escape mutants generated by selection pressure from 2B04 or 2H04 using a chimeric VSV expressing SARS-CoV-2 spike. FIG.
  • FIGS. 17B Identity and frequency of mutations seen in clinical isolates of SARS-CoV-2 at identical positions as the VSV-SARS-CoV-2 escape mutants.
  • the right axis shows the number of total sequences with mutations at each residue position, out of a total 106,606 sequences.
  • Logo plots were generated using dmslogo (Bloom lab).
  • FIG.17C RBD with ACE2 contacts and 2H04 escape mutants.
  • FIG.17D Ribbon diagram showing escape mutants at the binding interface of 2H04 on the RBD.
  • FIG. 17F Ribbon diagram showing escape mutants at the binding interface of 2B04 on the RBD.
  • FIG.18A Structural alignment of 2B04/RBD with ACE2/RBD complex (PDB 6M0J). RBD is displayed as a surface model, and 2B04 and ACE2 are also shown.
  • FIG.18B Structural alignment of 2H04/RBD with ACE2/RBD complex (PDB 6M0J). RBD, 2H04, and ACE2 are shown. Binding affinity of 2B04 (FIG.18C) and 2H04 (FIG.18D) for SARS- CoV-2 RBD to bacterially-derived SARS-CoV-2 RBD.
  • FIG.18E BLI traces of competitive binding of 2B04, 2H04 and ACE2 against SARS-CoV-2 RBD. mAbs were loaded onto anti-human IgG Fc biosensors, and recombinant RBD was captured, followed by either 2H04 Fab, 2B04 Fab, or monomeric hACE2.
  • FIG.18F Pre- and post- attachment inhibition assays.
  • FIG.18G Attachment blockade assay with authentic SARS-CoV-2. The fold change of viral RNA on cell surfaces was measured by qRT-PCR and compared to control cells infected with untreated virus. hE16 (anti-West Nile virus) was used as an isotype control.
  • FIG.18H Cartoon depicting different SARS-CoV-2 spike configurations. Spike trimers with three RBDs in the 'down' configuration cannot bind ACE2, whereas spike configurations with at least one RBD in the 'up' configuration can bind ACE2.
  • FIG.18I Cartoon model of SARS- CoV-2 neutralization by 2B04 and 2H04.2H04 binds to SARS-CoV-2 virions and inhibits viral accumulation at cellular surfaces, possibly by preventing heparan sulfate or other attachment factors from interacting with the RBD. 2B04 neutralizes by binding to the RBM, directly preventing RBD binding to ACE2, which is required for cellular entry.
  • Data in panel G are the mean of three independent experiments performed in duplicate. Error bars indicate SD.
  • FIGS. 19A-19D depict a comparison of 2B04 and 2H04 to germline Ig sequences.
  • FIG. 19A Sequence alignment of 2B04 VH to IgH2-9-1 (2B04 VH parent germline sequence) and IgHV3-53*02 (germline sequence of human antibodies targeting an overlapping RBM epitope). Residues are colored from black to white according to degree of conservation.2B04 contact residues on the SARS-CoV-2 RBD are shown as triangles.
  • FIG. 19A Sequence alignment of 2B04 VH to IgH2-9-1 (2B04 VH parent germline sequence) and IgHV3-53*02 (germline sequence of human antibodies targeting an overlapping RBM epitope). Residues are colored from black to white according to degree of conservation.2B04 contact residues on the SARS-CoV-2 RBD are shown as triangles.
  • FIG. 19A Sequence alignment of 2B04 VH to IgH2-9-1 (2B04 VH parent germline sequence
  • FIG.19A Sequence alignment of 2B04 VL to IgVL*01 (2B04 VL parent germline sequence), colored as in FIG.19A.
  • FIG.19C Sequence alignment of 2H04 VH to IgHV1-55*01 (2H04 VH parent germline sequence). Residues are colored from black to white according to degree of conservation. 2H04 contact residues on the SARS-CoV-2 RBD are shown as triangles.
  • FIG.19D Sequence alignment of 2H04 V L to IgKV5-48*01, colored as in FIG.19C.
  • DETAILED DESCRIPTION [0044] Aspects of the present invention relates to various antibodies and antigen- binding fragments thereof that show specificity to coronaviruses.
  • Antibodies and antigen- binding fragments thereof described herein can neutralize the virus.
  • the antibodies and antigen-binding fragments can comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 1–57 and 115–133; or an immunoglobulin light chain variable region comprising an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 58–114 and 134–152.
  • Specific light and heavy chains of various antibodies and antigen- binding fragments are described in more detail herein.
  • An additional aspect of the invention is a method of generating and isolating "escape mutants" of a coronavirus where the escape mutants comprise variants of the virus against which established antibodies to the coronavirus have reduced affinity. Methods of using these escape mutants to generate further antibodies to target them are also provided.
  • Coronavirus Specificity and Antibody Properties [0045] Applicants have discovered highly active antibodies that show high specificity for human coronaviruses (e.g., SARS-CoV-2). Accordingly, in various embodiments, the antibody or antigen-binding fragment thereof can selectively bind to a coronavirus.
  • the antibodies and antigen-binding fragments described herein can have important applications, for both therapeutic and prophylactic treatment of coronavirus infections (e.g., COVID-19).
  • coronavirus infections e.g., COVID-19.
  • mAbs were synthesized that are clonally related and bind coronaviruses (e.g., SARS CoV-2). These antibodies are highly active neutralizers of coronavirus (e.g., SARS CoV-2) in vitro and provide broad protection from mortality and morbidity in vivo.
  • SARS CoV-2 coronavirus
  • the discovery of these mAbs raises the hope that similar antibodies can be induced in the population if the right vaccination regimen is given. Knowledge about the binding mode and epitope of these mAbs may then guide the development of universal COVID-19 vaccines.
  • IgG antibody The general structure of an IgG antibody is well known. Briefly, there are two major subunits: the heavy chain and the light chain connected via disulfide bonds. Each heavy chain and light chain is further divided into a variable or a constant region. The variable regions interacts most directly with the antigen and further comprise three hyper variable regions (complementary determining domains, CDRs). Thus, a single antibody comprising two heavy chains and two light chains comprises a total of twelve CDRs (three for each heavy chain and each light chain). However, each of the variable regions, particularly the CDRs, possess some degree of affinity for the antigen and maximum affinity can be achieved with a single heavy chain coupled to a single light chain.
  • variable region of the antibody (both the heavy and light chains) is collectively known as the Fab fragment and can be cleaved from the constant region (known as the Fc portion) to form an antigen- binding fragment.
  • each of the CDRs possess some degree of affinity for the antigen, and can each be considered an antigen-binding fragment.
  • An antibody fragment can have an equivalent binding affinity for the target as the parent antibody. Both divalent and monovalent antibody fragments are included in the present invention.
  • the antibody or antibody binding fragment comprises a heavy chain variable region (or fragment thereof) and/or a light chain variable region (or fragment thereof).
  • the heavy chain variable region comprises three complementary defining regions (CDRs) classified as CDR H1 , CDR H2 , and CDR H3 .
  • the light chain variable region comprises three complementarity determining regions (CDRs) classified as CDR L1 , CDR L2 , and CDR L3 .
  • CDRs complementary defining regions
  • CDRs complementarity determining regions
  • Table 1 Illustrative CDR Sequences for Anti-SARS-CoV-2 antibodies
  • the CDRs are spaced out along the light and heavy chains and are flanked by four relatively conserved regions known as framework regions (FRs).
  • the heavy chain variable region comprises four framework regions (FRs) classified as FR H1 , FR H2 , FRH3, and FR H4
  • the light chain variable region comprises four framework regions (FRs) classified as FR L1 , FR L2 , FRL3, and FRL4.
  • Illustrative sequences for the framework regions in the antibodies described herein are shown in Table 2 below.
  • Table 2 Illustrative FR Sequences for Anti-SARS-CoV-2 Antibodies
  • any of the CDRH regions may be combined with one or more of the FRH sequences described above to form a heavy chain variable region.
  • suitable heavy chain variable regions can comprise any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 70% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 75% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 80% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 85% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 90% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 95% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 96% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 97% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 98% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 99% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 99.5% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising at least about 99.9% sequence identity to any one of SEQ ID NOs: 115–133.
  • the antibody or antibody binding fragment can comprise a heavy chain variable region comprising any one of SEQ ID NOs: 118, 119, 126, and 133.
  • any of the CDRL regions may be combined with one or more of the FR L sequences described above to form a light chain variable region.
  • suitable light chain variable regions can comprise any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 70% sequence identity of any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 75% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 80% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 85% sequence identity to any one of SEQ ID NOs: 134–152 [0068] For example, the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 90% sequence identity to any one of SEQ ID NOs: 134–152. [0069] For example, the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 95% sequence identity to any one of SEQ ID NOs: 134–152. [0070] For example, the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 96% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 97% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 98% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 99% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 99.5% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising at least about 99.9% sequence identity to any one of SEQ ID NOs: 134–152.
  • the antibody or antibody binding fragment can comprise a light chain variable region comprising any one of SEQ ID NOs: 137, 138, 145, and 152.
  • sequences for SEQ IDs 115–152 are described in Table 3 below. Table 3: Illustrative Heavy Chain or Light Chain Variable Regions for Anti-SARS- CoV-2 Antibodies [0078]
  • the various CDR sequences and FR sequences may be combined in various ways to form new antibodies.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising an amino acid sequence having at least 70% identity to any one of SEQ ID NO: 1–57 and 115–133.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin light chain variable region comprising an amino acid sequence having at least 70% identity to any one of SEQ ID NOs: 58–114 and 134–152.
  • the antibody or antigen-binding fragment thereof can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 1–19, a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 20–38, a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 39–57, or a combination of any thereof; (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 58–76, a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 77–95, a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 96–114, or a combination of any thereof; or (c) a combination thereof [0082]
  • the antibody or antigen-binding fragment thereof can comprise (a) an immunoglobulin heavy chain variable region compris
  • the antibody or antigen-binding fragment comprises immunoglobulin heavy chain variable region comprises a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 1–19, a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 20–38, a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 39–57, or a combination of any thereof.
  • the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 1–19.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 1.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 2.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 3.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 4.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 5. [0090] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 6. [0091] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 7. [0092] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 8.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 9. [0094] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 10. [0095] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 11. [0096] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 12.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 13. [0098] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 14. [0099] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 15. [0100] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 16.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 17. [0102] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 18. [0103] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 19. [0104] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 4, 5, 12, and 19.
  • the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 20–38.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 20.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 21 [0108]
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 22.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 23.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 24.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 25.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 26.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 27.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 28.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 29.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 30.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 31.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 32.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 33.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 34.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 35.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 36.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 37.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising SEQ ID NO: 38.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 23, 24, 31 and 38.
  • the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 39–57.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 39.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 40.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 41.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 42.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 43.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 44.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 45.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 46.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 47.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 48.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 49. [0138] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 50. [0139] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 51. [0140] The antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 52.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 53.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 54.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 55.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 56.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising SEQ ID NO: 57.
  • the antibody or antigen-binding fragment thereof can comprise an immunoglobulin heavy chain variable region comprising a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 42, 43, 50 and 57.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 1–19, a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 20–38, and a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 39–57.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 4, 5, 12, and 19, a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 23, 24, 31 and 38, and a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 42, 43, 50, and 57.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 22–28, a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 29–34, or a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 35–41.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising (a) a CDR H1 having an amino acid sequence comprising SEQ ID NO: 1, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 20, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 39; (b) a CDR H1 having an amino acid sequence comprising SEQ ID NO: 2, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 21, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 40; (c) a CDR H1 having an amino acid sequence comprising SEQ ID NO: 3, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 22, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 41; (d) a CDR H1 having an amino acid sequence comprising SEQ ID NO: 4, a CDR H2 having an amino acid sequence comprising SEQ ID NO:
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 4, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 23, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 42.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 5, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 24, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 43.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 12, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 31, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 50.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 19, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 38, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 57.
