WO2022051650A1 - Human monoclonal antibodies to sars-cov-2 and use thereof - Google Patents

Human monoclonal antibodies to sars-cov-2 and use thereof Download PDF

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Publication number
WO2022051650A1
WO2022051650A1 PCT/US2021/049112 US2021049112W WO2022051650A1 WO 2022051650 A1 WO2022051650 A1 WO 2022051650A1 US 2021049112 W US2021049112 W US 2021049112W WO 2022051650 A1 WO2022051650 A1 WO 2022051650A1
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amino acid
seq
antigen
cov
antibody
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PCT/US2021/049112
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French (fr)
Inventor
James KOBIE
Mark R. Walter
Michael PIEPENBRINK
Ashlesha DESHPANDE
Paul GOPEFERT
Nathaniel ERDMANN
Luis MARTINEZ-SOBRIDO
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The Uab Research Foundation
Texas Biomedical Research Institute
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Priority to CN202180065874.3A priority Critical patent/CN116615450A/en
Priority to EP21865212.1A priority patent/EP4208479A1/en
Priority to AU2021338380A priority patent/AU2021338380A1/en
Priority to JP2023514983A priority patent/JP2023540759A/en
Priority to CA3190311A priority patent/CA3190311A1/en
Publication of WO2022051650A1 publication Critical patent/WO2022051650A1/en

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    • 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
    • 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
    • A61K2039/507—Comprising a combination of two or more separate antibodies
    • 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
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A61K2039/544—Mucosal route to the airways
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • 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
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • Coronaviruses are members of the family Coronaviridae.
  • the Coronaviridae has 4 separate genera, the Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus with each genus having one or more subgenus.
  • the Alphacoronaviruses and Betacoronaviruses mainly infect bats, but they also infect other species such as humans, camels, rabbits, dogs and masked palm civets.
  • CoVs are enveloped viruses that possess extraordinarily large single-stranded RNA genomes ranging from 26 to 32 kilobases in length. CoVs were historically regarded as pathogens that only cause mild diseases. Currently, at least seven CoV species are known to cause diseases in humans.
  • HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKUl generally cause only mild common cold symptoms. Severe illness can be caused by the remaining three viruses, each in the Betacoronavirus genus. SARS-CoV resulted in the outbreak of sever acute respiratory syndrome (SARS) in 2002 and 2003. MERS-CoV was responsible for Middle East respiratory syndrome (MERS), which emerged in 2012 and remains in circulation in camels. A small outbreak of MERS-CoV was reported on June 2, 2020 in Saudi Arabia (9 case reported with 5 deaths). Finally, SARS-CoV-2, which emerged in December 2019 in Wuhan province of China, and causes COVID-19. The COVID-19 pandemic is impacting virtually every country around the world, with no immediate prospects for easing. The US is currently topping the world in the number of infected people with more cases in sight.
  • SARS-CoV-2 has a basic reproduction rate (Ro) of 3.3-5.5, which is higher than those of SARS-CoV and MERS-CoV (2.7-3.9), indicating a higher transmissibility of SARS-CoV-2 than other human coronaviruses.
  • Ro basic reproduction rate
  • COVID 19 has currently infected more than 16,000,000 individuals and has been responsible for over 650,000 deaths worldwide.
  • FIG. 1 shows the ELISA binding profile of antibodies of the disclosure for binding to recombinant SARS-CoV-2 spike proteins.
  • FIG. 2 shows binding of antibodies of the disclosure to SARS-CoV-2 infected cells.
  • FIG. 3 depicts the high affinity of certain mAbs for SARS-CoV-2 Spike Receptor Binding Domain. Binding to SARS-CoV-2 RBD was determined by surface plasmon resonance (SPR).
  • FIGS. 4A-4D depict rationally designed 1212C2 mAb variants with increased neutralization activity.
  • Fig. 4A shows the sequences of 1212C2 mAb and variants thereof.
  • Fig. 4B, 4C and 4D show viral neutralization by 1212C2, 1212C2-V1, and 1212C2-V2 mAbs, respectively.
  • FIG. 5A-5D shows prophylactic and therapeutic activity of 1212C2 hmAb in SARS-CoV- 2-infected hamsters.
  • Golden Syrian hamsters were (Fig. 5 A) prophylactically treated i.p. with 10 mg/kg indicated hmAb or PBS 6 h before intranasal (i.n.) challenge with 2 x 10 5 PFU SARS-CoV- 2 or (Fig. 5B) therapeutically treated by intraperitoneal (i.p.) injection with 25 mg/kg indicated hmAb or PBS 6 h following i.n. challenge with SARS-CoV-2. Virus present in nasal turbinates and lungs was determined by plaque assay. Dotted line indicates limit of detection.
  • FIGS. 6A - 6C show therapeutic activity of inhaled 1212C2 hmAb in SARS-CoV-2- infected hamsters.
  • Golden Syrian hamsters were infected i.n. with 2 x 105 PFU SARS-CoV-2 and 12 h later treated with indicated hmAb by i.p. or inhaled administration.
  • Virus present in nasal turbinates and lungs was determined by plaque assay at 2 dpi (Fig. 6A) and 4 dpi (Fig. 6B). Dotted line indicates limit of detection.
  • Fig. 6C Distribution of pathologic lesion were measured using ImageJ and represented as the percentage of the total lung surface area. *p ⁇ 0.0055 and **p ⁇ 0.00055 compared to PBS i.p. -treated group determined by 2-tailed t test.
  • FIG. 7 shows the preservation of body weight with inhaled therapeutic treatment of SARS-
  • FIGS. 8A-8H show prophylactic activity of 1212C2 in mice infected with SARS-CoV-2.
  • Figs. 8 A and 8B five-week-old KI 8 hACE2 transgenic mice were injected with isotype IgG control or 1212C2 MAbs and 12 h after treatment, mice were infected (10 5 PFU/mouse) with recombiant SARS-CoV-2/Nluc-2A (Nluc-2A). Mock-treated and mock-infected mice were included as controls. Mice were anesthetized at 1, 2, 4 and 6 dpi and retro-orbitally injected with the Nluc substrate.
  • Nluc expression was determined using an IVIS system (Fig. 8A) and quantitively analyzed by the Aura program (Fig. 8B). The lungs were excised and photographed at 1, 2, 4 and 6 dpi (Fig. 8C) and the gross lesions on the lung surfaces were quantitively analyzed by ImageJ (Fig. 8D). ns, not significant. Nluc activity in the nasal turbinate (left), lungs (middle) and brains (right) from infected mice were measured using a multi-plate reader (Fig. 8E). Viral titers in the nasal turbinate (left), lungs (middle) and brain (right) were determined by plaque assay (Fig. 8F).
  • mice Five-week-old K18 hACE2 transgenic mice were injected with isotype IgG control or 1212C2 MAbs and 12 h after treatment, mice were infected (10s PFU/mouse) with rSARS-CoV-2/Nluc-2A (Nluc-2A). Mock-treated and mock-infected mice were included as controls. Mice were monitored for 12 days for changes in body weight 1 (Fig. 8G) and survival (Fig. 8H).
  • FIGS. 9A and 9B show prophylactic activity of 1212C2 and 1213H7 against SARS-CoV- 2 WT and SARS-CoV-2 Beta (SA) in KI 8 hACE2 transgenic mice.
  • FIGS. 10A and 10B show inhibition of SARS-CoV-2 lung viral burden in mice by 1212C2 and 1213H7.
  • lungs were collected to determine Venus and mCherry fluorescence expression using an Ami HT imaging system (A).
  • A Ami HT imaging system
  • B Mean values were normalized to the autofluorescence in mock-infected mice at each time point and were used to calculate fold induction.
  • Gross pathological scores in the lungs of mock-infected and rSARS-CoV- 2-infected KI 8 hACE2 transgenic mice were calculated based on the % area of the lungs affected by infection.
  • FIGS. 11A-11D show viral titers in the lungs, nasal turbinate and brain of KI 8 hACE2 transgenic mice treated with 1212C2 or 1213H7 mAbs and infected with rSARS- CoV-2 WT and rSARS-CoV-2 Beta.
  • Viral titers in the lungs (top), nasal turbinate (middle) and brain (bottom) at days 2 and 4 pi were determined by plaque assay in Vero E6 cells. Bars indicates the mean and SD of lung virus titers. Dotted lines indicate the limit of detection.
  • Figure 11D shows quantification of rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) in the lungs (top), nasal turbinate (middle) and brain (bottom) from mice co-infected with both rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) at days 2 and 4 pi.
  • FIG. 12 shows mAb binding to RBD variants.
  • SARS-CoV-2 RBD variants 50nM were injected over mAbs captured on a biacore chip and their binding levels measured and normalized to the binding response to a reference (WT) Wuhan-1 RBD.
  • WT reference
  • Two WT RBD binding experiments were performed at the beginning and end of the experiment to validate the reproducibility of the assay.
  • mAb binding is presented in the matrix as % of WT binding.
  • FIGS. 13A-13D show mAb epitope mapping. Competition epitope mapping using SPR, combined with the previously described variant analysis were used to define the binding epitopes of the mAbs.
  • FIGS. 13A-13C represent 3 distinct competition mapping experiments performed with hmAbs assigned to different epitope classes Cl, CID, C2, or C4.
  • FIG. 13D shows structural models of the mAb/RBD complexes that explain the epitope mapping and variant binding data. Protein data bank (PDB) entries used to model the mAb/RBD complexes are underlined in the Figure.
  • PDB Protein data bank
  • FIG. 14 shows MARM analysis of SARS-CoV-2 mAbs.
  • Vero E6 cells were infected with SARS-CoV-2 WA-1 and incubated with decreasing concentration of mAb.
  • Virus that emerged resistant to the indicated mAb was tested for recognition by the mAb using immuno-fluorescence assay (top).
  • the spike of the MARMs was sequenced and mutation noted. Mutations within the RBD are indicated in bold (bottom).
  • FIG. 15 shows the results of study 2020-20, in particular differential biodistribution and titer of an IgG mAb in serum, bronchial tissue and lung at 0.5, 6, 12 and 24hrs post administration in mouse after administration by IV, IN and IH routes.
  • FIGS. 16A-16G show representative wells and mAh dose plotted against percent viral infection pre- and post-treatment for a neutralizing antibody assay involving treatment with 1212C2 (A), 1213H7 (B), 1215D1 (C), 1212C2+1213H7 (D), 1213H7+1215D1 (E), 1212C2+1215D1 (F), and 1212C2+1213H7+1215D1 (G).
  • the disclosure provides for broadly neutralizing anti-SARS-CoV-2 monoclonal antibodies and antigen-binding fragments thereof.
  • the disclosure further provides for pharmaceutical compositions comprising an antibody of the disclosure and methods of using the antibodies of the disclosure.
  • the antibodies of the disclosure are shown to be effective in preventing a SARS-CoV- 2 infection as well as in treating a SARS-CoV-2 infection.
  • the antibodies of the disclosure bind epitopes within the spike protein of SARS-CoV-2.
  • the antibodies of the disclosure bind an epitope within the receptor binding domain (RBD) of the SARS-CoV-2 spike protein.
  • the antibodies of the disclosure bind an epitope within the receptor binding moiety (RBM) of the SARS-CoV-2 spike protein.
  • the antibodies of the disclosure bind an epitope within the S 1 region of the SARS-CoV-2 spike protein. In certain embodiments, the antibodies of the disclosure bind an epitope within the S2 region of the SARS-CoV-2 spike protein.
  • the antibodies of the disclosure reduce binding of SARS-CoV-2 to a target cell. In certain embodiments, the antibodies of the disclosure reduce cellular fusion between SARS-CoV-2 and a target cell. In certain embodiments, the antibodies of the disclosure reduce release of infective SARS-CoV-2 from an infected cell. In certain embodiments, the antibodies of the disclosure reduce infection of a target cell by SARS-CoV-2.
  • the antibodies of the disclosure were isolated from a subject with a documented SARS-CoV-2 infection, wherein the subject subsequently recovered from the SARS-CoV-2 infection. As such, the subject mounted an effective immunological response to the SARS-CoV-2 infection. Using single-cell immunoglobulin cloning, neutralizing antibodies to SARS-CoV-2 were isolated from the subject.
  • the term “about” refers to within 10%, preferably within 5%, and more preferably within 1% of a given value or range. Alternatively, the term “about” refers to within an acceptable standard error of the mean, when considered by one of ordinary skill in the art.
  • affinity refers to the strength of the sum total of noncovalent interactions between a single binding site of an antibody or other molecule and its binding partner (such as, but not limited to, an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1 : 1 interaction between an antibody and an antigen or between members of a binding pair. Affinity is generally be represented by the dissociation constant (KD).
  • antibody includes whole antibodies and any antigen binding fragment thereof.
  • examples of an antibody include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) formed from at least two antibodies or antigen binding fragments thereof, chimeric antibodies, anti -idiotypic (anti-Id) antibodies, intrabodies, and antigen binding fragments of any of the foregoing,
  • Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain is comprised of a heavy chain variable (VH) region and a heavy chain constant (CH) region.
  • the CH region is comprised of three to four domains, CHI, CH2, CH3, and CH4.
  • Each light chain is comprised of a light chain variable (VL) region and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the VH and VL regions form a binding domain that interacts with an antigen in an antigen-specific manner.
  • the CH and CL regions mediate binding of the antibody to host tissues, cells or factors, including various cells of the immune system (e.g, effector cells) and the first component (Clq) of the classical complement system.
  • Antibodies may be of any type, including IgG, IgE, IgM, IgD, IgA and IgY and of any class, including, class IgGl, IgG2, IgG3, IgG4, IgAl and IgA2 or subclass.
  • the terms “antigen-binding fragment” or “antigen-binding portion” refer to one or more fragments derived from an antibody described herein (a parent antibody) that retain the ability to specifically bind to the same antigen as the parent antibody.
  • binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains), a F(ab)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fab' fragment (an Fab fragment comprising a portion of the hinge region), a F(ab’)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region and containing a portion of the hinge region) a Fd fragment (a monovalent fragment consisting of the VH and CHI domains), a Fv fragment (a monovalent fragment consisting of the VL and VH domains of a single arm of an antibody
  • the terms also include single chain Fv (scFv) which are created by recombinantly joining the VH and VL genes by a synthetic linker and expressed as a single polypeptide.
  • scFv include, but are not limited to, scFv-FC, scFv- CH, scFab, and scFv-zipper.
  • An antigen-binding fragment as described herein may be obtained using conventional methods known in the art and tested for binding as is done with conventional whole antibodies. Suitable antigen-binding fragments are described in Pluckthun (The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp.
  • an antibody of the disclosure means an antibody disclosed herein, and includes pharmaceutically acceptable forms thereof, such as, but not limited to, pharmaceutically acceptable salts, hydrates and/or solvates.
  • an antibody of the disclosure is an antigen-binding fragment.
  • antibody variant refers to any modified form of an antibody described herein, such as, but not limited to, an antibody having one or more substitutions, deletions or insertions relative to a parental antibody and an antibody linked to a protein or nonprotein moiety.
  • the terms “binds to an epitope” or “recognizes an epitope” with reference to an antibody refers to the epitope bound by the antibody. The term does not require the antibody to directly contact every amino acid within the epitope.
  • the term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same or overlapping amino acids, whether such amino acids are continuous or discontinuous segments. The term does not require the antibodies bind to or contact exactly the same amino acids. The precise amino acids which the antibodies contact can differ.
  • a first antibody can bind to a group of amino acids that is completely encompassed by the group of amino acids bound by a second antibody.
  • a first antibody can bind to a group of amino acids that overlap with a group of amino acids bound by a second antibody.
  • chimeric antibody refers to an antibody in which at least a portion of the variable region sequences (including CDR and FR sequences or just CDR sequences) are derived from one species (for example, a rat) and the constant region sequences are derived from another species (for example, a human).