  • the antibody or antigen-binding fragment comprises immunoglobulin light chain variable region a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 58–76, a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 77–95, a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 96–114, or a combination of any thereof.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 58–76.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 58.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 59.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 60.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 61.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 62.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 63.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 64.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 65.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 66.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 67.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 68.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 69.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 70.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 71.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 72.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 73.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 74.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 75.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 76.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 61, 62, 69 and 76. [0177] In various embodiments, the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 77–95. [0178] The antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 77.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 78. [0180] The antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 79. [0181] The antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 80. [0182] The antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 81.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 82.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 83.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 84.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 85.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 86.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 87.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 88.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 89.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 90.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 91.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 92.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 93.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 94.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising SEQ ID NO: 95.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 80, 81, 88 and 95 [0198] In various embodiments, the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 96–114. [0199] The antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 96.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 97.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 98.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 99.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 100.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 101.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 102.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 103.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 104.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 105.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 106.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 107.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 108.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 109.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 110.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 111.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 112.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 113.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising SEQ ID NO: 114.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 99, 100, 107 and 114.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 58–76 , a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 77–95, and a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 96–114.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 61, 62, 69, and 76, a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 80, 81, 88, and 95, and a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 99, 100, 107, and 114.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising: (a) a CDR L1 having an amino acid sequence comprising SEQ ID NO: 58, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 77, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 96; (b) a CDR L1 having an amino acid sequence comprising SEQ ID NO: 59, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 78, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 97; (c) a CDR L1 having an amino acid sequence comprising SEQ ID NO: 60, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 79, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 98; (d) a CDR L1 having an amino acid sequence comprising SEQ ID NO: 61
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 61, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 80, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 99.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 62, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 81, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 100.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 69, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 88, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 107.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO:76, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 95, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 114.
  • the antibody or antigen-binding fragment can comprise the immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 1–19, a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 20–38, a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 39–57; and an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 58–76, a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 77–95, a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 96–114.
  • the antibody or antigen-binding fragment can comprise the immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NOs: 4, 5, 12 and 19, a CDR H2 having an amino acid sequence comprising any one of SEQ ID NOs: 23, 24, 31 and 38, a CDR H3 having an amino acid sequence comprising any one of SEQ ID NOs: 42, 43, 50, and 57; and an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising any one of SEQ ID NOs: 61, 62, 69, and 76, a CDR L2 having an amino acid sequence comprising any one of SEQ ID NOs: 80, 81, 88, and 95, a CDR L3 having an amino acid sequence comprising any one of SEQ ID NOs: 99, 100, 107, and 114.
  • An illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 1, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 20, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 39; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 58, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 77, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 96.
  • a second illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising any one of SEQ ID NO: 2, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 21 and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 40; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 59, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 78, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 97.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 3, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 22, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 41; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 60, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 79, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 98.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 4, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 23, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 42; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 61, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 80, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 99.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 5, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 24, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 43; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 62, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 81, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 100.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 6, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 25, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 44; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 63, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 82, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 101.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 7, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 26, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 45; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 64, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 83, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 102.
  • Another illustrative antibody of the present invention can comprise an (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 8, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 27, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 46; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 65, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 84, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 103.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 9, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 28, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 47; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 66, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 85, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 104.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 10, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 29, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 48; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 67, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 86, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 105.
  • Another illustrative antibody of the present invention can comprise an (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 11, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 30, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 49; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 68, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 87, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 106.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 12, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 31, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 50; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 69, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 88, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 107.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 13, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 32, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 51; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 70, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 89, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 108.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 14, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 33, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 52; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 71, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 90, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 109.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 15, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 34, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 53; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 72, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 91, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 110.
  • Another illustrative antibody of the present invention can comprise an (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 16, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 35, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 54; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 73, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 92, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 111.
  • Another illustrative antibody of the present invention can comprise an (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 17, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 36, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 55; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 74, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 93, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 112.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 18, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 37, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 56; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 75, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 94, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 113.
  • Another illustrative antibody of the present invention can comprise (a) an immunoglobulin heavy chain variable region comprising a CDR H1 having an amino acid sequence comprising SEQ ID NO: 19, a CDR H2 having an amino acid sequence comprising SEQ ID NO: 38, and a CDR H3 having an amino acid sequence comprising SEQ ID NO: 57; and (b) an immunoglobulin light chain variable region comprising a CDR L1 having an amino acid sequence comprising SEQ ID NO: 76, a CDR L2 having an amino acid sequence comprising SEQ ID NO: 95, and a CDR L3 having an amino acid sequence comprising SEQ ID NO: 114.
  • the antibody or antigen-binding fragment comprises an immunoglobulin heavy chain variable region having at least about 70% sequence identity to SEQ ID NO: 115–133.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or at least about 99.9% sequence identity to SEQ ID NOs: 115– 133.
  • the antibody or antigen-binding fragment comprises an immunoglobulin light chain variable region having at least about 70% sequence identity to SEQ ID NO: 115–133.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin light chain variable region having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.5% or at least about 99.9% sequence identity to SEQ ID NOs: 134 – 152.
  • the antibody or antigen-binding fragment can comprise an immunoglobulin heavy chain variable region comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity to any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, or at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or at least about 99.5% sequence identity to any one of SEQ ID NOs: 134 – 152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134 – 152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 115 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 116 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 117 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 118 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 119 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 120 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 121 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 122 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 123 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 124 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 125 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 126 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 127 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 128 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 129 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 130 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 131 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 132 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 133 and an immunoglobulin light chain variable region comprising any one of SEQ ID NOs: 134–152.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 134.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 135.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 136.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 137.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 138.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 139.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 140.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 141.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 142.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 143.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 144.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 145.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 146.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 147.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 148.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 149.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 150.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 151.
  • the antibody or antibody-binding fragment can comprise an immunoglobulin heavy chain variable region comprising any one of SEQ ID NOs: 115–133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 152.
  • An illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 115 and an immunoglobulin light chain variable region comprising SEQ ID NO: 134.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO:116 and an immunoglobulin light chain variable region comprising SEQ ID NO: 135.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 117 and an immunoglobulin light chain variable region comprising SEQ ID NO: 136.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 118 and an immunoglobulin light chain variable region comprising SEQ ID NO: 137.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 119 and an immunoglobulin light chain variable region comprising SEQ ID NO: 138.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 120 and an immunoglobulin light chain variable region comprising SEQ ID NO: 139.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 121 and an immunoglobulin light chain variable region comprising SEQ ID NO: 140.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 122 and an immunoglobulin light chain variable region comprising SEQ ID NO: 141.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 123 and an immunoglobulin light chain variable region comprising SEQ ID NO: 142.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 124 and an immunoglobulin light chain variable region comprising SEQ ID NO: 143.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 125 and an immunoglobulin light chain variable region comprising SEQ ID NO: 144.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 126 and an immunoglobulin light chain variable region comprising SEQ ID NO: 145.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 127 and an immunoglobulin light chain variable region comprising SEQ ID NO: 146.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 128 and an immunoglobulin light chain variable region comprising SEQ ID NO: 147.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 129 and an immunoglobulin light chain variable region comprising SEQ ID NO: 148.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 130 and an immunoglobulin light chain variable region comprising SEQ ID NO: 149.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 131 and an immunoglobulin light chain variable region comprising SEQ ID NO: 150.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 132 and an immunoglobulin light chain variable region comprising SEQ ID NO: 151.
  • Another illustrative antibody or antibody binding fragment provided herein can comprise an immunoglobulin heavy chain variable region comprising SEQ ID NO: 133 and an immunoglobulin light chain variable region comprising SEQ ID NO: 152. Derivatives and Synthetically Synthesized Antibodies or Binding Moieties.
  • Also provided are peptides, polypeptides and/or proteins derived from any of the antibodies or antibody binding fragments described herein.
  • the derivatives provided here are substantially similar to the antibodies or antibody binding fragments described herein. For example, they may contain one or more conservative substitutions in their amino acid sequences or may contain a chemical modification.
  • the derivatives and modified peptides/polypeptides/proteins all are considered "structurally similar” which means they retain the structure (e.g., the secondary, tertiary or quarternary structure) of the parent molecule and are expected to interact with the antigen in the same way as the parent molecule.
  • a class of synthetically derived antibodies or antigen-binding moieties can be generated by conservatively mutating resides on the parent molecule to generate a peptide, polypeptide or protein maintaining the same activity as the parent molecule.
  • conservative substitutions are known in the art and are also summarized here. [0310] Generally, conservative substitutions can be made at any position so long as the required activity is retained. So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr.
  • amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
  • Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
  • Hydroxyl or sulfur/selenium-containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation.
  • a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell [0311]
  • a second way to generate a functional peptide/polypeptide or protein based on the sequences provided herein is through the use of computational, "in-silico" design.
  • computationally designed antibodies or antigen-binding fragments may be designed using standard methods of the art. For example, see Strauch EM et al., (Nat Biotechnol.2017 Jul;35(7):667-671), Fleishman SJ et al., (Science.
  • an antibody or antibody binding fragment thereof that binds a coronavirus (e.g., SARS-CoV-2) and is structurally similar to any of the antibodies described herein. That is, it has the same secondary, tertiary or quaternary structure as the antibodies or antigen-binding fragments described herein.
  • the antibody or antigen-binding fragment can have a tertiary structure that is structurally similar to a single CDR loop.
  • the antibody or antigen-binding fragment can have a tertiary structure that is structurally similar to a CDR H3 loop, e.g., a loop comprising SEQ ID NOs: 39 – 57 or any combination thereof.
  • the antibody or antigen-binding fragment can have a tertiary structure that is structurally similar to a CDR loop comprising any one of SEQ ID NOs: : 4, 5, 12, 19, 23, 24, 31, 38, 42, 43, 50, 57, 61, 62, 69, 76, 80, 81, 88, 95, 99, 100, 107 and 114.
  • the antibody can comprise at least one amino acid substitution, deletion, or insertion in a variable region, a hinge region or an Fc region relative to the sequence of a wild-type variable region, hinge region or a wild-type Fc region.
  • the antibody can comprise an Fc region that contains at least one amino acid substitution, deletion, or insertion relative to the sequence of a wild-type Fc region. In various embodiments, this substitution, deletion or insertion can prevent or reduce recycling of the antibody (e.g., in vivo).
  • the antibody or antigen-binding fragment can comprise a heavy chain variable region and/or light chain variable region comprising at least one amino acid substitution, deletion, or insertion as compared to any one of SEQ ID NOs: 1–152.
  • the present invention is directed to antibodies or antigen binding fragments that bind specific epitopes a coronavirus spike protein. Compositions and methods of expressing the antibodies or antigen-binding fragments are also provided.
  • An additional aspect of the invention is a method of generating and isolating "escape mutants" of a coronavirus where the escape mutants comprise variants of the virus against which established antibodies to the coronavirus have reduced affinity.
  • both 2B04 and 2H04 target epitopes shared by human mAbs (i.e., 2-4 for 2B04 and S309 for 2H04). This suggests that these epitopes are immunogenic in both humans and mice, and highlights the possibility of raising therapeutic antibodies in animals.
  • certain antibodies targeting the RBM epitope such as CC12.1 and B38, preferentially utilize the human IgHV3-53 heavy chain gene and display relatively little somatic hypermutation (Yuan et al., 2020b).
  • CC12.1 showed only 4 somatic mutations, whereas CC12.3 showed 3 substitutions and a deletion compared to its germline precursor (Yuan et al., 2020b).
  • IgBLAST analysis of 2B04 shows a similarly low number of somatic mutations, with only 3 amino acid substitutions in the heavy chain and 2 in the light chain. In contrast, 2H04 showed 6 heavy chain mutations and 5 light chain mutations. Also notable is the conservation of paratope contact residues. Only 2 of 15 contact residues in the heavy chain were mutated in 2B04, with one of the two bearing a functionally conservative S31N mutation.7 of these contact residues also were identical in the human IgHV3-53 gene, with a further 6 bearing conservative substitutions. Only two residues in 2B04 were completely non-conserved compared to human IgHV3-53, I30 and N58 (S30 and Y58 in IgHV3-53).
  • the epitope targeted by 2B04 also appears to be relatively immunodominant in mice, as clonally and functionally similar antibodies have been identified (Alsoussi et al., 2020; Hassan et al., 2020).
  • One notable feature of 2B04 compared to many other RBM epitope antibodies is its ability to bind RBD subunits in both the 'up' and 'down' conformation.
  • mAb 2-4 was only reported to bind 'down' RBDs, whereas antibodies targeting the opposite flank of the RBM, such as CC12.1, only bind to RBD in the 'up' conformation (Barnes et al., 2020; Liu et al., 2020; Wu et al., 2020a; Yuan et al., 2020b).
  • the antibody BD23 was observed to bind only one 'down' RBD per trimer (Cao et al., 2020), suggesting that binding orientation on the RBM ridge modulates antibody binding stoichiometry to SARS-CoV-2 spike trimers.
  • S309 and 47D11 are mAbs that bind RBD but cannot block spike binding to ACE2 (Pinto et al., 2020; Wang et al., 2020). We have shown that although 2H04 does not block ACE2 binding, it still impedes virus attachment as efficiently as the ACE2-blocking mAb 2B04. These data indicate there may be alternative attachment receptor(s) in addition to ACE2.
  • heparan sulfates and C-type lectins may have roles in SARS-CoV-2 infection and transmission (Amraie et al., 2020; Bermejo-Jambrina et al., 2020; Clausen et al., 2020; Partridge et al., 2020).
  • Vero E6-TMPRSS2 cells which we used for neutralization and attachment inhibition assays, do not express C-type lectins on their cell surface, but do express heparan sulfates (Mart ⁇ nez-Barragán and del Angel, 2001). Clausen et al.
  • Both S309 and 2H04 target an overlapping RBM-adjacent epitope with a relatively high degree of conservation of contact residues between SARS-CoV and SARS-CoV-2. Both antibodies also neutralize SARS-CoV- 2 without blocking ACE2 binding. As 2H04 neutralizes SARS-CoV-2 by reducing cellular attachment without blocking AEC2 engagement, we predict that S309 might also inhibit SARS-CoV-2 infection through a similar mechanism. [0323] The fact that VSV expressing the SARS-CoV-2 S protein can escape antibody-mediated neutralization, and that mutations at identical residues have been seen in circulating SARS-CoV-2 strains, suggests that SARS-CoV-2 might escape antibody- mediated neutralization when used as monotherapy.
  • antibody or antigen-binding fragment that binds to a coronavirus spike protein at an epitope comprising at least one residue selected from the group consisting of R346, Y351, R403, D405, E406, R408, Q409, T415, G416, K417, D420, Y421, K444, V445, G446, G447, N448, Y449, N450, L452, Y453, L455, F456, R457, K458, S459, N460, T470, I472, Y473, Q474, A475, G476, S477, T478, G482, V483, E484, G485, F486, N487, C488, Y489, F490, L492, Q493, S494, Y495, G496, Q498, T500, N501, G502, V503, G504, Y505 and/or at least one residue selected from the group consisting of T333,
  • SEQ ID NO: 305 (the RBD of the SARS-COV-2 virus) is provided herein in the table below for reference.
  • SEQ ID NO:306 (the RBD of SARS-COV) is also provided.
  • Table 4 SARS-COV-2 and SARS-COV amino acid sequences
  • the coronavirus spike protein comprises a SARS- CoV or SARS-CoV-2 spike protein.
  • the coronavirus spike protein comprises an RBD comprising SEQ ID NO: 305 or SEQ ID NO: 306 as provided above.
  • the antibody or antigen-binding fragment binds to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein.
  • the antibody or antigen-binding fragment sterically blocks ACE2 binding.
  • the antibody or antigen-binding fragment binds to at least one residue is selected from the group consisting of R346, Y351, R403, D405, E406, R408, Q409, T415, G416, K417, D420, Y421, K444, V445, G446, G447, N448, Y449, N450, L452, Y453, L455, F456, R457, K458, S459, N460, T470, I472, Y473, Q474, A475, G476, S477, T478, G482, V483, E484, G485, F486, N487, C488, Y489, F490, L492, Q493, S494, Y495, G496, Q498, T500, N501, G502, V503, G504, Y505 and combinations thereof according to reference SEQ ID NO:
  • the antibody or antibody-binding fragment does not bind to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein.
  • the antibody or antibody-binding fragment can bind adjacent to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein.
  • the antibody or antibody-binding fragment can bind to an epitope comprising at least one residue selected from the group consisting of T333, N334, L335, P337, G339, E340, V341, N343, A344, T345, R346, N354, K356, R357, I358, S359, N360, C361, N439, N440, L441, D442, S443, K444, V445, G446, G447, N448, Y449, N450, Q498, P499, T500, N501, R509 and combinations thereof, according to reference SEQ ID NO: 305.
  • the antibodies or antigen-binding fragments described herein can be expressed recombinantly (e.g., using a recombinant cell line or recombinant organism). Accordingly, the antibodies or antigen-binding fragments may comprise post-translational modifications (e.g., glycosylation profiles, methylation) that differs from naturally occurring antibodies. Derivatives and Synthetically Synthesized Antibodies or Binding Moieties. [0333] Also provided are peptides, polypeptides and/or proteins derived from any of the antibodies or antibody binding fragments described herein. Generally, as used herein, the derivatives provided here are substantially similar to the antibodies or antibody binding fragments described herein.
  • a class of synthetically derived antibodies or antigen-binding moieties can be generated by conservatively mutating resides on the parent molecule to generate a peptide, polypeptide or protein maintaining the same activity as the parent molecule. Representative conservative substitutions are known in the art and are also summarized here.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr.
  • amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
  • Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
  • Hydroxyl or sulfur/selenium-containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids. Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation.
  • a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell [0336]
  • a second way to generate a functional peptide/polypeptide or protein based on the sequences provided herein is through the use of computational, "in-silico" design.
  • computationally designed antibodies or antigen-binding fragments may be designed using standard methods of the art. For example, see Strauch EM et al., (Nat Biotechnol.2017 Jul;35(7):667-671), Fleishman SJ et al., (Science.
  • the antibody can comprise at least one amino acid substitution, deletion, or insertion in a variable region, a hinge region or an Fc region t relative to the sequence of a wild-type variable region, hinge region or a wild-type Fc region.
  • the antibody can comprise an Fc region that contains at least one amino acid substitution, deletion, or insertion relative to the sequence of a wild-type Fc region.
  • this substitution, deletion or insertion can prevent or reduce recycling of the antibody (e.g., in vivo).
  • the antibodies or antigen-binding fragments described herein can be expressed recombinantly (e.g., using a recombinant cell line or recombinant organism). Accordingly, the antibodies or antigen-binding fragments may comprise post-translational modifications (e.g., glycosylation profiles, methylation) that differs from naturally occurring antibodies.
  • binding and Function of the Antibodies and Antigen-Binding Fragments [0340] The antibodies and antigen-binding fragments thereof described herein have some measure of binding affinity to a coronavirus.
  • the antibody or antigen- binding fragment binds SARS-CoV-2 (that is, the coronavirus comprises SARS-CoV-2).
  • the antibodies and antigen-binding fragments thereof described herein can bind a receptor binding domain (RBD) expressed by the coronavirus (e.g., SARS-CoV- 2).
  • RBD receptor binding domain
  • the antibodies and antigen-binding fragments herein may have a certain affinity for a specific epitope on the coronavirus (e.g., an epitope on the receptor binding domain, RBD).
  • the binding of the antibody or antigen-binding fragment can neutralize the coronavirus (e.g., SARS-CoV-2).
  • the antibodies and/or binding fragment neutralize the coronavirus with an IC50 of about 0.0001 ⁇ g/ml to about 30 ⁇ g/ml.
  • the antibody or antigen-binding fragment can have an IC50 of about 0.001 ⁇ g/ml to about 30 ⁇ g/ml.
  • the neutralizing ability of the antibody or antigen-binding fragment can be determined by measuring, for example, the ability of the virus to replicate in the presence or absence of the antibody or antigen-binding fragment.
  • Humanized, Monoclonal and IgG antibodies [0343] In various embodiments, the antibody or antigen-binding fragment described herein is humanized.
  • “Humanized” antibodies are generally chimeric or mutant monoclonal antibodies from mouse, rat, hamster, rabbit or other species, bearing human constant and/ir variable region domains or specific changes. Techniques for generating a so-called “humanized” antibody are well known to those of skill in the art.
  • the antibody or antigen-binding fragment described herein is a monoclonal antibody.
  • the term “monoclonal antibodies” refer to antibodies or antigen-binding fragments that are expressed from the same genetic sequence or sequences and consist of identical antibody molecules.
  • the antibody or antigen-binding fragment described herein is an IgG type antibody.
  • the antibody or antigen-binding fragment can be an IgG1, IgG2, IgG3, or an IgG4 type antibody.
  • Antibody Production Methods for producing antibodies of the invention are known in the art. For example, DNA molecules encoding light chain variable regions and/or heavy chain variable regions can be chemically synthesized. Synthetic DNA molecules can be ligated to other appropriate nucleotide sequences, including, e.g., constant region coding sequences, and expression control sequences, to produce conventional gene expression constructs encoding the desired antibody. Production of defined gene constructs is within routine skill in the art.
  • Nucleic acids encoding desired antibodies can be incorporated (ligated) into expression vectors, which can be introduced into host cells through conventional transfection or transformation techniques.
  • Illustrative host cells are E. coli cells, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human embryonal kidney (HEK) cells and myeloma cells that do not otherwise produce IgG protein.
  • Transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the immunoglobulin light and/or heavy chain variable regions.
  • Specific expression and purification conditions will vary depending upon the expression system employed.
  • the engineered gene is to be expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon, and, optionally, may contain enhancers, and various introns.
  • This expression vector optionally contains sequences encoding all or part of a constant region, enabling an entire, or a part of, a heavy or light chain to be expressed.
  • the gene construct can be introduced into eukaryotic host cells using conventional techniques.
  • the host cells express VL or VH fragments, VL-VH heterodimers, VH-VL or VL-VH single chain polypeptides, complete heavy or light immunoglobulin chains, or portions thereof, each of which may be attached to a moiety having another function (e.g., cytotoxicity).
  • a host cell is transfected with a single vector expressing a polypeptide expressing an entire, or part of, a heavy chain (e.g., a heavy chain variable region) or a light chain (e.g., a light chain variable region).