  • the term also includes an antibody in which its variable region sequence or CDR(s) is derived from one source (e.g., an IgAl antibody) and the constant region sequence or Fc is derived from a different source (e.g., a different antibody, such as an IgG, IgA2, IgD, IgE or IgM antibody).
  • Chimeric antibodies are described in U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
  • the term “detectable label” refers to a molecule capable of being detected in a subject or an assay, including, but not limited to, radioactive isotopes, fluorescent compounds, chemiluminescent compounds, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands, intercalating dyes and the like.
  • epitope refers to an antigenic determinant that interacts with (is bound by) a specific antigen binding site in the variable region of an antibody molecule (the paratope).
  • a single antigen such as, but not limited to, a polypeptide
  • different antibodies may bind to different epitopes on an antigen and may have different biological effects depending on which epitope is bound.
  • epitope also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody.
  • Epitopes may be defined as a structural epitope (the portion of the antigenic determinant that is contacted by the CDR loops of an antibody) or a functional epitope (a subset of a structural epitope comprising those energetic residues centrally located in the structural epitope and directly contribute to the affinity of the antibody-epitope interaction). Epitopes may become immunologically available after fragmentation or denaturation of an antigen (a cryptotope). Epitopes may be linear or conformational (composed of non-linear amino acids brought together in a folded three-dimensional structure).
  • Epitopes may include residues that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics.
  • An epitope typically includes at least 3 to 15 amino acids.
  • epitope mapping refers to the process of identification of an epitope for antibody-antigen recognition.
  • the term “functional equivalent” with reference to an antibody disclosed herein refers to an antibody variant of a parent antibody that retains one or more characteristics (such as, but not limited to, binding to the same epitope) of the parent antibody as disclosed herein.
  • a functional equivalent may optionally differ in one or more characteristics of the parent antibody (such as, but not limited to, binding affinity, ADCC, and/or CDC).
  • human antibody refers to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term human antibody is not intended to include antibodies in which CDR sequences are derived from the germline of another mammalian species, such as a mouse, that have been grafted onto human framework sequence and/or constant regions.
  • human monoclonal antibody refers to a monoclonal antibody that is obtained from a human.
  • the term “immune response” refers to a biological response within a vertebrate against a foreign agent, which response protects the vertebrate, at least partially, against the foreign agent and diseases caused by the foreign agent.
  • An immune response is mediated by the action of a cell of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate’s body of invading foreign agent, cells or tissues infected with the foreign agent.
  • the term “immune response” does not include a response to a self-antigen.
  • isolated antibody refers to an antibody that is substantially free of other antibodies having different antigenic specificities.
  • An isolated antibody can optionally be substantially further free of other cellular material and/or reagents.
  • isotype refers to the antibody class (IgG, including IgGl-IgG4, IgM, and IgA. Including IgAl and IgA2, IgD and IgE) that is encoded by the heavy chain constant region genes.
  • K ass oc and “K a ” refer to the association rate of a particular antibody-antigen interaction.
  • Kdis or “Kd” refer to the dissociation rate of a particular antibody-antigen interaction.
  • KD refers to the dissociation constant, which is obtained from the ratio of Kd to K a and is expressed as a molar concentration (M).
  • the term “monoclonal antibody” refers to antibody molecules of a single molecular composition such that each of the antibody molecules displays a single binding specificity and binding affinity for a given epitope.
  • pharmaceutically acceptable refers to a compound that is compatible with an antibody of the disclosure or other ingredients of a composition and not deleterious to the subject receiving the antibody of the disclosure or composition.
  • pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • the term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium or an excipient which does not interfere with the effectiveness of an antibody of the disclosure or other active ingredient of the composition and which is not toxic to the subject at the concentrations at which it is administered.
  • the term includes, but is not limited to, a solvent, a stabilizer, a solubilizer, a tonicity enhancing agent, a structure-forming agent, a suspending agent, a dispersing agent, a chelating agent, an emulsifying agent, an anti-foaming agent, an ointment base, an emollient, a skin protecting agent, a gel-forming agent, a thickening agent, a pH adjusting agent, a preservative, a penetration enhancer, a complexing agent, a lubricant, a demulcent, a viscosity enhancer, a bio-adhesive polymer, or a combination thereof.
  • a solvent e. W. Martin, 18 th Ed., 1990, Mack Publishing Co.: Easton, Pa.
  • the term “pharmaceutically acceptable salt” refers to salts derived from inorganic or organic acids including, for example hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids.
  • Pharmaceutically acceptable salt forms may also include forms wherein the ratio of molecules comprising the salt is not 1: 1.
  • the salt may comprise more than one inorganic or organic acid molecule per molecule of antibody, such as two hydrochloric acid molecules per molecule of antibody.
  • the salt may comprise less than one inorganic or organic acid molecule per molecule of antibody, such as two molecules of compound of antibody per molecule of tartaric acid. Salts may also exist as solvates or hydrates.
  • a “pharmaceutical composition” refers to a mixture of one or more of the antibodies of the disclosure, with other components, such as, but not limited to, pharmaceutically acceptable carriers and/or excipients.
  • the purpose of a pharmaceutical composition is to facilitate administration of a compound of disclosure.
  • the term “recombinant antibody” includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as, but not limited to, antibodies isolated from an animal that is transgenic or transchromosomal or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody (a transfectoma), antibodies isolated from a recombinant or combinatorial antibody library, and antibodies prepared or created by any means involving the splicing of immunoglobulin gene sequences to other DNA sequences.
  • Such recombinant antibodies may be human recombinant antibodies.
  • Such recombinant antibodies can be subjected to in vitro mutagenesis or in vivo somatic mutagenesis.
  • the term “subject” refers to an animal.
  • the animal is a mammal.
  • a subject also refers to for example, primates (e.g, humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like.
  • the subject is a human.
  • the term “therapeutically effective amount” refers to an amount of an antibody of the disclosure that is sufficient to achieve a beneficial or desired result, including a clinical result.
  • the “therapeutically effective amount” may be sufficient, for example, to reduce or ameliorate the severity and/or duration of a SARS-CoV-2 infection, or one or more symptoms thereof, prevent the recurrence, development, or onset of one or more symptoms associated with a SARS-CoV-2 infection, prevent or reduce the replication or multiplication of SARS-CoV, prevent or reduce the production and/or release of a SARS-CoV-2 particle, or enhance or otherwise improve the prophylactic or therapeutic effect(s) of another therapy used in treating a SARS-CoV-2 infection.
  • a “therapeutically effective amount” is an amount of the antibody of the disclosure that avoids or substantially attenuates undesirable side effects.
  • the “therapeutically effective amount” in the context of a SARS- CoV-2 infection is an amount sufficient to reduce one or more of the following steps of a the life cycle of SARS-CoV-2: the docking of the virus particle to a cell, the introduction of viral genetic information into a cell, the expression of viral proteins, the translation of viral RNA, the transcription of viral RNA, the replication of viral RNA, the synthesis of new viral RNA, the production of new virus particles and the release of virus particles from a cell.
  • Such a reduction in any of the foregoing may be by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the “therapeutically effective amount” in the context of a SARS- CoV-2 infection reduces the replication, multiplication or spread of the virus by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • the “therapeutically effective amount” in the context of a SARS-CoV-2 infection increases the survival rate of infected subjects by at least 5%, preferably at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%.
  • a reduction of increase when a reduction of increase is specified, such reduction of increase may be determined with respect to a subject that has not been treated with an antibody of the disclosure and that has a diagnosed SARS-CoV-2 infection.
  • beneficial or desired clinical results in the context of a SARS-CoV-2 infection include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, a diminution of extent of disease, a stabilized (z.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable.
  • Treatment or “treating” can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • the disclosure provides the following neutralizing SARS-CoV-2 antibodies that specifically binds to the spike protein of SARS-CoV-2: 1212C2, 1212F5, 1213H7, 1212F2, 1206D1, 1212D5, 1207B4, 1213F2, 1212D4, 1206A5, 1212D6, 1206D12, 1207F10, 1206G12, 1212C8, 1212E9, 1214E9, and 1215D1.
  • an antigen binding fragment of the antibodies 1212C2, 1212F5, 1213H7, 1212F2, 1206D1, 1212D5, 1207B4, 1213F2, 1212D4, 1206A5, 1212D6, 1206D12, 1207F10, 1206G12, 1212C8, 1212E9, 1214E9, and 1215D1 is provided.
  • the antibodies are human monoclonal antibodies and/or the antigen binding fragments are derived from human monoclonal antibodies.
  • Table 1 provides the SEQ ID NOS: for the nucleotide sequence (NT) of the heavy chain variable regions and light chain variable regions, the amino acid (AA) sequence of the heavy and light chain variable regions, the AA sequence of the heavy chain CDR1, CDR2, and CDR3, and the AA sequence of the light chain
  • the neutralization titer 50 (NT50) of the antibodies of the disclosure ranges from less than 0.050 pg/ml to 1.61 gg/ml.
  • the NT50 value if the antibodies of the disclosure is less than or equal to 10 pg/ml, less than or equal to 8 pg/ml, less than or equal to 6 pg/ml, less than or equal to 4 pg/ml, less than or equal to 3 pg/ml, less than or equal to 2 gg/ml, or less than or equal to 1 gg/ml.
  • the NT50 value if the antibodies of the disclosure is less than or equal to 0.5 pg/ml, less than or equal to 0.25 pg/ml, less than or equal to 0.1 pg/ml, less than or equal to 0.075 pg/ml, less than or equal to 0.05 pg/ml, less than or equal to 0.025 pg/ml, or less than or equal to 0.01 pg/ml.
  • NT50 values are determined as described in Example 4 of the disclosure.
  • the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising: 1. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 38-40, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42-44, respectively (1212C2);
  • a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 46-48, respectively, and
  • a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50-52, respectively (1215F5);
  • a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 126-128, respectively, and
  • a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 130-132, respectively (1206D12);
  • a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 142-144, respectively
  • a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 146-148, respectively (1206G12), or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
  • the disclosure provides an isolated antibody, or an antigenbinding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising: 1. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 37, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 41, or an amino acid sequence at least 80% homologous thereto (1212C2);
  • a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 93, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 97, or an amino acid sequence at least 80% homologous thereto (1213F2); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 101, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 105, or an amino acid sequence at least 80% homologous thereto (1212D4); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 109, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 113, or an amino acid sequence at least 80% homologous thereto (1206
  • a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 155, or an amino acid sequence at least 80% homologous thereto
  • a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 156 (1212C8), or an amino acid sequence at least 80% homologous thereto, or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
  • the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising:
  • a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 158, 39 and 159, respectively
  • a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42, 43, and 161, respectively (1212 variant), or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
  • the amino acid at position 3 of SEQ ID NO: 158 is Thr (T) or He (I)
  • the amino acid at position 1 of SEQ ID NO: 159 is Thr (T) or Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid
  • the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser
  • S amino acid at position 10 of SEQ ID NO: 161 is Vai (V) or Phe (F).
  • amino acid at position 3 of SEQ ID NO: 158 is Thr
  • the amino acid at position 1 of SEQ ID NO: 159 is Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid (preferably Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), or Met (M))
  • the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser (S)
  • the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
  • the amino acid at position 3 of SEQ ID NO: 158 is Thr (T) or He (I)
  • the amino acid at position 1 of SEQ ID NO: 159 is Thr (T) or Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Leu (L)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid
  • the amino acid at position 6 of SEQ ID NO: 161 is Asn (N)
  • the amino acid at position 10 of SEQ ID NO: 161 is Phe (F) or Vai (V).
  • the amino acid at position 3 of SEQ ID NO: 158 is Thr (T)
  • the amino acid at position 1 of SEQ ID NO: 159 is Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Leu (L)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid (preferably Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), or Met (M))
  • the amino acid at position 6 of SEQ ID NO: 161 is Asn (N)
  • the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
  • the amino acid at position 3 of SEQ ID NO: 158 is He (I)
  • the amino acid at position 1 of SEQ ID NO: 159 is Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Phe (F)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid
  • the amino acid at position 6 of SEQ ID NO: 161 is Ser (S)
  • the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
  • the amino acid at position 3 of SEQ ID NO: 158 is He (I)
  • the amino acid at position 1 of SEQ ID NO: 159 is Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Phe (F)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid(preferably Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), or Met (M))
  • the amino acid at position 6 of SEQ ID NO: 161 is Ser (S)
  • the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
  • the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising:
  • a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 157, or an amino acid sequence at least 80% homologous thereto
  • a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 160, or an amino acid sequence at least 80% homologous thereto (1212 variant); or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
  • the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2A below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2B below.
  • the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2C below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2D below.
  • the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2E below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2F below.
  • the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2G below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2H below.
  • the antibody is provided as a pharmaceutically acceptable salt.
  • the antibodies bind an epitope within the RBD of the SARS-CoV-2 spike protein. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies bind an epitope within the RBM of the SARS-CoV-2 spike protein. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies bind an epitope within the SI region of the SARS-CoV-2 spike protein. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies bind an epitope within the S2 region of the SARS-CoV-2 spike protein.
  • the antibodies reduce binding of SARS-CoV-2 to a target cell. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies reduce cellular fusion between SARS-CoV-2 and a target cell. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies reduce release of infective SARS-CoV-2 from an infected cell. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies reduce infection of a target cell by SARS-CoV-2.
  • the antibody is an antibody variant. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antibody variant comprising one or more substitutions, deletions, and/or insertions relative to the parental antibody. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antibody variant comprising one or more substitutions relative to the parental antibody.
  • the antibody is an antibody variant that has a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
  • the antibody comprises a variant Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein a protein moiety or non-protein moiety is linked to the Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein a water soluble polymer is linked to the Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein a polyethylene glycol polymer is linked to the Fc constant region.
  • the antibody comprises a variant Fc constant region, wherein a polyoxazoline polymer is linked to the Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein the variant Fc constant region provides a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
  • the antibody is a human antibody. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is a chimeric antibody. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is a cl ass- switched antibody.
  • the antibody is linked to a therapeutic agent. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is linked to a detectable label. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is linked to an enzyme. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is linked to an enzyme inhibitor.
  • the antibody is an antigen-binding fragment. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antigen-binding fragment selected from the groups consisting of: a Fab fragment, a F(ab)2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a disulfide-linked Fv (sdFv), a dAb fragment, an isolated CDR, a nanobody or single domain antibody, a portion of the VH region containing a single variable domain and two constant domains, a diabody, a triabody, a tetrabody, scFv, scFv-FC, scFv-CH, scFab, and scFv-zipper.
  • the amino acid sequence has at least 85% homology to the reference sequence, at least 90% homology to the reference sequence, at least 95% homology to the reference sequence, at least 96% homology to the reference sequence, at least 97% homology to the reference sequence, at least 98% homology to the reference sequence, or at least 99% homology to the reference sequence.
  • the antibody is 100% homologous to the reference sequence across the CDRs and the amino acid sequence has at least 85% homology to the reference sequence across the FRs, the amino acid sequence has at least 85% homology to the reference sequence across the FRs, at least 90% homology to the reference sequence across the FRs, at least 95% homology to the reference sequence across the FRs, at least 96% homology to the reference sequence across the FRs, at least 97% homology to the reference sequence across the FRs, at least 98% homology to the reference sequence across the FRs, or at least 99% homology to the reference sequence across the FRs.
  • an antibody of the disclosure is an antigen-binding fragment.
  • Antigen-binding fragment include, but are not limited to, Fab, Fab', Fab'-SH, F(ab)2, F(ab’)2, Fv, Fd, sdFv, dAb scFv fragments (including, but not limited to, scFv-FC, scFv-CH, scFab, and scFv- zipper), diabodies, triabodies tetrabodies, nanobodies, and other fragments described here.
  • Fab and F(ab) fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.
  • Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells as described herein or as known in the art.
  • an antibody of the disclosure is a chimeric antibody.