  • a host cell is transfected with a single vector encoding (a) a polypeptide comprising a heavy chain variable region and a polypeptide comprising a light chain variable region, or (b) an entire immunoglobulin heavy chain and an entire immunoglobulin light chain.
  • a host cell is co-transfected with more than one expression vector (e.g., one expression vector encoding a polypeptide comprising an entire, or part of, a heavy chain or heavy chain variable region, and another expression vector encoding a polypeptide comprising an entire, or part of, a light chain or light chain variable region).
  • a polypeptide comprising an immunoglobulin heavy chain variable region or light chain variable region can be produced by growing (culturing) a host cell transfected with an expression vector encoding such variable region, under conditions that permit expression of the polypeptide. Following expression, the polypeptide can be harvested and purified or isolated using techniques known in the art, e.g., using affinity tags such as glutathione-S-transferase (GST) and histidine tags.
  • GST glutathione-S-transferase
  • a monoclonal antibody, or an antigen-binding fragment of the antibody can be produced by growing (culturing) a host cell transfected with: (a) an expression vector that encodes a complete or partial immunoglobulin heavy chain, and a separate expression vector that encodes a complete or partial immunoglobulin light chain; or (b) a single expression vector that encodes both chains (e.g., complete or partial heavy and light chains), under conditions that permit expression of both chains.
  • the intact antibody (or antigen-binding fragment of the antibody) can be harvested and purified or isolated using techniques known in the art, e.g., Protein A, Protein G, affinity tags such as glutathione-S-transferase (GST) and histidine tags.
  • a nucleic acid is provided, the nucleic acid comprising a nucleotide sequence encoding the antibody or antigen-binding fragment described herein.
  • the skilled man will appreciate that functional variants of these nucleic acid molecules are also intended to be a part of the present invention. Functional variants are nucleic acid sequences that can be directly translated, using the standard genetic code, to provide an amino acid sequence identical to that translated from the parental nucleic acid molecules.
  • Suitable nucleic acids that can encode portions of the inventive antibodies can be determined by one skilled in the art using standard techniques.
  • the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin heavy chain variable region of the antibody or antigen-binding fragment described herein. In various embodiments, the nucleic acid comprises a nucleotide sequence encoding an immunoglobulin light chain variable region of the antibody or antigen-binding fragment described herein. In some embodiments, the nucleic acids encode one or more complementary determining regions (CDR) having the amino acid sequences described herein. As described above, a single nucleic acid may be provided that encodes more than one protein product (e.g., the immunoglobulin light chain and the immunoglobulin heavy chain).
  • CDR complementary determining regions
  • an expression vector comprising one or more of the nucleic acids described herein.
  • Vectors can be derived from plasmids such as: F, F1, RP1, Col, pBR322, TOL, Ti, etc; cosmids; phages such as lambda, lambdoid, M13, Mu, P1, P22, Q ⁇ , T-even, T-odd, T2, T4, T7 etc; or plant viruses.
  • Vectors can be used for cloning and/or expression of the binding molecules of the invention and might even be used for gene therapy purposes.
  • Vectors comprising one or more nucleic acid molecules according to the invention operably linked to one or more expression-regulating nucleic acid molecules are also covered by the present invention.
  • the choice of the vector is dependent on the recombinant procedures followed and the host used. Introduction of vectors in host cells can be affected by inter alia calcium phosphate transfection, virus infection, DEAE-dextran mediated transfection, lipofectamin transfection or electroporation.
  • Vectors may be autonomously replicating or may replicate together with the chromosome into which they have been integrated.
  • the vectors contain one or more selection markers. The choice of the markers may depend on the host cells of choice.
  • vectors comprising one or more nucleic acid molecules encoding the human binding molecules as described above operably linked to one or more nucleic acid molecules encoding proteins or peptides that can be used to isolate the human binding molecules are also covered by the invention.
  • proteins or peptides include, but are not limited to, glutathione-S-transferase, maltose binding protein, metal-binding polyhistidine, green fluorescent protein, luciferase and beta-galactosidase.
  • the expression vector may be transfected into a host cell to induce the translation and expression of the nucleic acid into the heavy chain variable region and/or the light chain variable region. Therefore, a host cell is provided comprising any expression vector described herein. Host cells include, but are not limited to, cells of mammalian, plant, insect, fungal or bacterial origin.
  • Bacterial cells include, but are not limited to, cells from Gram-positive bacteria or Gram-negative bacteria such as several species of the genera Escherichia, such as E. coli, and Pseudomonas.
  • yeast cells are used in the group of fungal cells. Expression in yeast can be achieved by using yeast strains such as inter alia Pichia pastoris, Saccharomyces cerevisiae and Hansenula polymorpha.
  • insect cells such as cells from Drosophila and Sf9 can be used as host cells.
  • the host cells can be plant cells such as inter alia cells from crop plants such as forestry plants, or cells from plants providing food and raw materials such as cereal plants, or medicinal plants, or cells from ornamentals, or cells from flower bulb crops.
  • Transformed (transgenic) plants or plant cells are produced by known methods, for example, Agrobacterium-mediated gene transfer, transformation of leaf discs, protoplast transformation by polyethylene glycol- induced DNA transfer, electroporation, sonication, microinjection or bolistic gene transfer.
  • a suitable expression system can be a baculovirus system.
  • Expression systems using mammalian cells such as Chinese Hamster Ovary (CHO) cells, COS cells, BHK cells, NSO cells or Bowes melanoma cells are preferred in the present invention. Since the present invention deals with molecules that may have to be administered to humans, a completely human expression system would be particularly preferred. Therefore, even more preferably, the host cells are human cells.
  • the antibody or antigen-binding fragment can be expressed using a recombinant cell line or recombinant organism.
  • a method is provided for producing an antibody or antigen-binding fragment that binds a coronavirus, the method comprising growing a host cell as described herein under conditions so that the host cell expresses a polypeptide or polypeptides comprising the immunoglobulin heavy chain variable region and the immunoglobulin light chain variable region, thereby producing the antibody or antigen-binding fragment and purifying the antibody or antigen-binding fragment.
  • an assay for isolating an escape mutant of a SARS coronavirus, the method comprising: a) expressing a SARS-CoV-2 chimeric vesicular stomatitis virus (VSV-SARS-CoV-2) comprising a receptor binding domain of a SARS-CoV- 2 virus; b) applying a neutralizing antibody having an affinity for an epitope on the RBD to the virus; c) incubating in replication competent conditions for a period of time; and d) isolating the escape mutant, wherein the escape mutant comprises at least one mutation relative to the wild-type.
  • VSV-SARS-CoV-2 chimeric vesicular stomatitis virus
  • the at least one mutation decreases the affinity of the neutralizing antibody for the epitope on the RBD of the virus.
  • the at least one mutation is at a residue selected from the group consisting of R346, Y351, R403, D405, E406, R408, Q409, T415, G416, K417, D420, Y421, K444, V445, G446, G447, N448, Y449, N450, L452, Y453, L455, F456, R457, K458, S459, N460, T470, I472, Y473, Q474, A475, G476, S477, T478, G482, V483, E484, G485, F486, N487, C488, Y489, F490, L492, Q493, S494, Y495, G496, Q498, T500, N501, G502, V503, G504, Y505 and combinations thereof; or is
  • SARS-CoV-2 chimeric vesicular stomatitis virus is an example of a pseudo-virus, capable of expressing antigenic portions of pathogenic viruses, but without the pathogenicity.
  • SARS-CoV-2 chimeric vesicular stomatitis virus is described in more detail in Case et al., ("Neutralizing Antibody and Soluble ACE2 Inhibition of a Replication-Competent VSV-SARS-CoV-2 and a Clinical Isolate of SARS-CoV-2., 2020, Cell Host & Microbe 28, 475-485.e5), hereby incorporated by reference in its entirety.
  • replication competent refers to conditions suitable for viral replication. Such conditions are known to those skilled in the art, and are also referenced in Case et al., incorporated herein.
  • RBD refers to "receptor binding domain”. Additional Assays, Methods and Applications [0364] An assay is provided for evaluating an antibody provided herein. For example, the assay can comprise testing whether the antibody can effectively neutralize an escape mutant isolated as described above.
  • a method for generating an antibody selective for an escape mutant isolated as described above comprising either: a) immunizing an animal with the escape mutant isolated above and isolating the antibody, or b) recombinantly expressing the antibody by transfecting a recombinant organism with a nucleic acid encoding an amino acid sequence of the antibody, wherein the amino acid sequence comprises at least one mutation relative to a reference antibody, the mutation increasing the affinity of the antibody to the escape mutant as compared to the reference antibody.
  • the reference antibody can comprise an anti SARS- CoV-2 antibody described in the art and selected from the group consisting of: 2B04, 2H04, 2-4, B38, CB6, C105, CC12.1, CC12.3, COVA2-04, CV30, REGN10933, COVA2-39, BD23, P2B-2F6, S309, and REGN10987.
  • an anti SARS- CoV-2 antibody described in the art and selected from the group consisting of: 2B04, 2H04, 2-4, B38, CB6, C105, CC12.1, CC12.3, COVA2-04, CV30, REGN10933, COVA2-39, BD23, P2B-2F6, S309, and REGN10987.
  • Pharmaceutical Compositions [0367] Also provided are pharmaceutical compositions comprising at least one antibody or antigen-binding fragment described herein.
  • Pharmaceutical compositions containing one or more of the antibodies or antigen-binding fragments described herein can be formulated in any conventional manner.
  • Routes of administration include, but are not limited to parenteral (e.g., intravenous, intra-arterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal), topical (nasal, transdermal, intraocular), intravesical, intrathecal, enteral, pulmonary, intralymphatic, intracavital, vaginal, transurethral, intradermal, aural, intramammary, buccal, orthotopic, intratracheal, intralesional, percutaneous, endoscopical, transmucosal, sublingual and intestinal administration.
  • parenteral e.g., intravenous, intra-arterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal
  • topical nasal, transdermal, intraocular
  • intravesical, intrathecal enteral
  • pulmonary
  • the composition is administered parenterally or is inhaled (e.g., intranasal).
  • the pharmaceutical compositions can also be formulated for parenteral administration, e.g., formulated for injection via intravenous, intra-arterial, subcutaneous, rectal, subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal, intraperitoneal, or intrasternal routes. Dosage forms suitable for parenteral administration include solutions, suspensions, dispersions, emulsions or any other dosage form that can be administered parenterally.
  • the pharmaceutical composition can be formulated without blood, plasma or a major component of blood or plasma (e.g., blood cells, fibrin, hemoglobin, albumin, etc.).
  • the pharmaceutical composition can comprise from about 0.001 to about 99.99 wt.% of the antibody or antigen-binding fragment according to the total weight of the composition.
  • the pharmaceutical composition can comprise from about 0.001 to about 1%, about 0.001 to about 5%, about 0.001 to about 10%, about 0.001 to about 15%, about 0.001 to about 20%, about 0.001 to about 25%, about 0.001 to about 30%, about 1 to about 10%, about 1 to about 20%, about 1 to about 30%, about 10 to about 20%, about 10 to about 30%, about 10 to about 40%, about 10 to about 50%, about 20 to about 30%, about 20 to about 40%, about 20 to about 50%, about 20 to about 60%, about 20 to about 70%, about 20 to about 80%, about 20 to about 90%, about 30 to about 40%, about 30 to about 50%, about 30 to about 60%, about 30 to about 70%, about 30 to about 80%, about 30 to about 90%, about 40 to about 50%, about 40 to about 60%, about 40 to about 70%, about 40 to about 80%
  • compositions described herein can also comprise one or more pharmaceutically acceptable excipients and/or carriers.
  • pharmaceutically acceptable excipients and/or carriers for use in the compositions of the present invention can be selected based upon a number of factors including the particular compound used, and its concentration, stability and intended bioavailability; the subject, its age, size and general condition; and the route of administration.
  • Some examples of materials which can serve as pharmaceutically acceptable carriers in the compositions described herein are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil; and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; artificial cerebral spinal fluid (CSF
  • compositions of the invention are identified, for example, in The Handbook of Pharmaceutical Excipients, (American Pharmaceutical Association, Washington, D.C., and The Pharmaceutical Society of Great Britain, London, England, 1968). Additional excipients can be included in the pharmaceutical compositions of the invention for a variety of purposes. These excipients can impart properties which enhance retention of the compound at the site of administration, protect the stability of the composition, control the pH, facilitate processing of the compound into pharmaceutical compositions, and so on.
  • compositions further comprises at least one other therapeutic, prophylactic and/or diagnostic agent.
  • the therapeutic and/or prophylactic agents are capable of preventing and/or treating an coronavirus infection and/or a condition/symptom resulting from such an infection.
  • Therapeutic and/or prophylactic agents include, but are not limited to, anti-viral agents. Such agents can be binding molecules, small molecules, organic or inorganic compounds, enzymes, polynucleotide sequences, anti- viral peptides, etc.
  • the therapeutic and/or prophylactic agent can comprise an M2 inhibitor (e.g., amantadine, rimantadine) and/or a neuraminidase inhibitor (e.g., zanamivir, oseltamivir).
  • the anti-viral agent can comprise baloxavir, oseltamivir, zanamivir, peramivir, remdesivir, or any combination thereof.
  • the therapeutic and/or prophylactic agent can also include various anti-malarial such as chloroquine, hydroxychloroquine, and analogues thereof.
  • the additional antibodies or therapeutic/prophylactic and/or diagnostic agents may be used in combination with the antibodies and antigen-binding fragments of the present invention. "In combination" herein, means simultaneously, as separate formulations (e.g., co- administered), or as one single combined formulation or according to a sequential administration regiment as separate formulations, in any order.
  • coronavirus e.g., SARS-CoV-2
  • coronavirus e.g., SARS-CoV-2
  • Vaccine a vaccine is provided for preventing a coronavirus infection.
  • the vaccine can provide protection from SARS CoV-2 which can cause an infection known as COVID-19.
  • the vaccine may comprise a polypeptide comprising the epitope targeted by the antibodies or antigen-binding fragments described herein.
  • the vaccine further comprises an adjuvant to stimulate an immune response.
  • Suitable adjuvants are known in the art and can include, for example, alum, aluminum hydroxide, monophosphoryl lipid A (MPL) or combinations thereof.
  • the vaccine may be prepared using suitable carriers and excipients according to pharmaceutical compositions described herein above.
  • the vaccine can elicit an immunological response to prevent a coronavirus infection.
  • the infection may be caused by the SARS-CoV-2 virus.
  • the infection can comprise COVID-19.
  • Methods of Treating [0380]
  • a method of preventing or treating a coronavirus infection e.g., COVID-19 caused by SARS-CoV-2 in a subject in need thereof is provided.
  • the method can comprise administering any antibody or antigen-binding fragment (including any nucleic acid or expression vector that encodes the antibody or antigen-binding fragment), any vaccine, or any composition as described herein to the subject.
  • the composition is administered parentally (e.g., systemically).
  • the composition is inhaled orally (e.g., intranasally).
  • the composition is formulated (e.g., with carriers/excipients) according to its mode of administration as described above.
  • the composition is administered via intranasal, intramuscular, intravenous, and/or intradermal routes.
  • the composition is provided as an aerosol (e.g., for nasal administration).
  • Dosing regiments can be adjusted to provide the optimum desired response (e.g., a prophylactic or therapeutic response). Therefore, the dose used in the methods herein can vary depended on the intended use (e.g., for prophylactic vs. therapeutic use). Nevertheless, the compositions described herein may be administered at a dose of about 1 to about 100 mg/kg body weight, or from about 1 to about 70 mg/kg body weight. Furthermore, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic of the therapeutic situation. [0384] In various embodiments, the antibody or antigen-binding fragment is delivered using a gene therapy technique.
  • Such techniques are well known in the art and generally comprise administering a viral vector comprising a nucleic acid that codes for a gene product of interest to a subject in need thereof. Therefore, in certain embodiments, the antibody or antigen-binding fragment described herein is delivered to a subject in need thereof by administering a viral vector or vectors (e.g., an adenovirus) containing one or more of the necessary nucleic acids (such as, for example, the nucleic acids provided herein) for expressing the antibody or antibody binding fragment in vivo.
  • a viral vector or vectors e.g., an adenovirus
  • Similar delivery methods have successfully lead to the expression of protective antibodies in other disease contexts. For example, see Sofer-Podesta C.
  • the coronavirus infection to be treated is a SARS infection (e.g., severe acute respiratory syndrome caused by the coronavirus).
  • the coronavirus infection comprises COVID-19. Definitions [0386]
  • the term "antigen-binding fragment” means any antigen- binding fragment of an antibody, including an intact antibody or antigen-binding fragment that has been modified, engineered or chemically conjugated.
  • antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies.
  • Antigen-binding fragments include, inter alia, Fab, F(ab′), F(ab′)2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the (poly)peptide, etc.).
  • the antigen-binding fragment binds with the same antigen that is recognized by the intact immunoglobulin.
  • An antigen-binding fragment can comprise a peptide or polypeptide comprising an amino acid sequence of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 contiguous amino acid residues of the amino acid sequence of the binding molecule.
  • the above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineered by recombinant DNA techniques. The methods of production are well known in the art and are described, for example, in Antibodies: A Laboratory Manual, Edited by: E.
  • CDR complementarity determining regions
  • the term “complementarity determining regions” (CDR) as used herein means sequences within the variable regions of antibodies that usually contribute to a large extent to the antigen binding site which is complementary in shape and charge distribution to the epitope recognized on the antigen.
  • the CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e.g., by solubilization in SDS. Epitopes may also consist of posttranslational modifications of proteins.
  • the term “host”, as used herein, is intended to refer to an organism or a cell into which a vector such as a cloning vector or an expression vector has been introduced.
  • the organism or cell can be prokaryotic or eukaryotic.
  • the hosts are isolated host cells, e.g. host cells in culture.
  • the term “host cells” merely signifies that the cells are modified for the (over)-expression of the antibodies of the invention and include B-cells that originally express these antibodies and which cells have been modified to over-express the binding molecule by immortalization, amplification, enhancement of expression etc.
  • Amino acid sequence identity percent is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • percent sequence identity X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • operably linked refers to two or more nucleic acid sequence elements that are usually physically linked and are in a functional relationship with each other.
  • a promoter is operably linked to a coding sequence, if the promoter is able to initiate or regulate the transcription or expression of a coding sequence, in which case the coding sequence should be understood as being “under the control of” the promoter.
  • pharmaceutically acceptable excipient is meant any inert substance that is combined with an active molecule such as a drug, agent, or antibody and that facilitate processing of the active compounds into preparations which can be used pharmaceutically.
  • the “pharmaceutically acceptable excipient” is an excipient that is non-toxic to recipients at the used dosages and concentrations, and is compatible with other ingredients of the formulation comprising the drug, agent or binding molecule. Pharmaceutically acceptable excipients are widely applied and known in the art.
  • pharmaceutically acceptable carrier means a non- toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material, or formulation auxiliary of any type.
  • materials which can serve as pharmaceutically acceptable carriers are sugars such as lactose, glucose, and sucrose; starches such as corn starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; corn oil; and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; artificial cerebral spinal fluid (CSF), and phosphate buffer solutions, as well as
  • the term “specifically binding”, as used herein, in reference to the interaction of an antibody, and its binding partner, e.g. an antigen, means that the interaction is dependent upon the presence of a particular structure, e.g. an antigenic determinant or epitope, on the binding partner.
  • the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms.
  • the binding may be mediated by covalent or non-covalent interactions or a combination of both.
  • the term “specifically binding” means immunospecifically binding to an antigenic determinant or epitope and not immunospecifically binding to other antigenic determinants or epitopes.
  • An antibody that immunospecifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by, e.g., radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art.
  • Antibodies or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens, carrying the same epitope.
  • antibodies or fragments thereof that immunospecifically bind to an antigen do not cross-react with other antigens.
  • neutralizing refers to antibodies that inhibit a coronavirus from replication, in vitro and/or in vivo, regardless of the mechanism by which neutralization is achieved, or assay that is used to measure the neutralization activity.
  • therapeutically effective amount refers to an amount of the antibodies as defined herein that is effective for preventing, ameliorating and/or treating a condition resulting from infection with a coronavirus (e.g., COVID-19). Amelioration as used herein may refer to the reduction of visible or perceptible disease symptoms, viremia, or any other measurable manifestation of coronavirus infection.
  • treatment refers to therapeutic treatment as well as prophylactic or preventative measures to cure or halt or at least retard disease progress.
  • Those in need of treatment include those already inflicted with a condition resulting from infection with coroanvirus as well as those in which infection with coronavirus is to be prevented.
  • Subjects partially or totally recovered from infection with coronavirus e.g., SARS-CoV-2
  • Prevention encompasses inhibiting or reducing the spread of the coronavirus or inhibiting or reducing the onset, development or progression of one or more of the symptoms associated with infection with coronavirus.
  • vector denotes a nucleic acid molecule into which a second nucleic acid molecule can be inserted for introduction into a host where it will be replicated, and in some cases expressed.
  • a vector is capable of transporting a nucleic acid molecule to which it has been linked.
  • vectors include, but are not limited to, plasmids, cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC) and vectors derived from bacteriophages or plant or animal (including human) viruses.
  • Vectors comprise an origin of replication recognized by the proposed host and in case of expression vectors, promoter and other regulatory regions recognized by the host.
  • a vector containing a second nucleic acid molecule is introduced into a cell by transformation, transfection, or by making use of viral entry mechanisms.
  • Certain vectors are capable of autonomous replication in a host into which they are introduced (e.g., vectors having a bacterial origin of replication can replicate in bacteria).
  • Other vectors can be integrated into the genome of a host upon introduction into the host, and thereby are replicated along with the host genome.
  • polypeptide e.g., an antibody or antigen-binding fragment thereof
  • a polypeptide or protein "structurally similar" to another polypeptide or protein would be expected to have similar binding affinity to the reference protein's binding target.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • So-called conservative exchanges can be carried out in which the amino acid which is replaced has a similar property as the original amino acid, for example the exchange of Glu by Asp, Gln by Asn, Val by Ile, Leu by Ile, and Ser by Thr.
  • amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
  • Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
  • Hydroxyl or sulfur/selenium-containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • Deletion is the replacement of an amino acid by a direct bond. Positions for deletions include the termini of a polypeptide and linkages between individual protein domains. Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids.
  • Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation. On the basis of this artificially generated polypeptide sequences, a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
  • Example 1 Methods directed to Examples 2-5
  • Example 2-5 Cells, viruses, and recombinant proteins
  • Expi293F cells were cultured at 37°C in Expi293 Expression medium.
  • Vero E6 cells CRL-1586, Vero CCL81, and HEK293 were cultured at 37°C in Dulbecco’s Modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 mM HEPES pH 7.3, 1 mM sodium pyruvate, 1x non-essential amino acids, and 100 U/ml of penicillin–streptomycin.