  • a chimeric antibody comprises a non-human variable region (for example, a variable region derived from a mouse, rat, hamster, rabbit, or a monkey or other non-human primate) and a human constant region.
  • a chimeric antibody is a “class switched” antibody in which the class or subclass of the antibody has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.
  • a chimeric antibody is a humanized chimeric antibody.
  • an antibody of the disclosure is a human antibody.
  • Human antibodies can be produced using various techniques known in the art or using techniques described herein. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes.
  • Human antibodies can also be made by hybridoma-based methods.
  • Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are known in the art (Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)).
  • Human antibodies generated via human B-cell hybridoma technology are also known in the art (Li et al., Proc. Natl. Acad. Sci. USA, 103:3557- 3562 (2006)).
  • Human antibodies may also be generated by 1 isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.
  • amino acid sequence variants of the antibodies provided herein are contemplated. Such variants may be used to improve the binding affinity of an antibody, to improve a biological property of an antibody (such as, but not limited to, half-life), or a combination of the foregoing.
  • Amino acid variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis directly incorporating such amino acid change.
  • Antibody variants include, but are not limited to, fusion proteins including an antibody of the disclosure, substitutions of one or more amino acids, deletion of one or more amino acids, insertion of one or more amino acids, and any combination of the foregoing. In certain embodiments, the antibody variant retains the ability to bind to the same epitope.
  • antibody variants having one or more amino acid substitutions are provided.
  • the substitution may be made at any desired location.
  • the substitution occurs in the Sites of interest for substitutional mutagenesis include the VH region, the VL region, the heavy chain CDRs, the light chain CDRs, and/or the FR region. Such substitutions may be conservative or nonconservative. Conservative substitutions are defined herein.
  • an antibody of the disclosure comprises a conservative amino acid substitution.
  • such conservative amino acid substitution may be made in the VH region, the VL region, the heavy chain CDRs, the light chain CDRs, and/or the FR region.
  • the disclosure provides for an antibody variant of one or more of the polypeptides of SEQ ID NOS: 37 to 156.
  • the disclosure provides for an antibody variant comprising one or more substitutions, including conservative substitutions, of one or more of the polypeptides of SEQ ID NOS: 37 to 156.
  • an antibody variant comprising one or more substitutions, including conservative substitutions, of the heavy chain variable region (including CDRs 1-3) and/or light chain variable region (including CDRs 1-3), of one or more of the polypeptides of SEQ ID NOS: 37 to 156.
  • an antibody variant has an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a parent antibody.
  • the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences.
  • the percent identity between two amino acid sequences is preferably determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • an antibody variant includes a conservative modification.
  • conservative modifications refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody containing the conservative modification. Such conservative modifications include amino acid substitutions, additions, and deletions.
  • an antibody variant contains a conservative amino acid substitution.
  • Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (lysine, arginine, and histidine), acidic side chains (aspartic acid and glutamic acid), uncharged polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), beta-branched side chains (threonine, valine, and isoleucine), and aromatic side chains (tyrosine, phenylalanine, tryptophan, and histidine). Non-conservative substitutions will entail exchanging a member of one of these classes for
  • a conservative amino acid substitution may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity, steric bulk, charge, hydrophobicity and/or hydrophilicity of the amino acid residue at that position.
  • Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties. It will be appreciated by those of skill in the art that polypeptide described herein may be chemically synthesized as well as produced by recombinant means.
  • the hydropathic index of an amino acid may be considered.
  • Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. Hydropathic index values are resented by: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cy stine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In one embodiment, making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +/- 1; in an alternate embodiment, the hydropathic indices are within +/- 0.5; in yet another alternate embodiment, the hydropathic indices are within +/- 0.25.
  • the hydrophilicity may also be considered.
  • the greatest local average hydrophilicity of a polypeptide as governed by the hydrophilicity of its adjacent amino acids correlates with a biological property of the protein.
  • hydrophilic index values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
  • a skilled artisan will be able to determine suitable substitutions, insertions and deletions, including combinations thereof, of a polypeptide as set forth in any of SEQ ID NOS: 37 to 156 using techniques known in the art. For identifying suitable areas of a polypeptide that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity.
  • homologous polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a polypeptide described herein to such homologous polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a polypeptide described herein that are not conserved relative to such homologous polypeptide would be less likely to adversely affect the biological activity and/or structure of a polypeptide described herein. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (for example, conservative amino acid substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to such amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
  • deletions, insertions, and substitutions can be selected, as would be known to one of ordinary skill in the art, to generate a desired polypeptide variants. For example, it is not expected that deletions, insertions, and substitutions in a non-functional region of a polypeptide would alter activity. Likewise conservative amino acid substitutions and/or substitution of amino acids with similar hydrophilic and/or hydropathic index values is expected to be tolerated in a conserved region and a polypeptide activity may be conserved with such substitutions.
  • An exemplary substitution variant is an affinity matured antibody, which may be conveniently generated using phage display-based affinity maturation techniques such as those described in Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)).
  • Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue.
  • insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.
  • An antibody variant may be screened for a desired activity, including, but not limited to, retained epitope binding, improved epitope binding, decreased immunogenicity, improved antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or any combination of the foregoing.
  • a non-protein based moiety is a polymer, preferable a water soluble polymer.
  • Suitable water soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyoxazoline (POZ), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either as a homopolymer or a copolymer), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propylene glycol, poly oxy ethylated polyols (such as glycerol), polyvinyl alcohol, copolymers of ethylene glycol and propylene glycol, copolymers of propylene glycol, copolymers of propylene glycol, copolymers of ethylene glycol and propy
  • the copolymer may be present as a block copolymer or a random copolymer.
  • An amino acid substitution may be used to introduce a site for attachment of a water soluble polymer to the antibody.
  • the water soluble polymer may be of any molecular weight, and may be dendrimers, branched or unbranched.
  • the number of polymers attached to an antibody may vary.
  • the number average molecular weight of the water soluble polymer is from 2,500 to 75,000 Da, from 5,000 to 50,000 Da, from 7,500 to 40,000 Da, or from 10,000 to 30,000 Da.
  • 1 to 10 water soluble polymer chains are attached.
  • the water soluble polymers may be the same or different and may be of the same of different number average molecular weight.
  • the water soluble polymers increases the half-life of an antibody of the disclosure and/or decrease immunogenicity of an antibody of the disclosure.
  • Water soluble polymers may be linked to an antibody of the disclosure using conventional reactive groups on the polymer and the antibody.
  • water soluble polymers such as. But not limited to, POZ and PEG polymers, may be linked to an antibody using acylation alkylation reactions.
  • Water soluble polymers may be linked to the antibody in a site specific manner or randomly (for example, the e-amino group of a lysine residue or the thiol group of cysteine residue may be used in conjugation reactions with an appropriate functionality on the water soluble polymer).
  • a non-natural amino acid may be introduced into the antibody and used to link a water soluble polymer to the antibody.
  • a selenocysteine residue may be introduced into the antibody for reaction with a water soluble polymer containing an appropriate functionality (for example, a maleimide group or an iodoacetimide group).
  • the water soluble polymer may be linked to a therapeutic agent, a detectable label, an enzyme, or an enzyme inhibitor as described herein.
  • a non-protein based moiety is a therapeutic agent, for example a therapeutic agent useful in treating a SARS-CoV-2 infection as described herein or a cytotoxic agent.
  • a non-protein based moiety is a detectable label.
  • a non-protein based moiety is an enzyme.
  • a non-protein based moiety is an enzyme inhibitor, for example a serine protease inhibitor, such as, but not limited to, a TMPRSS2 inhibitor.
  • an antibody of the disclosure is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.
  • an aglycoslated antibody can be made. Glycosylation can be altered to for a variety of purposes, including, but not limited to, to increase the affinity of the antibody for an antigen.
  • one or more amino acid substitutions are made that result in elimination of one or more FR glycosylation sites, which may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861.
  • N297 in the Fc portion may be substituted with another residue (for example, alanine) and/or by mutating an adjacent amino acid to thereby reduce glycosylation on N297.
  • N297 in the Fc portion may be substituted with another residue (for example, alanine) and/or by mutating an adjacent amino acid to thereby reduce glycosylation on N297.
  • an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures.
  • altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with altered glycosylation machinery (such cells are described in EP 1,176,195; PCT Publication WO 03/035835; Shields, R. L. et al. (2002), J. Biol. Chem. 277:26733-26740; PCT Publication WO 99/54342; Umana et al. (1999), Nat. Biotech. 17: 176-180).
  • variable regions of the antibody described herein can be linked to an Fc region (such as, but not limited to, an IgGl, IgG2, IgG3 or IgG4 Fc), which may be of any allotype or isoallotype (including for IgGl : Glm, Glml(a), Glm2(x), Glm3(f), Glml7(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(gl), G3m28(g5), G3ml l(b0), G3m5(bl), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(t), G3m6(c3), G3m24
  • an Fc region such as, but not limited to, an IgGl, IgG2, IgG3 or IgG4 Fc
  • variable regions of the antibodies described herein are linked to an Fc that binds to one or more activating Fc receptors (FcR), and thereby stimulate ADCC.
  • the variable regions of the antibodies described herein are linked to an Fc region optimized to engage a wider range of Fc receptors.
  • Fc receptors for isotypes other than gamma exist on particular leukocytes. By creating an Fc region that can interact with multiple Fc receptors, such as FcyRI and FcaRI, an antibody with expanded, novel abilities to engage effector cells may be created. Neutrophils are the most abundant leukocyte in the body and engage Fc of IgA antibodies via the FcaRI.
  • the antibody variable regions described herein may be linked to an Fc comprising one or more modification, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, ADCC, and/or CDC.
  • the Fc region encompasses domains derived from the constant region of an antibody.
  • Suitable immunoglobulins include IgG 1, IgG2, IgG3, IgG4, IgAl, IgA2, and other classes such as IgD, IgE and IgM
  • the constant region of an antibody is defined as a naturally-occurring or synthetically-produced polypeptide homologous to the antibody C-terminal region, and can include a CHI domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in combination.
  • an antibody of the disclosure has an Fc region other than that of a wild type IgAl.
  • An antibody of the disclosure may have an Fc region from that of IgG (e.g., IgGl, IgG2, IgG3, and IgG4) or other classes such as IgA2, IgD, IgE and IgM.
  • An antibody of the disclosure may have an Fc region that is contains a substitution, deletion, or insertion of wild-type IgAl.
  • the Fc of an antibody is responsible for many important functions including FcR binding and complement fixation.
  • the serum half-life of an antibody is influenced by the ability of that antibody to bind to an FcR.
  • Antibody molecules interact with multiple classes of cellular receptors.
  • IgG molecules interact with three classes of FcyR specific for the IgG class of antibody, namely FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, and FcyRIIIb.
  • FcyRI FcyRI
  • FcyRIIa FcyRIIa
  • FcyRIIb FcyRIIb
  • FcyRIIIa FcyRIIIb
  • the Fc region is a variant Fc region (an Fc sequence that has been modified such as by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence to provide desirable structural features and/or biological activity, including, but not limited to, (i) increased or decreased ADCC; (ii) increased or decreased CDC; (iii) increased or decreased affinity for Clq; and/or (iv) increased or decreased affinity for a FcR (each of the foregoing relative to the parent Fc).
  • Such Fc region variants will generally comprise at least one amino acid substitution, deletion, and/or insertion in the Fc region.
  • an Fc region contains from 1 to 6 amino acid substitutions, deletions and/or insertions (preferably substitutions).
  • a variant Fc region may also comprise a sequence modification wherein an amino acid involved in disulfide bond formation are removed or replaced with another amino acid.
  • the Fc region may be modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc region, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase.
  • one or more glycosylation sites within the Fc domain are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues e.g., alanine).
  • sites involved in interaction with complement may be removed from the Fc region.
  • sites involved in interaction with complement such as the Clq binding site
  • sites that affect binding to Fc receptors may be removed, preferably sites other than salvage receptor binding sites.
  • an Fc region may be modified to remove an ADCC site known in the art. Specific examples of variant Fc domains are disclosed for example, in WO 1997/34631 and WO 1996/32478.
  • the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is increased or decreased (U.S. Pat. No. 5,677,425) to, for example, facilitate assembly of the light and heavy chains and/or to increase or decrease the stability of the antibody.
  • the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding (U.S. Pat. No. 6,165,745).
  • SpA Staphylococcal protein A
  • the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter an effector function of the antibody.
  • one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody.
  • the effector ligand to which affinity is altered can be, for example, an FcR or the CI component of complement (U.S. Pat. Nos. 5,624,821 and 5,648,260).
  • one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished CDC (U.S. Pat. No. 6,194).
  • one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement (PCT Publication WO 1994/29351). All references to Fc region amino acid numbering is made according to the EU index of Kabat (Kabat et al., (1983) "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services).
  • the Fc region may be modified to increase ADCC and/or to increase the affinity for an FcyR by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263,
  • substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E.
  • Exemplary variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F7324T.
  • Fc modifications that increase binding to an FcyR include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region.
  • Fc modifications that can be made are those for reducing or ablating binding to FcyR and/or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC.
  • Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index of Kabat.
  • Exemplary substitutions include but are not limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index of Kabat.
  • An Fc variant may comprise 236R/328R.
  • the Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Publication Nos: WO 2000/42072; WO 2001/58957; WO 2002/06919; WO 2004/016750; WO 2004/029207; WO 2004/035752; WO 2004/074455; WO 2004/099249; WO 2004/063351; WO 2005/070963; WO 2005/040217, WO 2005/092925 and WO 2006/020114).
  • Fc variants that enhance affinity for an inhibitory receptor FcyRIIb may also be used. Such variants may provide an Fc fusion protein with immune-modulatory activities related to FcyRIIb cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRIIb relative to one or more activating receptors. Modifications for altering binding to FcyRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index of Kabat.
  • Exemplary substitutions for enhancing FcyRIIb affinity include but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E.
  • Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y.
  • Fc variants for enhancing binding to FcyRIIb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.
  • the affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including but not limited to, equilibrium methods (enzyme-linked immune-absorbent assay, radioimmunoassay, or kinetics, and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer, gel electrophoresis and chromatography). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels.
  • a detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody -immunogen interactions.
  • the antibody is modified to increase its biological half-life.
  • this may be done by increasing the binding affinity of the Fc region for FcRn.
  • one or more of following residues can be mutated: 252, 254, 256, 433, 435, and 436, as described in U.S. Pat. No. 6,277,375.
  • Specific exemplary substitutions include one or more of the following: T252L, T254S, and/or T256F.
  • the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos.
  • variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H. 434F, 434Y, and 434M.
  • Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al. 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al.
  • hybrid IgG isotypes with particular biological characteristics may be used.
  • an IgGl/IgG3 hybrid variant may be constructed by substituting IgGl positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ.
  • a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F.
  • an IgGl/IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and/or CH3 region with amino acids from IgGl at positions where the two isotypes differ.
  • a hybrid variant IgG antibody may be constructed chat comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to an insertion of a glycine at position 236), and 321 h.
  • IgGl variants with strongly enhanced binding to FcyRIIIa have been identified, including variants with S239D/I332E and S239D/I332E/A330L mutations which showed the greatest increase in affinity for FcyRIIIa, a decrease in FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006).
  • IgGl mutants containing L235V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcyRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et al., 2011).
  • Other Fc mutants that may be used include, but are not limited to: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L,
  • an Fc is chosen that has reduced binding to FcyRs.
  • An exemplary Fc, e.g., IgGl Fc, with reduced FcyR binding comprises the following three amino acid substitutions: L234A, L235E and G237A.
  • an Fc is chosen that has reduced complement fixation.
  • An exemplary Fc e.g., IgGl Fc, with reduced complement fixation has the following two amino acid substitutions: A33OS and P331S.
  • an Fc is chosen that has essentially no effector function, i.e., it has reduced binding to FcyRs and reduced complement fixation.