  • DMEM Modified Eagle medium
  • FBS fetal bovine serum
  • SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 was obtained from the Centers for Disease Control and Prevention.
  • a p3 stock was passaged once in CCL81-Vero cells and titrated by focus-forming assay on Vero E6 cells.
  • AdV-hACE2-GFP construct and defective virus preparation was reported previously (Jia et al., J. Virol.79, 14614–14621 (2005)).
  • AdV-hACE2-GFP was propagated in 293T cells and purified by cesium chloride density- gradient ultracentrifugation. The number of virus particles was determined using optical density (260 nm) measurement and plaque assay, as previously described (Mittereder, et al., J. Virol.70, 7498–7509 (1996)).
  • the viral stock titer was determined to be 10 11 PFU/mL.
  • DNA fragments encoding ectodomain of spike from SARS-CoV1 (residues 14-1193, GenBank: AY278488.2), SARS-CoV2 (residues 14-1211, GenBank: MN908947.3) and MERS-CoV (residues 19-1294, GenBank: JX869059.2) were synthesized and placed into the mammalian expression vector pFM1.2 with N-terminal mu-phosphatase signal peptide.
  • the C-terminus of all DNAs were engineered with a HRV3C protease cleavage site linked by a foldon trimerization motif and an 8XHis Tag.
  • the S1/S2 furin cleavage sites were mutated in both SARS-CoV2 and MERS-CoV S, and all three S proteins were stabilized with the 2P mutations.
  • the plasmids were transiently transfected in Expi293F cells using FectoPRO reagent and cell supernatants containing target protein were harvested 96h after transfection. The soluble S proteins were recovered using 2mL cobalt-charged resin.
  • Mammalian SARS- CoV2 RBD (residues 331 ⁇ 524) was cloned into vector pFM1.2 with N-terminal mu- phosphatase signal peptide and C-terminal 6XHis Tag.
  • the protein was expressed as S protein and recovered by nickel agarose beads, further purified by passage over S75i Superdex.
  • the bacterial version of RBD was cloned into the pET21a vector and expressed as inclusion bodies in Escherichia coli BL21(DE3) and purified as previously described for ZIKV DIII, (Zhao et al., Cell.166, 1016–1027 (2016).).
  • Mouse immunization [0406] All procedures involving animals were performed in accordance with guidelines of Institutional Animal Care and Use Committee (IACUC) of Washington University in Saint Louis.
  • mice Female C57BL/6J mice (Jackson Laboratories) were immunized intramuscularly with 10 ⁇ g SARS-CoV-2 RBD resuspended in PBS emulsified with AddaVax. Two weeks later, mice were boosted with 5 ⁇ g SARS-CoV-2 S protein twice, at 10-day intervals. One control mouse received PBS emulsified with AddaVax according to the same schedule. Sera were collected 5 days after the final boost and stored at -20°C before use. Draining iliac and inguinal lymph nodes were also harvested on day 5 after the final boost for plasmablast sorting.
  • Staining for sorting was performed using fresh lymph node single cell suspensions in PBS supplemented with 2% FBS and 1mM EDTA (P2). Cells were stained for 30 min on ice with biotinylated recombinant SARS-CoV-2 RBD diluted in P2, washed twice, then stained for 30 min at 4°C with Fas-PE (Jo2), CD4-eFluor 780 (GK1.5,), CD138- BV421 (281-2), IgD-FITC (11-26c.2a), GL7-PerCP-Cy5.5, CD38-PE-Cy7 (90), CD19-APC (1D3), and Zombie Aqua diluted in P2.
  • VH, V ⁇ , and V ⁇ genes were amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and nested PCR from singly-sorted SARS-CoV-2 RBD + plasmablasts using cocktails of primers specific for IgG, IgM/A, Ig ⁇ , and Ig ⁇ using first round and nested primer sets (Von Boehmer et al., 2016; Ehlers et al., J. Exp.
  • Enzyme-linked immunosorbent assay Ninety-six-well microtiter plates were coated with 100 ⁇ L recombinant SARS-CoV-2 S or RBD at a concentration of 0.5 ⁇ g/mL and 1 ⁇ g/mL, respectively, in 1X PBS at 4 °C overnight; negative control wells were coated with 1 ⁇ g/mL BSA. Plates were blocked for 1.5 h at room temperature with 280 ⁇ L blocking solution (1X PBS supplemented with 0.05% Tween-20 and 10% FBS). The mAbs were diluted to a starting concentration of 10 ⁇ g/mL, serially diluted 1:3, and incubated for 1 h at room temperature.
  • the plates were washed three times with T-PBS (1X PBS supplemented with 0.05% Tween-20), and 100 ⁇ L anti-human IgG horseradish peroxidase (HRP) antibody (goat polyclonal) diluted 1:2,500 in blocking solution was added to all wells and incubated for 1 h at room temperature. Plates were washed 3 times with T-PBS and 3 times with 1X PBS, and 100 ⁇ L peroxidase substrate was added to all wells. The reaction was stopped after 5 min using 100 ⁇ L 1M hydrochloric acid, and the plates were read at a wavelength of 490 nm using a microtiter plate reader. The data were analyzed using Prism v8.
  • the cDNAs were prepared after the GEM generation and barcoding, followed by GEM RT reaction and bead cleanup steps. Purified cDNA was amplified for 10-14 cycles before being cleaned up using SPRIselect beads. Samples were then run on a Bioanalyzer to determine cDNA concentration. BCR target enrichments were done on the full length cDNA. GEX and enriched BCR libraries were prepared as recommended by 10x Genomics Chromium Single Cell V(D)J Reagent Kits user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. The cDNA Libraries were sequenced on Novaseq S4, targeting a median sequencing depth of 50,000 and 5,000 read pairs per cell for gene expression and BCR libraries, respectively.
  • the final list of 259 mm10 Ig genes included 113 Ighv genes, 17 Ighd genes, 4 Ighj genes, 100 Igkv genes, 5 Igkj genes, 3 Iglv genes, 5 Iglj genes, 8 Ighc genes, 1 Igkc gene, and 3 Iglc genes. Genomic sequences for these genes were retrieved based on their Ensembl IDs via the Ensembl REST API (release 13.0).
  • IMGT Ig reference alleles for Mus musculus [0413] Ig reference alleles (release 202011-3) for mouse were downloaded from the ImMunoGeneTics information system (IMGT) on 2020-04-02 under the “F+ORF+in frame P” configuration.
  • IMGT alleles for Mus musculus included 406 IGHV alleles, 38 IGHD alleles, 9 IGHJ alleles, 150 IGKV alleles, 10 IGKJ alleles, 14 IGLV alleles, 5 IGLJ alleles, 106 IGHC alleles, 3 IGKC alleles, and 3 IGLC alleles.
  • each mm10 Ig gene was aligned against the IMGT alleles for its corresponding gene segment using blastn (v2.9.0). For Ighd genes, blastn-short was also used to accommodate short sequence lengths. For each mm10 Ig gene, a search for the IMGT allele with 100% match for the full length of the allele was conducted. For each of 247 out of the 259 mm10 genes, one or more matching alleles were identified. For 18 of these 247 mm10 genes, two IMGT alleles were identified with identical nucleotide sequences and full-length 100% matches.
  • the allele with name matching that of the mm10 Ig gene was designated as the corresponding IMGT allele.
  • the identical IMGT alleles IGKV4-54*01 and IGKV4-52*01 both matched with mm10 gene Igkv4- 54; in this case, IGKV4-54*01 was noted as the corresponding C57BL/6 IMGT allele.
  • an allele was chosen based on the locus representation map. For Ighd5-7, which matched with IGHD6-1*01 and IGHD6-3*01, IGHD6-3*01 was chosen.
  • Ighd5-8 which matched with IGHD6-1*02 and IGHD6-4*01, IGHD6-4*01 was chosen.
  • the corresponding IMGT alleles were used as the curated reference alleles.
  • Ighv12-3, Ighv2-3, Ighv8-2, and Ighv8-4 evidence for putative RSS motifs were observed adjacent to the final nucleotides of the IMGT alleles. In these cases, the additional nucleotides in the IMGT alleles were included for the curated reference alleles.
  • Igkv3-7, Igkv9-120, Ighg2b, and Ighg3, IGKV3-7*01, IGKV9-120*01, and IGHG2B*02, and IGHG3*01 were identified as the closest IMGT alleles with, respectively, 1, 1, 3, and 3 nucleotide mismatches.
  • the curated reference alleles deferred to the nucleotides found in the corresponding mm10 genomic sequences.
  • mm10 genes Igkc, Iglc1, and Iglc3 length discrepancies were noted at the 5’ end, where the mm10 genomic sequences begin, in the form of 1 additional nucleotide each in the closest matching IMGT alleles: IGKC*01, IGLC*01, and IGLC*03.
  • the curated reference alleles deferred to the mm10 genomic sequences and did not include the additional nucleotide found in IMGT alleles.
  • the final curated set of C57BL/6 reference alleles included 113 IGHV alleles, 17 IGHD alleles, 4 IGHJ alleles, 100 IGKV alleles, 5 IGKJ alleles, 3 IGLV alleles, 5 IGLJ alleles, 8 IGHC alleles, 1 IGKC allele, and 3 IGLC alleles.
  • the 10x Genomics and mAb sequences were combined with paired heavy and light chain nested PCR sequences from 100 cells.
  • Germline V(D)J gene annotation was performed for all sequences using IgBLAST v1.15.0 with a curated set of immunoglobulin reference alleles specific for the C57BL/6 strain of Mus musculus (see above section).
  • IgBLAST output was parsed using Change-O v0.4.6. Additional quality control required sequences to be productively rearranged and have valid V and J gene annotations, consistent chain annotation (excluding sequences annotated with heavy chain V gene and light chain J gene), and a junction length that is a multiple of 3. Furthermore, only cells with exactly one heavy chain sequence paired with at least one light chain sequence were kept.
  • 6,262 cells with paired heavy and light chains including 83 cells with nested PCR sequences, 34 cells with mAb sequences, and 6,145 cells with 10x Genomics BCR sequences.
  • Clonal lineage inference [0420] B cell clonal lineages were inferred using hierarchical clustering with single linkage (Gupta et al., J. Immunol.198, 2489–2499 (2017)). Cells were first partitioned based on common heavy and light chain V and J gene annotations and junction region lengths, where junction was defined to be from IMGT codon 104 encoding the conserved cysteine to codon 118 encoding phenylalanine or tryptophan.
  • Mutation frequency was calculated for cells with 10x Genomics BCRs by counting the number of nucleotide mismatches from the germline sequence in the heavy chain variable segment leading up to the CDR3. Calculation was performed using the calcObservedMutations function from SHazaM v0.2.3. Processing of 10x Genomics single-cell 5’ gene expression data [0422] Demultiplexed pair-end FASTQ reads were preprocessed using the “cellranger count” command from 10x Genomics’ Cell Ranger v3.1.0 for alignment against the GRCm38 mouse reference v3.0.0 (refdata-cellranger-mm10-3.0.0). A feature UMI count matrix containing 7,485 cells and 31,053 features was generated.
  • the biotypes of the features were retrieved from the GTF annotation of Ensembl release 93. Additional quality control was performed as follows.1) To remove presumably lysed cells, cells with mitochondrial content greater than 15% of all transcripts were removed.2) To remove likely doublets, cells with more than 5,000 features or 80,000 total UMIs were removed.3) To remove cells with no detectable expression of common housekeeping mouse genes, cells with no transcript for any of Actb, Gapdh, B2m, Hsp90ab1, Gusb, Ppih, Pgk1, Tbp, Tfrc, Sdha, Ldha, Eef2, Rpl37, Rpl38, Leng8, Heatr3, Eif3f, Chmp2a, Psmd4, Puf60, and Ppia were removed.4) The feature matrix was subset, based on their biotypes, to protein-coding, immunoglobulin, and T cell receptor genes that were expressed in at least 0.1% of the cells.