  • An exemplary Fc, IgGl Fc that is effectorless comprises the following five mutations: L234A, L235E, G237A, A33OS and P331S.
  • substitution S228P which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules.
  • the disclosure also provides for nucleic acid sequences encoding the antibodies of the disclosure.
  • the nucleic acid sequences may code for an antigen-binding fragment.
  • the nucleic acids are codon optimized based on how the antibodies are produced.
  • the nucleic acid sequence comprises one or more of SEQ ID NOS: 1 to 36.
  • the nucleic acid sequence has at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, or greater than 95% homology with one or more of SEQ ID NOS: 1 to 36.
  • the disclosure also provides a vector comprising a nucleic acid sequence coding for an antibody of the disclosure.
  • a vector comprises a nucleic acid sequence coding for a variable heavy chain region and a nucleic acid sequence coding for a variable light chain region. Any nucleic acid sequence of the disclosure (SEQ ID NOS: 1-36) may be combined with a vector as described herein.
  • a nucleic acid sequence coding for a variable heavy chain is on the same vector as a nucleic acid sequence coding for a variable light chain.
  • a nucleic acid sequence coding for a variable heavy chain region is on a different vector than a nucleic acid sequence coding for a variable light chain region.
  • the vector is a plasmid. In some embodiments, the vector is a phage vector, such as, but not limited to, X-phage. In some embodiments, the vector is a viral vector, such as, but not limited to, non-replicating adenoviral vector, lentiviral vector, pSV, pCMV, and retroviral vectors. In some embodiments, the vector is a cosmid. In some embodiments, the vector is a recombinant chromosome. In some embodiments, the combinations of the foregoing vectors are employed. The expression of different nucleic acid sequences may occur at the same time generally or be temporally separated. The expression of one or more nucleic acid sequences may be inducible.
  • the vector may comprise a nucleic acid coding for an intact antibody or an antigen binding fragment, particularly those antigen-binding fragments disclosed herein.
  • the vector comprises a nucleic acid sequence encoding an immunoglobulin constant region, such as, but not limited to, an IgG (e.g. IgGl, IgG2, IgG3, and IgG4) constant region.
  • an immunoglobulin constant region comprises an IgGl constant region.
  • the disclosure provides for a vector comprising nucleotide sequences that code for the heavy and/or light chain variable regions of an antibody selected from the groups consisting of 1212C2, 1212F5, 1213H7, 1212F2, 1206D1, 1212D5, 1207B4, 1213F2, 1212D4, 1206A5, 1212D6, 1206D12, 1207F10, 1206G12, 1212C8, 1212E9, 1214E9, and 1215D1.
  • the vector may be a mammalian expression vectors, such that the vector may be transfected into mammalian cells and the DNA may be integrated into the genome by homologous recombination in the case of stable transfection, or alternatively the cells may be transiently transfected.
  • Common to most engineered vectors are origin of replications, multicloning sites, and selectable markers.
  • Common promoters for mammalian expression vectors include CMV and SV40 promoters, and non-viral promoters such as, but not limited to, EF-1 promoters.
  • the disclosure provides a vector comprising one or more nucleic acid sequences encoding one or more CDRs of one or more heavy and/or light chains of one or more of the antibodies of the disclosure (for example, a nucleic acid encoding an amino acid sequences of SEQ ID NOS: 38-40, 42-44, 46- 48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98- 100102-104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-135, 138- 140, 142-144, and 146-148, or an amino acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% homology with SEQ ID NOS: 38-40, 42-44, 46-48, 50-
  • the disclosure provides a vector comprising one or more nucleic acid sequences encoding one or more heavy and/or light chain variable regions of one or more of the antibodies of the disclosure (for example, a nucleic acid encoding an amino acid sequences of SEQ ID NOS: 37, 42, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101,
  • the nucleic acid sequences may encode for an antibody variant as described herein, including an antibody containing a conservative substitution.
  • a vector that codes for one or both variable region(s) of an antibody of the disclosure may contain one of or both of SEQ ID NOS: required to generate such antibody, or a nucleotide sequence that shares a degree minimum of homology with such SEQ ID NOS: (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% homology).
  • a vector that encodes the heavy and light chain variable region of antibody 1212C2 may comprise SEQ ID NOS: 1 and 2, or a nucleotide sequence that shares a degree minimum of homology with such SEQ ID NOS: 1 and 2 (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% homology).
  • the disclosure also provides for a cell transformed with a vector described herein.
  • a cell transformed with a vector described herein may lead to different antibody products and may possibly impact the therapeutic efficacy of the antibody products, e.g. through having distinct variations in glycosylation patterns, especially N-linked glycosylation patterns.
  • Such discussions may be found in Liu L, J Pharm Sci. 2015 June; 104(6): 1866-84; Rosenlocher et al., J Proteomics. 2016 Feb. 16; 134:85-92; Mimura et al., J Immunol Methods. 2016 January; 428:30-6; and Croset et al., Journal of Biotechnology, 161(3), Oct. 31, 2012.
  • the cell is a bacterial cell, a yeast cell, a plant cell, or a mammalian cell.
  • the mammalian cell is one of a Chinese hamster ovary (CHO) cell, including DUXB11, DG44 and CHOK1 lineages, a NSO murine myeloma cell, aPER.C6 cell, and a human embryonic kidney (HEK) cell, including HEK293 lineages.
  • CHO Chinese hamster ovary
  • HEK human embryonic kidney
  • Other less common host cells include plant cells, for example, those based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens.
  • Cell-free expression systems also exist, for example, based on E.
  • Eukaryotic and mammalian cell-free systems are also known in the art, for example wheat germ cell-free expression system. Some recombinant antibody production systems express the recombinant antibodies on the surface of the host cell before harvesting, others simply release the antibodies into a medium for collection. Such variations are intended to be within the scope of the disclosure.
  • the disclosure also provides for a method of making a recombinant antibody of the disclosure.
  • the antibody is an antigen binding fragment.
  • the host cell comprising a vector described herein is induced to produce the recombinant antibodies and the host cell assembles the antibodies from heavy/light chains in the host cell and then transport the antibodies out of the cell, or the antibodies may self-assemble outside the host cell and be exported as heavy/light chains.
  • An overview of cell culture processes for recombinant monoclonal antibody production may be found in Li et al., Mabs. 2010 September-October; 2(5): 466-477.
  • the disclosure provides for a method of making a recombinant antibody, or antigenbinding fragment thereof, that specifically binds to SARS-CoV-2 spike protein, the method comprising providing a cell comprising a vector comprising a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of any one of SEQ ID NOS: 1-36, expressing at least one nucleic acid sequence in the vector to create at least one of a heavy chain, a light chain, or combinations thereof, and collecting a formed antibody or the antigen-binding fragment, thereof.
  • Antibodies of the disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics (Hoogenboom et al. in Methods in Molecular Biology 178: 1- 37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001); McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol.
  • repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage.
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • scFv single-chain Fv
  • Fab fragments fragments from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas.
  • the naive repertoire can be cloned (e.
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro (Hoogenboom and Winter, I. Mol. Biol., 227: 381-388 (1992)).
  • Antibodies, including antigen-binding fragments, isolated from human antibody libraries are considered human antibodies.
  • the disclosure provides a method for treating a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2 either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • a combination of 1212C2 and 1213H7 mAbs are used.
  • the disclosure provides a method for suppressing a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the disclosure provides a method for preventing a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the disclosure provides a method for treating, suppressing and/or preventing a disease or condition relating to a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the disease or condition is Guillain- Barre Syndrome.
  • the disease or condition is multisystem inflammatory syndrome, particularly when the subject is under the age of 25 years of age.
  • the disease or condition is organ injury, such as, but not limited to, lung injury, liver injury, and/or heart injury. In certain embodiments of the fourth aspect, the disease or condition is acute respiratory distress syndrome. In certain embodiments of the fourth aspect, the disease or condition is increased inflammation resulting from an imbalance in the renin-angiotensin system (such as, but not limited to, excess production of angiotensin II and/or the decreased production of angiotensin 1-7).
  • organ injury such as, but not limited to, lung injury, liver injury, and/or heart injury.
  • the disease or condition is acute respiratory distress syndrome.
  • the disease or condition is increased inflammation resulting from an imbalance in the renin-angiotensin system (such as, but not limited to, excess production of angiotensin II and/or the decreased production of angiotensin 1-7).
  • the disclosure provides a method of reducing or preventing cellular entry of SARS-CoV-2 in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, or a combination of the foregoing, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the disclosure provides a method of reducing or preventing binding of SARS-CoV-2 to a cellular ACE2 in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the disclosure provides a method for reducing viral titer of a SARS- CoV-2 in a bodily fluid, tissue or cell of a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the transmission of the SARS-CoV-2 (for example, from a subject infected with SARS-CoV-2 to a subject that is not yet infected) is reduced as a result of a reduced viral titer.
  • the disclosure provides a method for reducing or preventing the transmission of a SARS-CoV-2 infection from a first subject to a second subject, the method comprising administering to said first subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • such reduction or prevention is obtained, at least in part, by reducing the cellular entry of a SARS-CoV-2 in the first subject.
  • administration to the first subject occurs before the first subject has been infected with SARS-CoV-2, after the first subject has been infected with the SARS-CoV-2, or after the first subject has been infected with the SARS-CoV-2 and before the SARS-CoV-2 infection can be detected.
  • the disclosure provides a method for reducing or preventing the transmission of a SARS-CoV-2 infection from a first subject to a second subject, the method comprising administering to the second subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV- 2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the second subject may be at risk for SARS-CoV-2 infection.
  • such reduction or prevention is obtained, at least in part, by preventing or reducing SARS-CoV-2 cellular entry in the second subject.
  • such reduction or prevention is obtained, at least in part, by preventing or suppressing a SARS-CoV-2 infection in the second subject.
  • a SARS-CoV-2 infection occurs in the second subject, it can be eliminated physiologically (for example, by the immune system) by the second subject, either with or without the administration of additional therapeutic compounds.
  • administration to the second subject before the second subject has been infected with the SARS-CoV-2, after the second subject has been infected with the SARS-CoV-2, or after the second subject has been infected with the SARS-CoV-2 and before the SARS-CoV-2 infection can be detected.
  • the disclosure provides a method of neutralizing a SARS-CoV-2 in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
  • the antibody, or an antigen-binding fragment thereof binds to SARS-CoV-2 viral particles before they are able to interact with cellular ACE2, thereby reducing or preventing SARS-CoV-2 viral particles from entering the cell.
  • the antibody, or an antigen-binding fragment thereof reduces or prevents SARS-CoV-2 viral particles from binding to cellular ACE2.
  • the antibody, or an antigen-binding fragment thereof reduces cleavage of the SARS-CoV-2 spike protein.
  • the antibody, or an antigen-binding fragment thereof reduce binding of SARS-CoV-2 to a target cell. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces cellular fusion between SARS-CoV-2 and a target cell. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces release of infective SARS-CoV-2 from an infected cell. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduce infection of a target cell by SARS-CoV-2.
  • the methods of the first to tenth aspects may further comprise one or more of the steps: i) identifying a subject in need or treatment, prevention, suppression, reduction, or inhibition; and (ii) providing an antibody, or an antigen-binding fragment thereof, of the disclosure or a pharmaceutical composition comprising the foregoing.
  • the antibody or antibodies, or an antigen-binding fragment(s) thereof is any one or more antibodies or antigen binding fragment(s) described herein, or a pharmaceutically acceptable form thereof.
  • the antibody or antibodies, or an antigen-binding fragment(s) thereof comprises:
  • a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 102-104, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 106-108, respectively (1212D4);
  • a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 126-128, respectively, and
  • a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 130-132, respectively (1206D12);
  • a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 142-144, respectively, and
  • a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 146-148, respectively (1206G12), or a pharmaceutically acceptable form of any of the foregoing.
  • the antibody, or an antigen-binding fragment thereof comprises:
  • a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 101, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 105, or an amino acid sequence at least 80% homologous thereto (1212D4); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 109, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 113, or an amino acid sequence at least 80% homologous thereto (1206 A5); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 117, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 121, or an amino acid sequence at least 80% homologous thereto (1212D4);
  • a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 155, or an amino acid sequence at least 80% homologous thereto
  • a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 156 (1212C8), or an amino acid sequence at least 80% homologous thereto, or a pharmaceutically acceptable form of any of the foregoing.
  • the antibody, or an antigen-binding fragment thereof comprises: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 158, 39 and 159, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42, 43, and 161, respectively (1212 variant), or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
  • the amino acid at position 1 of SEQ ID NO: 158 is Thr (T) or He (I)
  • the amino acid at position 1 of SEQ ID NO: 159 is Thr (T) or Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid
  • the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser
  • S amino acid at position 10 of SEQ ID NO: 161 is Vai (V) or Phe (F).
  • amino acid at position 1 of SEQ ID NO: 158 is Thr
  • the amino acid at position 1 of SEQ ID NO: 159 is Ala (A)
  • the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F)
  • the amino acid at position 1 of SEQ ID NO: 161 is any amino acid (preferably Ala (A))
  • the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser (S)
  • the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
  • the amino acid at position 1 of SEQ ID NO: 158 is Thr (T)
  • the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Thr (T) and Leu (L), respectively
  • the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid, Asn (N), and Phe (F), respectively.
  • the amino acid at position 1 of SEQ ID NO: 158 is Thr (T)
  • the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Ala (A) and Leu (L), respectively
  • the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid (preferably Ala (A)), Asn (N), and Vai (V), respectively.
  • the amino acid at position 1 of SEQ ID NO: 158 is lie (I)
  • the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Ala (A) and Phe (F), respectively
  • the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid, Ser (S), and Vai (V), respectively.
  • the amino acid at position 1 of SEQ ID NO: 158 is lie (I)
  • the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Ala (A) and Phe (F), respectively
  • the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid (preferably Ala (A)), Ser (S), and Vai (V), respectively.
  • the antibody, or an antigen-binding fragment thereof comprises: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 157, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 160, or an amino acid sequence at least 80% homologous thereto (1212 variant); or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
  • amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2A and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2B.
  • amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2C and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2D.
  • amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2E and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2F.
  • the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2G below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2H below.
  • the antibody, or an antigen-binding fragment thereof comprises a mixture of any of the foregoing.
  • the antibody, or an antigen-binding fragment thereof is present as a pharmaceutically acceptable salt.
  • the antibody, or an antigen-binding fragment thereof binds an epitope within the RBD of the SARS-CoV-2 spike protein. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds an epitope within the RBM of the SARS-CoV-2 spike protein. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds an epitope within the S 1 region of the SARS-CoV-2 spike protein. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds an epitope within the S2 region of the SARS-CoV-2 spike protein.
  • the antibody, or an antigen-binding fragment thereof reduces binding of SARS-CoV-2 to a target cell. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces cellular fusion between SARS-CoV-2 and a target cell. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces release of infective SARS-CoV-2 from an infected cell. In certain embodiments of the first to tenth aspects, the antibody, or an antigenbinding fragment thereof, reduces infection of a target cell by SARS-CoV-2.
  • the antibody, or an antigen-binding fragment thereof is an antibody variant. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antibody variant comprising one or more substitutions, deletions, and/or insertions relative to the parental antibody. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antibody variant comprising one or more substitutions relative to the parental antibody.
  • the antibody, or an antigen-binding fragment thereof is an antibody variant that has a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
  • the antibody, or an antigen-binding fragment thereof comprises a variant Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region, wherein a protein moiety or non-protein moiety is linked to the Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region, wherein a water soluble polymer is linked to the Fc constant region.
  • the antibody, or an antigenbinding fragment thereof comprises a variant Fc constant region, wherein a polyethylene glycol polymer is linked to the Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region, wherein a polyoxazoline polymer is linked to the Fc constant region.
  • the antibody, or an antigen-binding fragment thereof comprises a variant Fc constant region, wherein the variant Fc constant region provides a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
  • the antibody, or an antigen-binding fragment thereof is a human antibody. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is a chimeric antibody. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is a class- switched antibody.
  • the antibody, or an antigen-binding fragment thereof is linked to a therapeutic agent. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is linked to a detectable label. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is linked to an enzyme. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is linked to an enzyme inhibitor.
  • the antibody, or an antigen-binding fragment thereof is an antigen-binding fragment.
  • the antibody, or an antigen-binding fragment thereof is an antigen-binding fragment selected from the groups consisting of: a Fab fragment, a F(ab)2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a disulfide-linked Fv (sdFv), a dAb fragment, an isolated CDR, a nanobody or single domain antibody, a portion of the VH region containing a single variable domain and two constant domains, a diabody, a triabody, a tetrabody, scFv, scFv-FC, scFv-CH, scFab, and scFv-zipper.
  • the antibody, or an antigen-binding fragment thereof, the amino acid sequence of the antibody, or an antigen-binding fragment thereof has at least 85% homology to the reference sequence, at least 90% homology to the reference sequence, at least 95% homology to the reference sequence, at least 96% homology to the reference sequence, at least 97% homology to the reference sequence, at least 98% homology to the reference sequence, or at least 99% homology to the reference sequence.
  • the antibody, or an antigen-binding fragment thereof, the amino acid sequence of the antibody, or an antigen-binding fragment thereof is 100% homologous to the reference sequence across the CDRs and has at least 85% homology to the reference sequence across the FRs, at least 85% homology to the reference sequence across the FRs, at least 90% homology to the reference sequence across the FRs, at least 95% homology to the reference sequence across the FRs, at least 96% homology to the reference sequence across the FRs, at least 97% homology to the reference sequence across the FRs, at least 98% homology to the reference sequence across the FRs, or at least 99% homology to the reference sequence across the FRs.
  • the antibody, or an antigen-binding fragment thereof is used in combination with other anti-viral agents as described herein, such as inhibitors of viral RNA polymerase activity and/or other serine and non-serine protease inhibitors.
  • a subject is infected with SARs-CoV- 2 and by one or more additional viruses.
  • the antibody, or an antigen-binding fragment thereof is administered in an effective amount. Suitable effective amounts are described in more detail herein.
  • the administering step may comprise administering a single dose of an antibody, or an antigen-binding fragment thereof, according to a course of treatment (where the dose may contain an effective amount).
  • the administering step may comprise administering more than one dose of the antibody, or an antigen-binding fragment thereof, according to a course of treatment (where one or more doses may contain an effective amount).
  • the antibody, or an antigen-binding fragment thereof, in each dose administered during a course of treatment is not required to be the same.
  • the administering step may comprise administering at least one loading dose and at least one maintenance dose during a course of treatment.
  • the administering step comprises administering a single dose or a plurality of doses comprising the antibody, or an antigen-binding fragment thereof, according to a course of treatment. In certain embodiments of the first to tenth aspects, the administering step comprises administering a dose or a plurality of doses comprising the antibody, or an antigen-binding fragment thereof, by intravenous administration according to a course of treatment. In certain embodiments of the first to tenth aspects, the administering step comprises administering a dose or a plurality of doses comprising the antibody, or an antigenbinding fragment thereof, by intranasal administration according to a course of treatment. In certain embodiments of the first to tenth aspects, the administering step comprises administering a dose or a plurality of doses comprising the antibody, or an antigen-binding fragment thereof, by pulmonary administration according to a course of treatment.
  • the subject is suffering from or suspected of suffering from a SARS-CoV-2 infection.
  • compositions and/or medicaments comprising the antibody, or an antigen-binding fragment thereof, may be administered according to the methods described herein.
  • the subject is a mammal. In certain embodiments of the first to tenth aspects, the subject is a human.
  • the administering step occurs before the subject has been infected with SARS-CoV-2 (/. «, the subject is at risk for infection), after the subject has been infected with SARS-CoV-2 (but before an infection can be detected), or after a subject has been infected with SARS-CoV-2 and the infection can be detected.
  • the antibody, the subject is a healthcare worker, a first responder (for example, a policeman or a fireman), or a member of the military as such individuals may be required to undertake activities that place them at a higher risk of SARS-CoV-2 infection.
  • a first responder for example, a policeman or a fireman
  • a member of the military as such individuals may be required to undertake activities that place them at a higher risk of SARS-CoV-2 infection.
  • the subject has travelled to a region where SARS-CoV-2 infections have been documented, the subject has had contact with a person who has travelled to a region where SARS-CoV-2 infections have been documented, the has had contact with a person who has a SARS-CoV-2 infection (including a SARS-CoV-2 infection that has not been detected) or is suspected of having a SARS-CoV-2 infection, the subject is a family member or acquaintance of a person who has a SARS-CoV-2 infection (including a CoV infection that has not been detected) or is at risk of having a SARS- CoV-2 infection, the subj ect is an infant or child (for example, a subject under the age of 18 years) who has a caregiver or parent who has a SARS-CoV-2 infection or is at risk of having a SARS- CoV-2 infection.
  • the subject may be suffering from pulmonary disease, cardiovascular disease, diabetes mellitus, bacterial superinfection, sepsis syndrome, hypertension, chronic lung disease (inclusive of asthma, chronic obstructive pulmonary disease, and emphysema), chronic renal disease, chronic liver disease, immunodeficiency, an immunocompromised condition, neurologic disorder, neurodevelopmental, or intellectual disability.
  • chronic lung disease inclusive of asthma, chronic obstructive pulmonary disease, and emphysema
  • chronic renal disease chronic liver disease
  • immunodeficiency an immunocompromised condition
  • neurologic disorder neurodevelopmental, or intellectual disability.
  • the antibody, or an antigen-binding fragment thereof is administered parenterally, such as by intravenous administration, intramuscular administration, or subcutaneous administration, orally, or via the respiratory tract (for example, by pulmonary or intranasal administration).
  • the antibody, or an antigen-binding fragment thereof is administered intravenously.
  • the antibody, or an antigen-binding fragment thereof is administered intramuscularly.
  • the antibody, or an anti gen -binding fragment thereof is administered intranasally.
  • compositions or methods described herein may further comprise one or more additional anti-viral agents in combination with an antibody of the disclosure.
  • additional anti-viral agents include, but are not limited to, those agent that inhibit replication of SARS-CoV-2, such by inhibition of a RNA polymerase activity of SARS- CoV-2, and protease inhibitors, including but not limited to, serine protease inhibitors (for example, inhibitors of TMPRSS2) and cysteine protease inhibitors.
  • Representative agents include, but are not limited to, galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscarnet, laninamivir, oseltamivir, zanamivir, favipiravir,
  • the polypeptides of the disclosure are administered to the subject (or are contacted with cells of the subject) in an effective amount.
  • an effective amount decreases the viral titer of SARS-CoV-2 in the subject and/or limits or prevents an increase in the viral titer of SARS-CoV-2 viral particles in the subj ect.
  • an effective amount decreases viral entry of SARS-CoV-2 subject.
  • an effective amount reduces binding of SARS-CoV-2 to a target cell.
  • an effective amount reduces cellular fusion between SARS-CoV-2 and a target cell.
  • an effective amount reduces release of infective SARS-CoV-2 from an infected cell of the subject.
  • an effective amount reduces infection of a target cell by SARS-CoV-2.
  • the effective amount of an antibody of the disclosure ranges from about 0.01 mg/kg to about 100 mg/kg.
  • the effective amount of an antibody of the disclosure ranges from: i) about 1 mg/kg to about 5 mg/kg; ii) about 1 mg/kg to about 4 mg/kg; iii) about 1 mg/kg to about 4 mg/kg; or iv) about 1 mg/kg to about 4 mg/kg.
  • the effective amount described herein are administered every day, every other day, every three days, every 4 days, every 5 days, every six days or every seven days. In certain embodiments, the effective amount described herein are administered in weekly intervals (such as every week, every 2 weeks, every three weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer). In certain embodiments, the effective amount described herein are administered in monthly intervals (such as every month, every 2 months, every three months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or longer).
  • an effective amounts is administered every day during a course of treatment.
  • the effective amount per day may be administered in a single dose or in more than 1 dose per day (such as two to three doses per day).
  • the effective amount per day is administered as a single dose per day.
  • the effective amount per day is administered in two doses each day (/.c., b i d ), wherein the amount of the antibody in each dose need not be the same.
  • the effective amount described above is administered every other day, every three days, every 4 days, every 5 days, every six days, or every seven days during a course of treatment.
  • the effective amount in the dosing schedules may be administered in a single dose or in more than 1 dose (such as two to three doses).
  • the effective amount in the dosing schedules is administered as a single dose on the day of administration.
  • the effective amount in the dosing schedules is administered in two doses on the day of administration, wherein the amount of the antibody in each dose need not be the same.
  • the effective amounts described above are administered in weekly intervals (such as every week, every 2 weeks, every three weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer), during a course of treatment.
  • the effective amount per weekly interval may be administered in a single dose or in more than 1 dose.
  • the effective amount per week is administered as a single dose on the day of administration.
  • the effective amount per week is administered in two doses on the day of administration, wherein the amount of the antibody in each dose need not be the same.
  • the effective amounts described above are administered in monthly intervals (such as every month, every 2 months, every three months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or longer), during a course of treatment.
  • the effective amount per monthly interval may be administered in a single dose or in more than 1 dose.
  • the effective amount per month is administered as a single dose on the day of administration.
  • the effective amount per month is administered in two doses on the day of administration, wherein the amount of the antibody in each dose need not be the same.
  • the term “dose” refers to an amount of an antibody of the disclosure administered at a given time point. For example, if a course of treatment for an antibody of the disclosure is b.i.d (2 times/admini strati ons per day) for 7 days, each administrations on each of days 1-7 would comprise administering a dose (for 2 doses each day).
  • each dose administered may contain the same amount of an antibody of the disclosure or one or more of doses administered may contain a greater or lesser amount of an antibody of the disclosure as compared to another dose administered on that day.
  • the first dose administered may contain a first amount (z.e., 10 mg/kg) and the second dose administered may contain a second amount (i.e., 5 mg/kg).
  • the first dose administered on day 1 may contain a first amount (i.e., 10 mg/kg)
  • the second dose administered on day 1 may contain a second amount i.e., 5 mg/kg
  • the two doses administered on each of days 2-4 may contain the second amount
  • the two doses administered on each of days 5-7 may contain a third amount (i.e., 2 mg/kg).
  • the administration schedule for the effective amount is not required to be the same.
  • a course of treatment may call for an effective amount to be delivered monthly (either as a single dose or multiple doses) for the first 6 months, and then every three months (either as a single dose or multiple doses) for the next 12 months.
  • the effective amount delivered at each administration during a course of treatment is not required to be the same.
  • a course of treatment may call for an effective amount to be delivered monthly (either as a single dose or multiple doses) for the first 6 months, and then every two months (either as a single dose or multiple doses) for the next 6 months, wherein the effective amount for the first monthly administration is 10 mg/kg and the effective amount for the remaining monthly administration and the bi-monthly administrations is 2.5 mg/kg.
  • any given dose may be delivered in a single unit dose form or more than one unit dose form.
  • a dose when given by IV administration may be provided as a single IV infusion (z.e., a single 20 mg/kg IV infusion) or as two or more IV infusions administered one after the other and be considered a single dose (z.e., two 10 mg/kg IV infusions).
  • a dose may be further divided into a sub-dose.
  • a sub-dose might be, for example, a number of discrete loosely spaced administrations, such as multiple inhalations from an inhaler, by application of a plurality of drops into the eye, or multiple tablets for oral administration.
  • the methods may comprise the administration of a single dose of an effective amount of an antibody of the disclosure during the entire course of treatment. In certain embodiments, more than one dose of an antibody of the disclosure is administered during a course of treatment. Therefore, the methods may comprise the administration of multiple doses of an antibody of the disclosure during the course of treatment. In certain embodiments, the course of treatment may range from 2 days to 1 month, from 2 days to 3 weeks, from 2 days to 2 weeks, or from 2 days to 1 week.
  • the course of treatment may range from 1 month to 6 months, from 1 month to 12 months, from 1 month to 24 months, or from 1 month to 36 months. In certain embodiments, the course of treatment may range from 2 days to 6 days, from 2 days to 5 days, from 2 days to 4 days, or from 2 days to 3 days. In certain embodiments, a dose is delivered at least 1 time per day (z.e., 1 to 3 times) during the course of treatment. In certain embodiments, the course of treatment is continuous. In certain embodiments, a dose is not administered every day during the course of treatment (for example, a dose is be administered at least 1 time per day every other day during the course of treatment). Furthermore, the amount of an antibody of the disclosure in each dose need not be the same as discussed above. In certain embodiments, of the foregoing, one or more doses, preferably all of the doses, contain an effective amount of an antibody of the disclosure.
  • a course of treatment may comprise administering at least one dose as a loading dose and at least one dose as a maintenance dose, wherein the loading dose contains a greater amount of an antibody of the disclosure as compared to the maintenance dose (such as, but not limited to, 2 to 10 times higher).
  • the loading dose is administered initially, followed by administration of one or more maintenance doses through the remaining course of treatment.
  • a loading dose of 10 mg/kg may be administered as the first dose on day 1, followed by maintenance doses of 2 mg/kg for the remainder of the course of treatment.
  • a loading dose of 20 mg/kg may be administered as the first dose on day 1, followed by maintenance doses of 5 mg/kg as the second dose on day 1 and each dose on days 2-4, followed by maintenance doses of 2 mg/kg for the remainder of the course of treatment.
  • a loading dose may be given as a dose that is not the first dose administered during a course of treatment.
  • a loading dose may be administered as the first dose on day 1 and as a dose on one or more additional days (for example, day 4).
  • a loading dose of 10 mg/kg may be administered as the first dose on day 1, followed by a maintenance dose of 2 mg/kg as the second dose on day 1 and each dose on days 2-3, followed by a loading dose of 10 mg/kg as the first dose on day 4, followed by a maintenance dose of 2 mg/kg for the remainder of the course of treatment.
  • the loading dose may be the same (z.e., 10 mg/kg) or different (z. e. , 20 mg/kg for the first loading dose and 10 mg/kg for each other loading dose).
  • the loading dose comprises 2 to 15 times more of an antibody of the disclosure as compared to a maintenance dose administered during the same course of treatment. In certain embodiments, the loading dose comprises 2 to 10 times more of an antibody of the disclosure as compared to a maintenance dose administered during the same course of treatment. In certain embodiments, the loading dose comprises 2 to 5 times more of an antibody of the disclosure as compared to a maintenance dose administered during the same course of treatment.
  • one or more of the loading and maintenance doses preferably all of the loading and maintenance doses, contain an effective amount of an antibody of the disclosure.
  • administration of one or more loading and/or maintenance doses may comprise administering one or more sub-doses and/or administering one or more unit dose forms.
  • the course of treatment is initiated (i.e., the first dose administered) after a subject has been infected with SARS-CoV-2. In certain embodiments, the course of treatment is initiated any time after a subject has been infected with SARS-CoV-2. In certain embodiments, the course of treatment is initiated any time after a subject has been infected with SARS-CoV-2 and before an active SARS-CoV-2 infection can be detected (i.e., by laboratory diagnosis or other methods). In certain embodiments, the course of treatment is initiated any time during which a subject has an active SARS-CoV-2 infection (i.e., by laboratory diagnosis or other methods). In certain embodiments, the course of treatment is initiated 1-5 days after a subject has been infected with SARS-CoV-2. In certain embodiments, the course of treatment is initiated 5- 10 days after a subject has been infected with SARS-CoV-2.
  • the course of treatment is initiated before a subject is infected with SARS-CoV-2 (i.e., a prophylactic administration).
  • SARS-CoV-2 i.e., a prophylactic administration
  • the subject may undergo a course of treatment with an antibody of the disclosure prior to travel to the region or prior to potential exposure.