  • Single-cell gene expression analysis was performed using Seurat v3.1.1. UMI counts measuring gene expression were log-normalized. The top 2,000 highly variable genes (HVGs) were identified using the “FindVariableFeatures” function with the “vst” method. Mouse homologs for a set of 293 immune-related, “immunoStates” human genes (Vallania et al., Nat. Commun.9 (2018)) were added to the HVG list, while immunoglobulin and T cell receptor genes were removed.
  • the mouse homologs were obtained by first looking up the Human and Mouse Homology Class report from Mouse Genome Informatics (MGI), accessed on 2020-04-06, and then manually searching NCBI Gene for the human genes for which MGI reported no mouse homolog. The data was then scaled and centered, and principal component analysis (PCA) was performed based on the expression of the HVGs. PCA-guided t-distributed stochastic neighbor embedding (tSNE) was performed using the top 20 principal components.
  • Gene expression-based clusters were identified using the “FindClusters” function with resolution 0.05. Differentially expressed genes for each cluster were identified via the “FindAllMarkers” function using Wilcoxon Rank Sum tests, followed by Bonferroni correction for multiple testing.
  • the identities of the clusters were assigned by examining the expression of canonical marker genes and differentially expressed genes.
  • the plasmablast clusters were based on high expression of Cd79a, Cd79b, Xbp1, Sdc1, and Fkbp11.
  • One of the plasmablast clusters was highly proliferating based on high expression of Mki67, Top2a, Cdk1, Ccna2, and Cdca3.
  • the T cell cluster was based on high expression of Cd8b1, Ms4a4b, Cd3d, Cd3e, Ccr7, and Il7r.
  • SARS-CoV-2 neutralization assay [0425] The SARS-CoV-2 neutralization assay was performed as follows.
  • ACE2 competition assay [0426] The ACE2 competition binding assay was performed at 25°C on an Octet Red bilayer interferometry (BLI) instrument using anti-human IgG Fc biosensors to capture target antibody.
  • antibodies were loaded onto anti-human IgG Fc pins for 3 min at 10 ⁇ g/mL in assay buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant with 3% BSA). Unbound antibodies were washed away, and the IgG-loaded tips were dipped into RBD- containing wells for 1 min or 3 min, followed by immersion into wells containing 1 ⁇ M ACE2 protein. The mAbs were considered competing if no additional BLI signal was observed compared to control mAb hE16 (humanized West Nile virus-specific mAb), whereas increased signal indicated inability of mAbs to block RBD binding to ACE2.
  • assay buffer 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant with 3% BSA. Unbound antibodies were washed away, and the IgG-loaded tips were dipped into RBD- containing
  • mice Eight-week old BALB/cJ mice were administered 2 mg of anti-IFNAR1 (MAR1-5A3) via intraperitoneal injection 24 hours prior to intranasal administration of 2.5 x 10 8 PFU of AdV-hACE2. Five days later, mice were inoculated intranasally with 4x10 5 FFU of SARS-CoV-2. Weight was monitored daily, animals were euthanized 4 days post infection, and tissues were harvested to measure viral burden. Collected tissues were weighed and homogenized with zirconia beads in a MagNA Lyser instrument in 1mL of DMEM media supplemented with 2% heat-inactivated FBS.
  • Example 2 Generating monoclonal antibodies specific for SARS-2 RBD.
  • mice Two mice were immunized intramuscularly (i.m.) with 10 ⁇ g of recombinant SARS-CoV-2 RBD in squalene-based adjuvant. Fourteen days after primary immunization, mice were boosted twice with 5 ⁇ g of recombinant SARS-CoV-2 S protein, at a 10-day interval (FIG.1A). Serum antibody binding to SARS-CoV-2 recombinant trimeric S protein or RBD was measured by enzyme-linked immunosorbent assay (ELISA) 5 days after the final booster immunization. Serum from both mice demonstrated potent binding to both SARS-CoV-2 RBD and S protein (FIG.1B).
  • ELISA enzyme-linked immunosorbent assay
  • Serum samples from the immunized mice were also evaluated for neutralization of a SARS-CoV-2 isolate (2019 n-CoV/USA_WA1/2020). Potent neutralizing activity against SARS-CoV-2 was found for both mice in a focus reduction neutralization test (FRNT) (FIG.1C). These results show that this immunization induced RBD and S protein-specific and neutralizing antibody responses.
  • FRNT focus reduction neutralization test
  • PBs plasmablasts
  • scRNA-seq single-cell RNA-seq
  • IGHV immunoglobulin heavy
  • IGKV kappa
  • IGLV lambda
  • Example 3 Cross-reactivity and neutralization of anti-RBD mAbs
  • SARS-CoV-2, SARS-CoV, and MERS-CoV S proteins were tested for their binding to SARS- CoV-2, SARS-CoV, and MERS-CoV S proteins to determine if the epitopes they recognize.
  • the 19 mAbs bound recombinant SARS-CoV- 2 S protein, with five (2C02, 2E06, 1C05, 1C07, and 2E10) recognizing SARS-CoV, but none binding to MERS-CoV S protein (FIG.3A, FIG.3B, FIG.3C).
  • the five cross-reactive mAbs recognized the SARS-CoV RBD (FIG. 4A, FIG. 4B, FIG.4C).
  • Example 4 Characterization of RBD+/RBD- sorted plasmablasts
  • Bulk-sorted total PBs were analyzed using scRNA-seq to further characterize the transcriptional profile, isotype distribution and somatic hypermutations (SHM) among responding PBs.
  • SHM somatic hypermutations
  • Gene expression-based clustering of PBs revealed two populations, Ki67hi and Ki67low, corresponding to proliferation states among responding PBs (FIG.5A, FIG. 6A-FIG.6B).
  • BCR B cell receptor
  • Example 5 In vivo protection by mAb 2B04 [0432] A mouse model of SARS-CoV-2 infection in which hACE2 is transiently expressed via a non-replicating adenoviral vector (hACE2-AdV) (Jia et al., J. Virol.79, 14614–14621 (2005)) was used to assess the protective capacity of 2B04 in vivo.
  • hACE2-AdV non-replicating adenoviral vector
  • mice were transduced with hACE2-AdV via intranasal (i.n.) administration to establish receptor expression in lung tissues. Animals then received 10 mg/kg 2B04 or isotype control via intraperitoneal (i.p.) injection one day before infection with the SARS-CoV-2 strain 2019 n-CoV/USA_WA1/2020 (FIG.7A). Mice receiving 2B04 lost significantly less body weight compared to those receiving isotype control mAb (FIG.7B). Compared to the isotype control mAb-treated mice, animals receiving 2B04 had 31- and 11-fold lower median levels of viral RNA in the lung and spleen, respectively (FIG.7C).
  • Example 6 Methods for Examples 7-12 [0433] The following methods are directed to Examples 7-12.
  • Protein production and purification method [0434] Genes encoding SARS-CoV-2-spike (residues 1-1213, GenBank: MN908947.3) and RBD (residues 319-541) were cloned into a mammalian expression vector with a C-terminal hexahistidine tag.
  • RBD variants were made using a Q5 Site-Directed Mutagenesis Kit (NEB), and were expressed and purified as described for WT RBD.
  • Genes encoding human ACE2 (hACE2 residues 1-615) were synthesized (IDT) and placed into a mammalian expression vector with a C-terminal HRV-3C protease cleavage site and a human Fc fragment, as previously reported (Case et al., Cell Host & Microbe 28, 475-485.e5 (2020)).
  • the vector was transfected into Expi293F cells using FectoPRO (Poly-plus) and hACE2-hFc was purified from culture supernatants 4 days post transfection by affinity chromatography using protein A resin. Monomeric hACE2 was generated by incubating hACE2-hFc with HRV-3C protease overnight at 4°C. hACE2 was subsequently purified by passage over a protein A column to remove cleaved Fc and further purified through size exclusion chromatography.
  • Cryo-EM sample preparation, data collection, and data processing method [0436] For standard lacey carbon grid datasets, trimeric SARS-CoV-2 spike at a concentration of 1 mg/mL in 20 mM HEPES pH 7.5, 150 mM NaCl was combined with 1 molar equivalent of a papain-cleaved Fab form of either 2B04 or 2H04 in 20 mM HEPES pH 7.5, 150 mM NaCl, and incubated for 10 minutes before flash-freezing on lacey carbon grids using a Vitrobot Mk IV.
  • SARS-CoV-2 spike was diluted to 0.2 mg/mL prior to mixing with 1 molar equivalent of either 2B04 or 2H04 Fab and then vitrified on thin-film lacey carbon grids using a Vitrobot Mk IV. Both sets of grids were glow discharged prior to sample application in a GloQube for at least 20 seconds under vacuum. [0437] Frozen grids were transferred to a Cs-corrected FEI Titan Krios 300KV microscope equipped with a Gatan K2 Summit detector mounted on a BioQuantum 968 energy filter operating in zero loss mode with a slit width of 20 eV.
  • Movies were collected at a nominal magnification of 105,000X resulting in a pixel size of 1.1 ⁇ /pixel, with 45 frames per movie at 200ms each with a dose of 1.49 e-/ ⁇ 2 /frame, resulting in a total dose of 66.9 e- / ⁇ 2 /movie.
  • Raw movies for both datasets were motion corrected using MotionCor2 v1.3.1.
  • Micrograph contrast transfer function correction parameters were estimated using GCTF v1.06.
  • Particles were picked on each dataset using CrYOLO v1.7.1 employing a general model.
  • Particles were first subjected to 2D classification in relion 3.1 (Scheres, J Mol Biol 415, 406–418 (2012); Zivanov et al., ELife 7, e42166 (2016)). Good 2D classes from both holey lacey carbon and thin-film lacey carbon particle sets were picked and merged for both 2B04 and 2H04. The merged particle datasets were then subjected to 3D classification using a 7.5 degree angular search for 25 iterations followed by 25 iterations of local searches at 1.8 degree sampling to separate spike conformational states, as previously described (Walls et al., Cell 181, 281-292.e6 (2020)).
  • the initial model for the RBD for 2B04 was taken from the crystal structure of Fab CR3022 bound to SARS-CoV-2 RBD (PDB 6W41), while for 2H04 it was derived from the cryo-EM structure of SARS-CoV-2 spike (PDB 6VXX). All starting model components were combined and rigid body fit into their respective maps. Model building and refinement were performed with Coot 0.91, Isolde v0.93, and phenix.
  • Density maps used to build models were deposited in the EMDB with the following accession numbers: 2B04 U/D/D as EMD-22748, 2B04/RBD locally refined as EMD-22749, 2H04 D/D/D as EMD-22750, and 2H04/RBD locally refined as EMD-22751. Models built with these maps were deposited in the PDB with the following accession codes: 2B04 U/D/D as 7K9H, 2B04/RBD locally refined as 7K9I, 2H04 D/D/D as 7K9J, and 2H04/RBD locally refined as 7K9K.
  • the pins were dipped into running buffer (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant with 3% BSA) containing 500 nM recombinant RBDs (RBD343-346, RBD486-490, RBD500-505 and RBDWT, respectively) to measure association, followed by a dissociation step in running buffer alone.