  • a subject may be someone that is not initially exposed to SARS-CoV-2 infection from a non-human vector source.
  • the spouse or partner of someone who has been exposed to SARS-CoV-2 or who is at risk for exposure to SARS-CoV-2 may undergo a course of treatment with an antibody of the disclosure as well.
  • Such a prophylactic use of the antibodies of the disclosure are beneficial not only to protect the subject that is administered an antibody of the disclosure, but also in protecting those the subject comes into contact with (for example, family members and co-workers).
  • the dose may comprise an antibody of the disclosure alone or an antibody of the disclosure in a pharmaceutical composition.
  • each dose is delivered by parenteral administration.
  • each dose is delivered by IV administration.
  • each dose is delivered by IM administration.
  • each dose is delivered by subcutaneous administration.
  • each dose is delivered by via the pulmonary route.
  • each dose is delivered by pulmonary administration.
  • each dose is delivered by intranasal administration.
  • each dose contains an amount of an antibody of the disclosure in a pharmaceutically acceptable form, such as a pharmaceutically acceptable salt.
  • the antibodies of the disclosure may be formulated into pharmaceutical compositions for administration to subjects in a pharmaceutically acceptable form suitable for administration in vivo.
  • the disclosure provides a pharmaceutical composition comprising antibodies of the disclosure in combination with a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutically acceptable carrier and/or excipient is chemically inert toward the active compounds and is non-toxic under the conditions of use.
  • the pharmaceutically-acceptable carrier and/or excipient employed herein may be selected from various organic or inorganic materials that are used as materials for pharmaceutical compositions and which are incorporated as analgesic agents, buffers, wetting agents, emulsifying agents, pH adjusting agents, binders, disintegrants, diluents, emulsifiers, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles, viscosity-increasing agents, antioxidants, colorants, flavor-improving agents, preservatives, and sweeteners.
  • analgesic agents buffers, wetting agents, emulsifying agents, pH adjusting agents, binders, disintegrants, diluents, emulsifiers, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles, viscosity-increasing agents, antioxidants, colorants, flavor-improving agents, pre
  • the disclosure provides a pharmaceutical composition comprising an antibody of the disclosure, wherein the pharmaceutical composition comprises 1 to 6,000 mg of the antibody.
  • the pharmaceutical composition may contain 1 to 2,000 mg, 1 to 600 mg, 1 to 500 mg, 1 to 400 mg, 1 to 300 mg, 1 to 200 mg, or 1 to 100 mg of the antibody.
  • the antigen of the disclosure is present in the pharmaceutical composition at a concentration of at least 1 mg/mL, 5 mg/mL, 10 mg/mL, 50 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/mldress or 500 mg/ml (with the concentration in each of the foregoing being less than 750 mg/ml).
  • the amount of an antibody of the disclosure in a pharmaceutical composition may very as is known in the art. Generally, the amount of an antibody of the disclosure will range from about 0.01% to about 99% by total weight of the pharmaceutical composition, preferably from about 0.1% to about 70%, from about 0.5% to 50%, or from about 1% to about 30%.
  • Examples of pharmaceutically acceptable carriers may include, for example, water or saline solution, polymers such as polyethylene glycol, carbohydrates and derivatives thereof, oils, fatty acids, or alcohols.
  • the pharmaceutically acceptable carrier is saline or water.
  • the pharmaceutically acceptable carrier is saline.
  • the pharmaceutically acceptable carrier is water or a saline solution.
  • Surfactants such as, but not limited to, detergents, are also suitable for use in the formulations.
  • Specific examples of surfactants include polyvinylpyrrolidone, polyvinyl alcohols, copolymers of vinyl acetate and of vinylpyrrolidone, polyethylene glycols, benzyl alcohol, mannitol, glycerol, sorbitol or polyoxyethylenated esters of sorbitan; lecithin or sodium carboxymethylcellulose; or acrylic derivatives, such as methacrylates and others, anionic surfactants, such as alkaline stearates, in particular sodium, potassium or ammonium stearate; calcium stearate or triethanolamine stearate; alkyl sulfates, in particular sodium lauryl sufate and sodium cetyl sulfate; sodium dodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or fatty acids, in particular those derived from coconut
  • the pharmaceutical composition When administered to a subject, the pharmaceutical composition is preferably sterile.
  • the pharmaceutical compositions of the disclosure are prepared by methods well-known in the pharmaceutical arts.
  • the antibodies of the disclosure are brought into association with a carrier and/or excipient, as a suspension or solution.
  • one or more accessory ingredients e.g, buffers, flavoring agents, surface active agents, and the like.
  • the choice of carrier and/or excipient is determined by the solubility and chemical nature of the antibodies, chosen route of administration and standard pharmaceutical practice.
  • the pharmaceutical composition comprises an antibody of the disclosure and water.
  • the formulation comprises an antibody of the disclosure and saline.
  • a pharmaceutical composition of the disclosure may be presented as capsules, tablets, powders, granules, or as a suspension or solution.
  • Capsule formulations may be gelatin, soft-gel or solid. Tablets and capsule formulations may further contain one or more adjuvants, binders, diluents, disintegrants, excipients, fillers, or lubricants, each of which are known in the art.
  • Such include carbohydrates such as lactose or sucrose, dibasic calcium phosphate anhydrous, corn starch, mannitol, xylitol, cellulose or derivatives thereof, microcrystalline cellulose, gelatin, stearates, silicon dioxide, talc, sodium starch glycolate, acacia, flavoring agents, preservatives, buffering agents, disintegrants, and colorants.
  • Orally administered pharmaceutical compositions may contain one or more optional agents such as, but not limited to, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preservative agents, to provide a pharmaceutically palatable preparation.
  • the antibodies of the disclosure may be combined with a sterile aqueous solution that is isotonic with the blood of the subject.
  • a sterile aqueous solution that is isotonic with the blood of the subject.
  • Such a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile.
  • the formulation may be presented in unit dose form, such as sealed ampules or vials.
  • the formulation may be delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, inhalation, intranasal, parenchymatous, subcutaneous, or sublingual or by way of catheter into the subject's body.
  • a preferred mode of administration is intravenous, intramuscular, or intranasal.
  • Parenteral administration includes aqueous and non-aqueous based solutions.
  • aqueous and non-aqueous based solutions examples include, for example, water, saline, aqueous sugar or sugar alcohol solutions, alcoholic (such as ethyl alcohol, isopropanol, glycols), ethers, oils, glycerides, fatty acids, and fatty acid esters.
  • water is used for parenteral administration.
  • saline is used for parenteral administration.
  • Oils for parenteral injection include animal, vegetable, synthetic or petroleum based oils.
  • sugars for solution include sucrose, lactose, dextrose, mannose, and the like.
  • oils include mineral oil, petrolatum, soybean, corn, cottonseed, peanut, and the like.
  • fatty acids and esters include oleic acid, myristic acid, stearic acid, isostearic acid, and esters thereof.
  • water is the excipient and/or carrier when the antibody of the disclosure is administered intravenously.
  • the excipient and/or carrier is a saline solution when the antibody of the disclosure is administered intravenously.
  • the excipient and/or carrier is a lactated Ringer’s solution when the antibody of the disclosure is administered intravenously.
  • Aqueous dextrose and glycerol solutions may also be employed as an excipient and/or carrier when the antibody of the disclosure is administered intravenously.
  • the antibodies of the disclosure can be formulated into aerosol formulations to be administered via the respiratory tract (for example, pulmonary or nasal administration). These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen. Such aerosol formulations may be administered by metered dose inhalers. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
  • the antibodies of the disclosure may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art.
  • the formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Pat. No. 4,511,069.
  • Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the active agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or a mixture thereof.
  • Nasal and pulmonary solutions of the disclosure typically comprise the drug or drug to be delivered, optionally formulated with a surface-active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers.
  • a surface-active agent such as a nonionic surfactant (e.g., polysorbate-80)
  • the nasal spray solution further comprises a propellant.
  • the pH of the nasal spray solution is optionally between about pH 3.0 and 6.0, preferably 4.5+-0.5.
  • Suitable buffers for use within these compositions are as described above or as otherwise known in the art.
  • Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases.
  • Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thimerosal, chlorobutanol, benzylalkonimum chloride, and the like.
  • Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphatidyl cholines, and various long chain diglycerides and phospholipids.
  • Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like.
  • gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like.
  • nasal and pulmonary formulations are administered as dry powder formulations comprising the active agent in a dry, usually lyophilized, form of an appropriate particle size, or within an appropriate particle size range, for intranasal delivery.
  • Minimum particle size appropriate for deposition within the nasal or pulmonary passages is often about 0.5 pm. mass median equivalent aerodynamic diameter (MMEAD), commonly about 1 pm MMEAD, and more typically about 2 pm MMEAD.
  • Maximum particle size appropriate for deposition within the nasal passages is often about 10 pm MMEAD, commonly about 8 pm MMEAD, and more typically about 4 pm MMEAD.
  • Intranasally and pulmonary respirable powders within these size ranges can be produced by a variety of conventional techniques, such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation, and the like.
  • These dry powders of appropriate MMEAD can be administered to a patient via a conventional dry powder inhaler (DPI), which relies on the patient's breath, upon pulmonary or nasal inhalation, to disperse the power into an aerosolized amount.
  • DPI dry powder inhaler
  • the dry powder may be administered via air-assisted devices that use an external power source to disperse the powder into an aerosolized amount, e.g., a piston pump.
  • the active agent can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for dispersion of the active agent(s).
  • Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, etc.
  • local anesthetics e.g, benzyl alcohol
  • isotonizing agents e.g., sodium chloride, mannitol, sorbitol
  • adsorption inhibitors e.g., Tween 80
  • solubility enhancing agents e.g., cyclodextrins and derivatives thereof
  • stabilizers e.g., serum albumin
  • reducing agents e.g., glutathione
  • the tonicity of the formulation is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the nasal mucosa at the site of administration.
  • the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7.
  • the antibodies of the disclosure may be dispersed in a base or vehicle, which may comprise a hydrophilic compound having a capacity to disperse the active agent and any desired additives.
  • the base may be selected from a wide range of suitable carriers, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof.
  • suitable carriers including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., male
  • a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof.
  • synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. can be employed as carriers.
  • Hydrophilic polymers and other carriers can be used alone or in combination, and enhanced structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking and the like.
  • the carrier can be provided in a variety of forms, including, fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa.
  • the use of a selected carrier in this context may result in promotion of absorption of the active agent.
  • the antibodies of the disclosure are in unit dose form such as a tablet, capsule, infusion bag for intravenous administration, or single-dose vial.
  • Suitable unit dose forms may contain and effective amount (including specific examples of an effective amount described herein), The effective amount may be determined and/or modified during clinical trials designed appropriately for each of the conditions for which administration of an antibody of the disclosure is indicated and will, of course, vary depending on the desired clinical endpoint.
  • the disclosure also provides articles of manufacture for treating and preventing disorders, such as a SARS-CoV-2 infection, in a subject.
  • the articles of manufacture comprise an antibody of the disclosure or a pharmaceutical composition comprising an antibody of the disclosure, optionally further containing at least one additional antiviral compound, as described herein.
  • the articles of manufacture may be packaged with indications for various disorders that the pharmaceutical compositions are capable of treating and/or preventing.
  • the articles of manufacture may comprise a unit dose of an antibody of the disclosure that is capable of treating or preventing a certain disorder, and an indication that the unit dose is capable of treating or preventing a certain disorder, for example a SARS-CoV-2 infection.
  • the antibodies of the disclosure may be used for detection and diagnosis of SARS-CoV-2. Detection may occur by any known means in the art, for example, by immunoassay, including ELISA.
  • immunoassays include, but are not limited to, enzyme immune assays (EIA), ELISPOT (enzyme-linked immunospot), radioimmunoassays (RIAs), immunofluorescence, and other assays known in the art, including but not limited to Western Blot analysis and/or immunoprecipitation methods.
  • a buffered solution of an antigen or a sample containing an antigen is added to a well of a microtiter plate.
  • a solution of non-reacting protein is then added to the well to prevent non-specific binding.
  • An antibody of the disclosure, or antigen-binding fragment thereof is added.
  • Such antibody or antigen binding fragment may be typically conjugated to a reporter molecule, such as, but not limited to, luciferase, horse-radish peroxidase, alkaline phosphatase, or P-D-galactosidase.
  • the antibody of the disclosure is not conjugated to a reporter molecule
  • secondary antibody that recognized the antibody of the disclosure may be added that is conjugated to a reporter molecule.
  • a substrate for the reporter molecule is then added, which leads to a detectable signal.
  • ELISAs may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. A competitive ELISA may also be used.
  • an unlabeled antibody of the disclosure, or antigen-binding fragment thereof, (the primary antibody, which may be conjugated to a reporter molecule or unlabeled) is incubated in the presence of a sample containing the antigen, which mixture is then added to a microtiter plate which is coated with the same antigen (the reference antigen) antigen-coated well. The plate is washed so as to remove unbound antibodies. If the primary antibody is not conjugated to a reporter molecule, a secondary antibody conjugated to a reporter molecule that is specific to the primary antibody is added to generate the detectable signal. Depending on the amount of antigen in the sample, more or less primary antibody will be available to bind the reference antigen.
  • a known quantity of an antigen is linked to a radioactive tracer, such as, but not limited to, 1-125, which is then mixed with a known amount of antibody of the disclosure to bind to an antigen (such as SARS-CoV-2 spike protein).
  • an antigen such as SARS-CoV-2 spike protein.
  • a sample containing unknown quantity of the antigen is added and as the concentration of unlabeled antigen is increased, the binding between the antibodies and the labeled standard is decreased, which is directly measurable by measuring radioactivity.
  • the present disclosure provides a method for the determination (such as an immunoassay determination) of SARS-CoV-2 in a patient, the method comprising: a) incubating a bodily sample from the patient with at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37 and a detectable label, wherein the detectable label is present on the antibody, or antigen binding fragment thereof, or the detectable label is present on a binding partner for either the SARS-CoV-2 or the antibody, or the antigen binding fragment thereof, to form an immunological complex containing the determinable group; and b) determining the presence of the detectable label in the sample, wherein the presence of the detectable label indicates SARS-CoV-2 is present in the sample.
  • such method may further comprise isolating the immunological complex from the sample and determining the presence of the detectable label in either in the isolated immunological complex or in the sample remaining.
  • the bodily sample is a serum sample, a blood sample, a plasma sample, a throat swab sample, a nasopharyngeal swab sample, a sputum sample, a fecal sample, a urine sample, a saliva sample, or a bronchoalveolar lavage fluid sample.
  • any assay described herein may be used for diagnostic purposes. Therefore, the disclosure provides for methods of diagnosing SARS-CoV-2 infection in a subject using the antibodies of the disclosure. Such a diagnostic use may be directed to determining if a SARS-CoV-2 infection is present, monitoring recovery from a SARS-CoV-2 infection, evaluating the efficacy of a therapeutic treatment for treating a SARS-CoV-2 infection, and for other purposes known in the art.
  • Kits The disclosure also provides a kit for use in the methods described herein, the kit comprising an antibody of the disclosure, or a pharmaceutically acceptable form thereof, and at least one of the following: (i) at least one other therapeutic agent; (ii) a buffer; (iii) instructions for administering the antibody of the disclosure, or pharmaceutically acceptable form thereof to a subject to treat a SARS-CoV-2 infection in the subject or detect SARS-CoV-2 in a sample (such as a sample from a subject).
  • the subject is a human.
  • the antibody is an antibody from Table 1 or Tables 2A-H, an antibody of the first to fourth embodiments, or any of the specific aspects of the first to fourth embodiments described above.
  • Example 1 Identification. Purification. Sequencing, and Recombinant Production of SARS-CoV- 2 Specific Antibodies
  • Peripheral blood samples were obtained from a single individual at the time of or following verified SARS-CoV-2 infection.
  • Peripheral blood mononuclear cells PBMC were isolated by density gradient purification.
  • PBMC peripheral blood mononuclear cells
  • RBD SAR-CoV-2 Spike Receptor Binding Domain
  • Single CD3- CD14- CD19+ RBD+ cells were sorted using a FACSMelody cell sorter (BD Biosciences) directly into 96-well PCR plates (Bio-Rad, Hercules, Calif.) containing 4 pL/well 0.5> ⁇ PBS with 10 mM DTT (Invitrogen), and 8 U RiboLock (ThermoFisher) RNAse inhibitor. Plates were sealed with MICROSEAL F FILM (Bio-Rad) and immediately frozen at -80° C until used for RT-PCR. cDNA was synthesized and semi-nested RT-PCR for IgH, IgX, and IgK V gene transcripts was performed as described in Kobie et al.
  • IgGl expression vector cloning and transfection of human HEK293T cells were performed as previously described in Kobie et al. Monoclon Antib Immunodiagn Immunother. 2015 Apr. 1; 34(2): 65-72 and Tiller et al., J Immunol Methods. 2008 Jan. 1; 329(1-2): 112-124.
  • PCR products were sequenced at Genewiz Sequences and analyzed by IgBlast and IMGT/V-QUEST to identify germline V(D)J gene segments with highest identity and to determine sequence properties (shown in Table 3).
  • IgG was purified from culture supernatant using MAGNA PROTEIN G beads (Promega, Madison, Wis.).
  • Table 3 provides a description of the SARS-CoV-2 hmAbs of the disclosure indicating the germline V(D)J for the heavy chain and light chain variable regions for each antibody as well as the original isotype of the antibody.
  • Example 2 The Antibodies of the Disclosure Bind to SARS-CoV-2 Proteins To determine if the antibodies of the disclosure bound to SARS-CoV-2 spike protein, binding experiments were performed. ELISA plates were coated with the following recombinant SARS-CoV-2 polypeptides: i) SARS-CoV-2 receptor binding domain (RBD) polypeptide (SEQ ID NO: 162); ii) SARS-CoV-2 SI domain polypeptide (containing the RBD) (SEQ ID NO: 163); iii) SARS-CoV-2 SI and S2 domain polypeptide (SEQ ID NO: 164); and iv) SARS-CoV-2 nucleoprotein (N; negative control) (SEQ ID NO: 165).
  • RBD SARS-CoV-2 receptor binding domain
  • SEQ ID NO: 163 SARS-CoV-2 SI domain polypeptide (containing the RBD)
  • SEQ ID NO: 163 SEQ ID NO: 163
  • the CR3022 antibody (recombinant human anti-SARS-CoV monoclonal antibody that specifically binds to spike protein) served as a positive control while wells incubated with PBS only (no recombinant SARS-CoV-2 polypeptide) also served as a negative control.
  • Recombinant SARS-CoV-2 polypeptides were coated at a concentration of 1 pg/ml and plates were blocked with 3% bovine serum albumin (BSA) in PBS.
  • BSA bovine serum albumin
  • the antibodies of the disclosure were diluted in PBS and tested at 1 and 10 pg/ml. Antibody binding was detected using horseradish peroxidase-conjugated anti-human IgG. The results are shown in FIG. 1, with the values expressed as area under the curve of the OD reading for both the 1 and 10 pg/ml antibody concentrations.
  • the antibodies of the disclosure bound recombinant SARS- CoV-2 Spike protein polypeptides specifically, with no significant binding to the negative control SARS-CoV-2 N polypeptide.
  • FIG. 1 also shows the antibodies of the disclosure showed more favorable binding to the recombinant SARS-CoV-2 RBD polypeptide as compared to the SARS- CoV-2 SI polypeptide and S1+S2 polypeptide.
  • Antibodies 1213H7, 1206D1, 1212D4, 1206D12, 1207F10, and 1206G12 demonstrated 2-fold or greater binding to recombinant SARS-CoV-2 RBD polypeptide as compared to recombinant SARS-CoV-2 SI polypeptide, suggesting that at least a portion of the epitope may be masked in the full length SARS-CoV-2 Spike protein.
  • antibodies 1212C2, 1212F2, 1212D5, 1206A5, 1212D6, and 1206D12 showed the least different in binding to recombinant SARS-CoV-2 RBD polypeptide as compared to recombinant SARS- CoV-2 SI polypeptide and S1+S2 polypeptide.
  • Example 3 The Antibodies of the Disclosure Bind to SARS-CoV-2 Infected Cells
  • Vero E6 cells were infected with SARS-CoV-2 virus (USA- WA1/2020 (Gen Bank: Accession No. MN985325.1) obtained from BEI Resources (NR-52281)) or mock infected and incubated for different times post-infection. Cells were then fixed with 4% neutral formaldehyde for 30 minutes followed by permeabilization using 0.5% triton X-100 for 15 minutes. Cells were then blocked with 2.5% Bovine Serum Albumin (BSA) at 37° C for 1 h.
  • BSA Bovine Serum Albumin
  • Example 4 The Antibodies of the Disclosure Neutralized SARS-CoV-2 Virus by SARS-CoV-2 Antibodies
  • the ability of the antibodies of the disclosure to neutralize SARS-CoV-2 virus was analyzed using a plaque reduction microneutralization (PRMNT) assay. Since serial dilutions of the Ab containing samples are used, the PRMNT assay measures the neutralizing activity of Abs in a concentration dependent manner. The ability of a neutralizing antibody to inhibit virus infection is manifested in the reduced capacity of the virus to produce visible plaques when compared to virus-only infected control cells.
  • the PRMNT assay is similar to a plaque reduction neutralization assay with the only difference that the PRMNT utilizes 96-well plates.
  • the ability of the antibodies to neutralize SARS-CoV-2 virus was determined using two different approaches, a pre-treatment approach and a post-treatment approach.
  • a pre-treatment approach antibodies of the disclosure are pre-incubated with SARS-CoV-2 at 37°C for Ih. This enables the antibodies to bind to the Spike protein blocking virus attachment, and subsequently interfering with the process of virus entry.
  • virus adsorption is allowed to progress for Ih at 37°C. This gives the virus an opportunity to initiate viral entry by binding to the cell surface receptor (ACE2).
  • ACE2 cell surface receptor
  • Antibodies incubated with the infected cells may interfere with active viral replication partly by blocking later steps of the virus entry into the cell and/or by inhibiting the cell-to-cell spread of virus progeny.
  • NT50 neutralization titer 50
  • the post-treatment PRMNT assay offers a therapeutic evaluation of the neutralizing activity of Abs against SARS-CoV-2.
  • the pretreatment PRMNT assay focuses on identifying antibodies targeting the SARS-CoV-2 Spike protein RBD
  • the post-treatment PRMNT assay allows the identification of antibodies targeting other regions on the viral Spike protein involved in the proteolytic cleavage step (for example, the SI region), the fusion event (for example, the S2 region), or antibodies affecting other steps in the replication cycle of the virus (for example, virus assembly and/or budding).
  • a potent SARS-CoV-2 neutralizing antibody will show a low NT50 titer whereas a weak neutralizing antibody will give a high NT50 value.
  • 100 PFU/well of SARS-CoV-2 (USA-WA1/2020 from BEI Resources (NR-52281)) was mixed with decreasing concentrations of the antibodies of the disclosure (2-folds dilutions, starting concentration 25,000 ng/well for antibodies of the disclosure in 100 pl of infection media (DMEM with 1% penicillin-streptomycin-L-glutamate) and incubated at 37° C, 5% CO2 for Ih in infection media.
  • Vero HL cells 96-well plate format, 4 x 10 4 cells/well, quadruplicate
  • virus-antibody mixture were infected with virus-antibody mixture and incubated (virus adsorption) for Ih. After Ih incubation, the media was replaced with post-infection media (DMEM containing 2% FBS and 1% Avicel).
  • Vero HL cells (96-well plate format, 4 x 10 4 cells/well, quadruplicate) in infection media were infected with 100 PFU/well of SARS-CoV-2 (USA- WA1/2020 from BEI Resources (NR-52281))). After 1 h of viral adsorption, the infection media was changed with the 100 pl of post-infection media containing decreasing concentrations of the antibodies of the disclosure (2-folds dilutions, starting 50,000 or 25,000 ng/ml for antibodies of the disclosure.
  • infected cells (both pre-treatment and post-treatment) were fixed with 10% neutral formalin for 24 h and were immunostained using anti-NP (nucleoprotein) monoclonal 1C7C7 antibody.
  • Virus neutralization was evaluated and quantified using ELISPOT, and the percentage of infectivity calculated using sigmoidal dose response curves. Mock-infected cells and viruses in the absence of the antibodies of the disclosure were used as internal controls.
  • the results are shown in Table 4 below.
  • the results show the antibodies of the disclosure are neutralizing SARS-CoV-2 antibodies.
  • Nine of the antibodies tested displayed similar activity in the pre-treatment and post-treatment conditions (with NT50 values for the pre-treatment and post-treatment conditions being within 2-fold of one another).
  • Eight of the antibodies tested displayed different activity in the pre-treatment and post-treatment conditions (with NT50 values for the pre-treatment and post-treatment conditions being greater 2-fold of one another).
  • antibody 1212C2 exhibited potent neutralizing activity in both the pre-treatment and post-treatment conditions, with NT50 values ⁇ 50 ng/ml in each assay.
  • Antibody 1212C2 exhibited strong binding to recombinant SARS-CoV-2 RBD polypeptide (FIG. 1).
  • antibodies 1212F5 and 1213H7 exhibited potent neutralizing activity in both the pre-treatment and post-treatment conditions, with NT 50 values ⁇ 100 ng/ml in each assay.
  • RBD was injected over the NmAbs at four concentrations (80nM, 20nM, 5nM, and 1.25nM) for 90 s, followed by 300 s dissociation phase. After each injection, the surface was regenerated with a 30 s pulse of 3M MgC12, followed by capture of fresh hmAb.
  • the buffer flowrate for affinity analysis was 50 pL/min.
  • Sensorgrams were globally fit to a 1 :1 model, without a bulk index correction, using Biacore T-200 evaluation software version 1.0. Standard errors for the rate constants were obtained from the fitted sensorgram data.
  • Replicate KD errors were estimated by repeating the experiments for four of NmAb-RBD interactions (1212C2, 1206D1, 1215D1 and CB6/JS016). Standard deviations from these experiments were used to define an average KD error of 11%.
  • Example 6 Broad and distinct neutralization of SARS-CoV-2 variants by SARS-CoV-2 antibodies SARS-CoV-2 has undergone substantial mutation, including within the Spike protein with such mutations impacting the ability of antibodies to recognize these emerging variants.
  • Variants of Concern are particularly threatening to human health and have driven multiple waves of the SARS-CoV-2 pandemic.
  • the defining mutations in spike for the VoC are as follows as reported by the US Center for Diseases Control and Prevention and Nexstrain. Beta: D80A, D215G, del241-243, K417N, E484K, N501Y, D614G, A701V.
  • infected cells were fixed with 10% neutral formalin for 24 h and were immune- stained using anti-NP monoclonal 1C7C7 antibody.
  • Virus neutralization was evaluated and quantified using ELISPOT, and the percentage of infectivity calculated using sigmoidal dose response curves.
  • the formula to calculate percent viral infection for each concentration is given as [(Average # of plaques from each treated wells - average # of plaques from “no virus” wells)/(average # of plaques from “virus only” wells - average # of plaques from “no virus” wells)] x 100.
  • a non-linear regression curve fit analysis over the dilution curve can be performed using GraphPad Prism to calculate NT50. Mock-infected cells and viruses in the absence of hmAb were used as internal controls.
  • the SARS-CoV-2 antibodies demonstrate unique neutralization profiles against the SARS- CoV-2 variants.
  • 1212C2 demonstrates potent neutralization with NT50 ⁇ 50 ng/ml for WAI, Delta, Epsilon, and rK417. Neutralization by 1212C2 is somewhat diminished for rN501 and is not evident for Beta, Gamma, rE484K, or the rK417N E484K N501Y triple mutation variant. This pattern is distinct from CB6/JS016, RGN10933, and RGN10987.
  • 1213H7 is able to neutralize all the viruses tested which is in contrast to CB6/JS016 and RGN10933. 1213H7 has superior neutralizing activity against WAI and the rK417N E484K N501Y triple mutation variant compared to RGN1087.
  • Example 7 Improved neutralization of SARS-CoV-2 by modifying 1212C2 heavy chain variable residues
  • 1212C2-V1 (Fig. 4C) was created by introducing the I28T, I34M, H93Y, T96A, S114Y changes in to 1212C2 and resulted in 33% increase in neutralization potency.
  • 1212C2-V2 (Fig. 4D) was created by introducing the Q1E, Q6E, I28T, D31A, I34L, H93Y, T96A, SI 14Y and resulted in at least a 44% increase in neutralization potency.
  • Example 8 SARS-CoV-2 antibodies have prophylactic and therapeutic activity in hamsters
  • SARS-CoV-2 antibodies 1212C2 and 1206D1 The in vivo activity of SARS-CoV-2 antibodies 1212C2 and 1206D1 was evaluated in the golden Syrian hamster model of SARS-CoV-2 infection.
  • 10 mg/kg hmAb was administered by intraperitoneal (i.p.) injection 6 h before intranasal (i.n.) challenge with SARS-CoV-2.
  • dpi In contrast, at 2 dpi, hamsters that received 1212C2 already started to exhibit meaningful viral load reduction in their nasal turbinate and lungs (Fig. 5A).
  • virus was detected in the nasal turbinates and lungs of all of the hamsters in the PBS and isotype control groups, although an overall decrease compared to 2 dpi, consistent with the viral dynamics of SARS-CoV-2 infection in hamsters.
  • prophylactically treated 1212C2 hamsters exhibited the eradication of viral loads in the nasal turbinates and lungs in 3 of 4 animals.
  • 1206D1 hmAb exhibited modest activity, noted by 50% of hamsters having no detectable virus in the nasal turbinates and all 1206D1 hamsters having detectable virus in the lungs, although trending to lower titers compared to PBS and isotype control groups.
  • 1212C2 demonstrated substantial prophylactic activity as seen in sterilizing protection in 63% of the hamsters.
  • Therapeutic activity was tested by treatment with 25 mg/kg of 1212C2 6 h following i.n. infection of hamsters with SARS-CoV-2 and evaluating viral burden at 4 dpi only due to the limited availability of hmAb.
  • SARS-CoV-2 was detected in the nasal turbinates in 3 of 4 animals in the PBS and isotype control groups. No virus was detected in the nasal turbinates of the 1212C2 treated hamsters.
  • SARS-CoV-2 was detected in the lungs of all PBS and isotype control treated hamsters, but only in 1 of 4 of the 1212C2 treated hamsters (Fig. 5B).
  • 1212C2 demonstrated substantial therapeutic activity, reducing virus to undetectable levels in 75% of the treated hamsters.
  • SARS-CoV-2 infection in hamsters results in gross lung lesions, including consolidation, congestion, and atelectasis.
  • Therapeutically, significantly less lung pathology was also observed in the 1212C2 treated hamster group compared to the PBS-treated group (p 0.0136), and ⁇ 58% reduction in lung pathology in the 1212C2 group compared to the control hamsters (Fig. 5D).
  • these differences observed in lung lesions are consistent with the viral burden seen in the upper and lower respiratory tract (A+B).
  • A+B upper and lower respiratory tract
  • Therapeutic mAbs are typically delivered systemically through intravenous infusion or intramuscular injection. Such delivery typically results in ⁇ 0.1% of the injected dose in the lung epithelial lining fluid at Cmax, thus highly inefficient in achieving optimal concentrations in the respiratory tract for the protection or treatment of respiratory infections, such as SARS-CoV-2.