  • running buffer 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.005% P20 surfactant with 3% BSA
  • 500 nM recombinant RBDs (RBD343-346, RBD486-490, RBD500-505 and RBDWT, respectively)
  • Anti-human IgG Fc biosensors were loaded with 2B04 or 2H04 IgG (10 ⁇ g/mL), with a parallel control of buffer alone to monitor non-specific binding. After a 30 second wash, the sensors were immersed into RBD-containing wells for 1 minute to capture RBD molecules, followed by immersion into buffer containing either 2H04 Fab, 2B04 Fab, or monomeric ACE2 for 1 minute, with binding curves recorded in real time. The two tested subunits were considered competitive if no increase in BLI signal was observed.
  • SARS-CoV-2 (2019 n-CoV/USA_WA1/2020) was obtained from the Centers for Disease Control and amplified in Vero CCL81 cells. Chimeric VSV-SARS-CoV-2 stocks were generated as previously described (Case et al., Cell Host & Microbe 28, 475-485.e5 (2020)). The chimeric virus was amplified on MA104 cells, and neutralization assays were performed on Vero E6-TMPRSS2 cells. [0444] For the pre-attachment assay, serially diluted 2B04 or 2H04 were incubated with GFP expressing chimeric VSV-SARS-CoV-2 for 1 hour at 4°C.
  • the virus-mAb mixture was subsequently added to pre-cooled Vero E6-TMPRSS2 cells and incubated for 1 hour.
  • Cells were washed three times with cold DMEM to remove unbound virus, and the plates were transferred to a 37°C incubator with 5% CO2.8 hours post-infection, the cells were trypsinized (Trypsin-EDTA) and fixed with 4% paraformaldehyde.
  • Infection frequency was quantified by measuring the number of GFP-positive infected cells via flow cytometry.
  • the virus was first incubated with chilled cells at 4°C.
  • Diluted mAbs (2B04 or 2H04) were premixed with SARS- CoV-2 (MOI of 0.01) and incubated for 1 hour at 4°C, followed by addition of the mAb-virus mixture to chilled Vero E6-TMPRSS2 cells for 1 hour at 4°C.
  • Virus alone and a control antibody humanized anti-West Nile virus mAb, hE16 were included.
  • Cells were then rinsed 4 times with chilled DMEM and once with PBS on ice before total cellular RNA extraction using a MagMAXTM mirVanaTM Total RNA Isolation Kit. Viral RNA levels were measured by qRT-PCR on an ABI 7500 Real Time-PCR system and normalized to an internal GAPDH control.
  • a focused classification using a mask enclosing one 'down' RBD and variable fragment (F V ) of the Fab in the case of 2B04, or all three subunits in the case of 2H04 was performed to identify populations of particles that had well-ordered RBD/FV regions relative to the rest of the spike protein (FIG. 10A- FIG.10B).
  • Local non-uniform refinement of the particles from the focused classification in cryoSPARC led to a 3.3 ⁇ reconstruction of the RBD/FV complex of 2B04, and a 3.14 ⁇ map for the RBD/FV complex of 2H04 (FIG.12A- FIG.12B).
  • CDR1 and CDR2 of the heavy chain spread across the ridge formed by the ⁇ 5 and ⁇ 6 strands (residues 445-454 and 491-498, respectively) of the RBD and press up against the flexible RBM loop (residues 472-490), while CDR-H3 mainly contacts the RBM loop (FIG.13A).
  • CDR1 and CDR3 of the light chain also interact with the flexible RBM loop (FIG.13A), although the majority of contacts derive from the heavy chain of 2B04 (FIG.13B).
  • the total buried surface area of the complex is 656 ⁇ 2 , with 92% of the area covered by the heavy chain.
  • the epitope is formed by a discontinuous set of strands adjacent to the RBM.2H04 CDR-H1, -H2, and - H3 contact a loop adjacent to the RBM encoded by residues 439-450 (FIG.13C).
  • CDR-L1, - L2, and -L3 target the region encoded by residues 339-353 (FIG.13C).
  • CDR-L3 and residues 60-62 of the heavy chain also make extensive contacts with an ⁇ -1,6-fucose linked to the glycan on N343 (FIG.14).
  • 2H04 shows more extensive utilization of the light chain (FIG.13D).
  • the total buried surface area at the interface of 2H04 and the RBD is 914 ⁇ 2 , with 60% coming from the interaction of the heavy chain.
  • Alignment of the 2H04 bound RBD with the 2B04 bound RBD showed that the Fabs have largely distinct epitope footprints, with only one overlapping residue, N450 (FIG.13E). Binding of 2B04, but not 2H04, was significantly reduced when residues 486 FNCYF 490 of the RBD were mutated to 486 AACAA 490 (FIG.13F). In contrast, binding of 2H04, but not 2B04, to the RBD was eliminated by mutating residues 343-346 to alanines (FIG.13F).
  • Example 9 2B04 and 2H04 share epitopes with human antibodies [0451]
  • Several groups have identified epitopes on the RBD of the SARS-CoV-2 spike protein (Barnes et al., Cell 182, 828-842.e16 (2020); Cao et al., Cell 182, 73-84.e16 (2020); Hansen et al., Science 369, 1010–1014 (2020); Hurlburt et al., BioRxiv (2020); Ju et al., Nature 584, 115–119 (2020); Liu et al., Nature 584, 450–456 (2020); Lv et al., Science 369, 1505–1509 (2020); Pinto et al., Nature 583, 290–295 (2020); Shi et al., Nature 584, 120–124 (2020); Wu et al., Cell Reports 108274 (2020a); Wu et al., Science 368, 1274–12
  • the epitopes of 2B04 and 2H04 were compared with those of other antibodies to identify conserved modes of engagement.
  • the structures of all human antibody-bound SARS-CoV-2 spike or RBD structures published in the RCSB protein databank were aligned with our 2B04/RBD and 2H04/RBD complexes.
  • RBM antibodies there were five distinct clusters of epitopes on RBD (FIG.15A), and 2B04 greatly overlapped with only one other antibody, 2-4 (FIG. 15A) (Liu et al., Nature 584, 450–456 (2020)).
  • Antibodies targeting the RBM ridge and loop were differentiated by their orientation of binding on the ridge but shared a common mechanism of action and tend to display potent neutralization.
  • Antibodies targeting RBM-adjacent (i.e., 2H04, S309, REGN-10987, and H014) or RBM-distal (i.e., CR3022 and EY6A) epitopes were differentiated by their rotation about the core RBM beta sheet, and generally showed less potent neutralization than RBM antibodies along with non-ACE2-competitive mechanisms of inhibition.
  • SARS-CoV-2 can escape neutralization by both 2B04 and 2H04 [0455] Escape from antibody-mediated neutralization by mutation of key binding residues is a feature of RNA viruses that hinders therapeutic development. A wide variety of amino acid substitutions in the spike protein of SARS-CoV-2 have been observed in human infections, suggesting that SARS-CoV-2 might be able to escape antibody-mediated neutralization.
  • T345, R346, L441, and K444 were found to lie outside the binding site for ACE2 (FIG.17C) and were all contained within the epitope for 2H04 (FIG.17D), whereas E484 and F486 were found to lie in the 2B04 footprint, with F486 also acting as an ACE2 contact residue (FIG.17E, FIG.17F). Although these variants were found to be present at low frequency in human populations (FIG.17B), these results show that SARS- CoV-2 can generate resistance to 2B04, 2H04, and other antibodies when used as monotherapies, which could result in expansion of resistant strains under prolonged selection pressure. Table 7 is shown below for reference. Table 7: SARS-CoV-2 isolates with mutations at 2B04 and 2H04 escape residues
  • Example 11 Mechanism of SARS-CoV-2 neutralization by 2B04 and 2H04 [0456] Structural analysis shows 2B04 potently neutralizes SARS-CoV-2 by blocking ACE2 binding, while 2H04 neutralizes less potently without blocking ACE2 (FIG.18A, FIG.18B). To determine why 2H04 is less efficient at neutralizing SARS2-CoV-2 compared to 2B04, the binding affinities of these two mAbs to recombinant RBD expressed in E.
  • coli were found by biolayer interferometry (BLI).2B04 tightly bound RBD with a kinetic KD value of 1.05 nM, and a relatively slow dissociation rate (half-life of 23.70 min) (FIG.18C). While 2H04 bound RBD strongly, it displayed weaker affinity (KD of 40.05 nM) and a faster off rate (half-life of 1.27 min) than 2B04 (FIG.18D). Since 2H04 recognizes the glycan of residue N343 (FIG.14), the binding of these two mAbs to RBD expressed in mammalian cells was also assessed (FIG.16A, FIG.16B).
  • Pre- and post- attachment neutralization assays were performed using a GFP- expressing replication-competent VSV-SARS-CoV-2 chimeric virus on Vero E6-TMPRSS2 cells.2B04 and 2H04 were incubated with VSV-SARS-CoV-2 before or after virus adsorption to the cell surface, and infection was monitored 8 hours post-infection by flow cytometry.2B04 efficiently inhibited VSV-SARS-CoV-2 infection when mixed with the virus before or after cell attachment (FIG. 18F).2H04, which did not compete with ACE2 for the binding of RBD (FIG.18E), neutralized infection more efficiently when added before virus attachment to cells.
  • FIG. 18H Structural (FIG. 18H) and mechanistic (FIG. 18I) are also shown.
  • Example 12 Comparison of 2B04 and 2H04 to germline Ig sequences. [0460] IgBLAST analysis of 2B04 shows a similarly low number of somatic mutations, with only 3 amino acid substitutions in the heavy chain and 2 in the light chain (FIG.19A, FIG.19B). In contrast, 2H04 showed 6 heavy chain mutations and 5 light chain mutations (FIG.19C, FIG.19D). Also notable is the conservation of paratope contact residues.

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Abstract

La présente invention concerne des anticorps ou des fragments de liaison à l'antigène qui sont utiles pour le traitement des infections au coronavirus (p.ex., COVID-19 provqué par SARS-CoV-2). La présente invention concerne également diverses compositions pharmaceutiques et des méthodes de traitement du coronavirus à l'aide des anticorps ou des fragments de liaison à l'antigène.
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WO2023070310A1 (fr) * 2021-10-26 2023-05-04 中国科学院深圳先进技术研究院 Anticorps monoclonal entièrement humanisé anti-sars-cov-2 et son procédé de préparation ainsi que son application
WO2023077429A1 (fr) * 2021-11-05 2023-05-11 Shanghaitech University Anticorps trimères dirigés contre la protéine de spicule de sars-cov-2
EP4183800A1 (fr) * 2021-11-19 2023-05-24 Medizinische Hochschule Hannover Nouveaux anticorps neutralisants du sars-cov-2
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WO2023122786A3 (fr) * 2021-12-23 2023-09-14 Novavax, Inc. Anticorps de spicule (s) anti-sars-cov-2 et leur utilisation dans le traitement du covid-19
WO2023133360A3 (fr) * 2022-01-10 2023-11-16 Phenomic Ai Anticorps anti-protéine 1 à répétition triple hélice de collagène (cthrc1) et méthodes d'utilisation associées
CN114790240A (zh) * 2022-06-10 2022-07-26 郑州大学 SARS-CoV-2中和性单克隆抗体及应用
CN114790240B (zh) * 2022-06-10 2023-06-06 郑州大学 SARS-CoV-2中和性单克隆抗体及应用
WO2024021839A1 (fr) * 2022-07-26 2024-02-01 北京昌平实验室 Anticorps monoclonal humanisé ayant une stabilité améliorée et son utilisation

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