  • Example 10 Inhaled delivery of 1212C2 provides more efficient therapeutic activity against SARS-CoV-2 infection in hamsters as compared to intraperitoneal delivery
  • the crystallizable fragment (Fc) of 1212C2 was modified with the LALA mutation (z.e., a double mutation of Leu234Ala together with Leu235Ala) to reduce Fc receptor (FcR) binding and subsequent Fc-mediated effector functions, and further modified to increase half-life (referred to as 1212C2-HLE-LALA with the mutations M252Y/S254T/T256E (YTE)).
  • LALA mutation z.e., a double mutation of Leu234Ala together with Leu235Ala
  • FcR Fc receptor binding and subsequent Fc-mediated effector functions
  • 1212C2-HLE-LALA with the mutations M252Y/S254T/T256E (YTE)
  • Virus was not detectable in the lungs of any hamsters treated via IH with 1212C2-HLE-LALA at 16.3 mg/kg, and virus was lower in their nasal turbinates compared to controls. In contrast, virus was detectable at 2 dpi in the lungs of 50% of the hamsters treated i.p. with 1212C2-HLE-LALA at 25 mg/kg, and not significantly decreased in nasal turbinates compared to controls. At 2 dpi, virus was only detectable in the lungs of 25% and 50% of the hamsters treated via IH with lung-delivered doses of 3.2 mg/kg and 0.6 mg/kg 1212C2-HLE- LALA, respectively.
  • inhaled 1212C2-HLE-LALA was superior in reducing viral titer in the lungs compared to i.p. administration.
  • virus was detected in the nasal turbinates and lungs of all infected control hamsters (saline and isotype hmAb), with a trend toward modest non-specific reduction in viral titer in nasal turbinates only with isotype control hmAb (Fig. 6B).
  • Viral titers were only sporadically detected in the nasal turbinates of the 1212C2 treated groups, with the exception of detectable virus in all of the 0.6 mg/kg inhaled 1212C2-HLE- LALA group.
  • Example 11 SARS-CoV-2 antibodies prevent SARS-CoV-2 in mice
  • mice 8C and 8D Nluc expression (Fig. 8E) and virus titers (Fig. 8F) in the nasal turbinate, lungs, and brains of 1212C2-treated mice.
  • 1212C2 was able to protect mice from clinical symptoms of rSARS-CoV-2/Nluc-2A infection as determined by changes in body weight (Fig. 8G) and survival rate (Fig. 8H).
  • mice were treated intraperitoneally (i.p.) with 25 mg/kg of 1212C2, 1213H7, or an IgG isotype control 24 h prior to challenge with 104 PFU of rSARS-CoV-2 Venus, rSARS-CoV-2 mCherry Beta, or both rSARS- CoV-2 Venus and rSARS-CoV-2 mCherry Beta together.
  • Body weight (Fig. 9A) and survival (Fig. 9B) were evaluated for 12 days post-infection (pi).
  • 1212C2 and 12137H7 similarly limited lung, nasal, and brain viral burden consistent with their activity against SARS-CoV-2 wild-type, or wild-type and Beta, respectively.
  • lungs were collected to determine Venus and mCherry fluorescence expression using an Ami HT imaging system (Fig. 10A).
  • BF bright field.
  • Venus and mCherry radiance values were quantified based on the mean values for the regions of interest in mouse lungs (Fig. 10B).
  • Mean values were normalized to the autofluorescence in mock-infected mice at each time point and were used to calculate fold induction.
  • Gross pathological scores in the lungs of mock-infected and rSARS-CoV- 2-infected KI 8 hACE2 transgenic mice were calculated based on the % area of the lungs affected by infection.
  • Viral titers in the lungs (top), nasal turbinate (middle) and brain (bottom) at days 2 and 4 pi were determined by plaque assay in Vero E6 cells. Bars indicates the mean and SD of lung virus titers. Dotted lines indicate the limit of detection. As shown in Fig. 1 ID, quantification of rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) in the lungs (top), nasal turbinate (middle) and brain (bottom) from mice co-infected with both rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) at days 2 and 4 pi.
  • WT rSARS-CoV-2 Venus
  • Beta rSARS-CoV-2 mCherry SA
  • Example 14 Classification of SARS-CoV-2 antibodies by competition epitope mapping
  • 1215D1, 1207B4, and 1207F10 mAbs were assigned to the C386-2/RBD complex, while 1212C2 was assigned to the C121/RBD complex.
  • 1212C2 (modeled by C121-RBD) overlaps with Cl (modeled by P2C-1F11/RBD) and C2 NAbs, while 1215D1 (modeled by C386-2/RBD) can simultaneously bind with several Cl (1214D4, 1212C8, 1212D5 etc.) and CID (1213H7) mAbs.
  • Constant exposure of SARS-CoV-2 to suboptimal levels of mAbs in infected individuals or in vitro can promote the selection of monoclonal antibody resistant mutant (MARM) SARS- CoV-2.
  • MARMs were selected and identified using an in vitro model (Vero E6 cells) of SARS-CoV-2 infection (Fig. 14). Culture of SARS-CoV-2 in the presence of 1212C2, 1213H7, or 1215D1 resulted in the generation of MARM that had developed mutations within the Spike protein, resulting in loss of recognition by the mAb.
  • the 1212C2 MARM has the E484K mutation in RBD.
  • the 1212C2/RBD model identifies HCDR2 N53 and HCDR3 R103 as residues that interaction with RBD E484 and sterically prevents 1212C2 from binding to RBD E484K, These and additional residues form a pocket optimized for glutamic acid and smaller residues, but occlude lysine and Arginine sidechains at RBD E484.
  • the 1213H7 MARM has RBD mutations G476D and F490S.
  • Our current model of the 1213H7/RBD complex is of lower resolution than 1212C2 and 1215D1 interactions. Nonetheless, the 1213H7 has been assigned as having a CID epitope that binds on the opposite side of the RBD tip, relative to the 1215D1 epitope.
  • This model is consistent with the competition epitope mapping.
  • F490 sits in the center of the heavy chain binding site of the 1215D1/RBD model, which provides an initial explanation of 1215D 1 ’s sensitivity to the F490S MARM and validates the general location of the 1215D1/RBD model. Hydrophobic packing interactions, contributed from HCDR3 Fi l l is expected to be an important contributor to the 1215D1/RBD interactions.
  • the mutations are described below in Table 8.
  • the goals of this study were to compare and contrast the efficiency and kinetics of delivery of a human mAb to the lung tissues following IV, IN and IH routes of delivery. Measurements of delivery were determined by quantitating BAL, lung and serum titer that were evaluated for human IgG using a quantitative ELISA assay. The comparative analysis is shown in Fig. 15 and indicate IgG titer in serum, BAL and lung over a 24 hour of evaluation at 30mins, 6hrs, 12hrs and 24hrs following dosing by IV, IN or IH routes of delivery. It should be noted that the concentration of drug use in IV, IN and IH was different for each route and reflect differences in the efficiency of delivery.
  • Example 17 Neutralization Assay for hmAbs alone or in combination
  • the 1212C2, 1213H7, and 1215D1 mAbs were assessed both alone and in combination in a neutralizing antibody assay against WAI P7 (1.7x105 pfu/mL).
  • 96-well plates were inoculated with lOOPFU/well (MOI 0.05), except for control wells containing no virus.
  • Wells containing virus were treated with lOOng, 50.
  • Ong of one or more antibodies for 24 hours in 1% Avicel media and NT50 was assessed pre- and posttreatment.
  • NT50 for 1212C2 was 4.5ng and 3.2ng pre- and post-treatment, respectively.
  • NT50 for 1213H7 was 6.6ng and 4.8ng pre- and post-treatment, respectively.
  • NT 50 for 1215D1 was 22.6ng and 5.9ng pre- and post-treatment, respectively.

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Abstract

The disclosure is based, at least in part, on certain human monoclonal antibodies, or antigen binding fragments thereof, having unexpected broad neutralizing activities against SARS-CoV-2. The disclosed antibodies and/or antigen-binding fragments thereof are therapeutic agents for the treatment of SARS-CoV-2 infections and are suitable for use in therapeutic methods to protect individuals from SARS-CoV-2 infections.

Description

HUMAN MONOCLONAL ANTIBODIES TO SARS-COV-2 AND USE THEREOF
BACKGROUND OF THE DISCLOSURE
Coronaviruses (CoVs) are members of the family Coronaviridae. The Coronaviridae has 4 separate genera, the Alphacoronavirus, Betacoronavirus, Deltacoronavirus, and Gammacoronavirus with each genus having one or more subgenus. The Alphacoronaviruses and Betacoronaviruses mainly infect bats, but they also infect other species such as humans, camels, rabbits, dogs and masked palm civets. CoVs are enveloped viruses that possess extraordinarily large single-stranded RNA genomes ranging from 26 to 32 kilobases in length. CoVs were historically regarded as pathogens that only cause mild diseases. Currently, at least seven CoV species are known to cause diseases in humans. HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKUl generally cause only mild common cold symptoms. Severe illness can be caused by the remaining three viruses, each in the Betacoronavirus genus. SARS-CoV resulted in the outbreak of sever acute respiratory syndrome (SARS) in 2002 and 2003. MERS-CoV was responsible for Middle East respiratory syndrome (MERS), which emerged in 2012 and remains in circulation in camels. A small outbreak of MERS-CoV was reported on June 2, 2020 in Saudi Arabia (9 case reported with 5 deaths). Finally, SARS-CoV-2, which emerged in December 2019 in Wuhan province of China, and causes COVID-19. The COVID-19 pandemic is impacting virtually every country around the world, with no immediate prospects for easing. The US is currently topping the world in the number of infected people with more cases in sight.
The fatality rate for SARS was approximately 10%, with the risk for a fatal outcome increasing with age, while the mortality of SARS-CoV-2 infection is estimated to be around 1.5 to 10%, with the risk for fatal outcome increasing with age and certain comorbidities. The fatality rate for MERS-CoV was approximately 37%. In addition, SARS-CoV-2 has a basic reproduction rate (Ro) of 3.3-5.5, which is higher than those of SARS-CoV and MERS-CoV (2.7-3.9), indicating a higher transmissibility of SARS-CoV-2 than other human coronaviruses. Unlike SARS and MERS which each infected less than 10,000 individuals, COVID 19 has currently infected more than 16,000,000 individuals and has been responsible for over 650,000 deaths worldwide.
No treatments are currently approved for the treatment of CoV infections, including SARS- CoV-2 infections. As such the art is in need of new therapies for the treatment of CoV infections, particularly SARS-CoV-2 infections. The disclosure provides a solution to the shortcomings of the art by providing new therapies for CoV infection, specifically including SARS-CoV-2 infection.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the ELISA binding profile of antibodies of the disclosure for binding to recombinant SARS-CoV-2 spike proteins.
FIG. 2 shows binding of antibodies of the disclosure to SARS-CoV-2 infected cells.
FIG. 3 depicts the high affinity of certain mAbs for SARS-CoV-2 Spike Receptor Binding Domain. Binding to SARS-CoV-2 RBD was determined by surface plasmon resonance (SPR).
FIGS. 4A-4D depict rationally designed 1212C2 mAb variants with increased neutralization activity. Fig. 4A shows the sequences of 1212C2 mAb and variants thereof. Fig. 4B, 4C and 4D show viral neutralization by 1212C2, 1212C2-V1, and 1212C2-V2 mAbs, respectively.
FIG. 5A-5D shows prophylactic and therapeutic activity of 1212C2 hmAb in SARS-CoV- 2-infected hamsters. Golden Syrian hamsters were (Fig. 5 A) prophylactically treated i.p. with 10 mg/kg indicated hmAb or PBS 6 h before intranasal (i.n.) challenge with 2 x 105 PFU SARS-CoV- 2 or (Fig. 5B) therapeutically treated by intraperitoneal (i.p.) injection with 25 mg/kg indicated hmAb or PBS 6 h following i.n. challenge with SARS-CoV-2. Virus present in nasal turbinates and lungs was determined by plaque assay. Dotted line indicates limit of detection. (C and D) Distribution of pathologic lesion, including consolidation, congestion, and pneumonic lesions, was measured using ImageJ and represented as the percentage of the total lung surface area in prophylactically treated (Fig. 5C) and therapeutically treated (Fig. 5D) animals. Each symbol represents an individual animal, p values determined by 2-tailed t test.
FIGS. 6A - 6C show therapeutic activity of inhaled 1212C2 hmAb in SARS-CoV-2- infected hamsters. Golden Syrian hamsters were infected i.n. with 2 x 105 PFU SARS-CoV-2 and 12 h later treated with indicated hmAb by i.p. or inhaled administration. Virus present in nasal turbinates and lungs was determined by plaque assay at 2 dpi (Fig. 6A) and 4 dpi (Fig. 6B). Dotted line indicates limit of detection. (Fig. 6C) Distribution of pathologic lesion were measured using ImageJ and represented as the percentage of the total lung surface area. *p < 0.0055 and **p < 0.00055 compared to PBS i.p. -treated group determined by 2-tailed t test.
FIG. 7 shows the preservation of body weight with inhaled therapeutic treatment of SARS-
CoV-2 infected hamsters. FIGS. 8A-8H show prophylactic activity of 1212C2 in mice infected with SARS-CoV-2. As shown in Figs. 8 A and 8B, five-week-old KI 8 hACE2 transgenic mice were injected with isotype IgG control or 1212C2 MAbs and 12 h after treatment, mice were infected (105 PFU/mouse) with recombiant SARS-CoV-2/Nluc-2A (Nluc-2A). Mock-treated and mock-infected mice were included as controls. Mice were anesthetized at 1, 2, 4 and 6 dpi and retro-orbitally injected with the Nluc substrate. Nluc expression was determined using an IVIS system (Fig. 8A) and quantitively analyzed by the Aura program (Fig. 8B). The lungs were excised and photographed at 1, 2, 4 and 6 dpi (Fig. 8C) and the gross lesions on the lung surfaces were quantitively analyzed by ImageJ (Fig. 8D). ns, not significant. Nluc activity in the nasal turbinate (left), lungs (middle) and brains (right) from infected mice were measured using a multi-plate reader (Fig. 8E). Viral titers in the nasal turbinate (left), lungs (middle) and brain (right) were determined by plaque assay (Fig. 8F). Five-week-old K18 hACE2 transgenic mice were injected with isotype IgG control or 1212C2 MAbs and 12 h after treatment, mice were infected (10s PFU/mouse) with rSARS-CoV-2/Nluc-2A (Nluc-2A). Mock-treated and mock-infected mice were included as controls. Mice were monitored for 12 days for changes in body weight 1 (Fig. 8G) and survival (Fig. 8H).
FIGS. 9A and 9B show prophylactic activity of 1212C2 and 1213H7 against SARS-CoV- 2 WT and SARS-CoV-2 Beta (SA) in KI 8 hACE2 transgenic mice. Six-to-eight-week-old female KI 8 hACE2 transgenic mice (n=5) were treated (i.p.) with 25 mg/kg of IgG isotype control, hMAb 1212C2, or hMAb 1213H7 and infected with 104 PFU of rSARS-CoV-2 Venus (left), rSARS- CoV-2 mCherry SA (Beta) (middle) or both, rSARS-CoV-2 Venus and rSARS-CoV-2 mCherry SA (Beta) (right). Mice were monitored for 12 days for changes in body weight (Fig. 9A) and survival (Fig. 9B). Data represent the means and SD of the results determined for individual mice.
FIGS. 10A and 10B show inhibition of SARS-CoV-2 lung viral burden in mice by 1212C2 and 1213H7. Six-to-eight-week-old female K18 hACE2 transgenic mice (n=3) were injected (i.p.) with 25 mg/kg of an IgG isotype control, mAb 1212C2, or mAb 1213H7 and infected with 104 PFU of rSARS-CoV-2 Venus (top), rSARS-CoV-2 mCherry SA (middle), or both, rSARS-CoV- 2 Venus and rSARS-CoV-2 mCherry SA (Beta) (bottom). At days 2 and 4 pi, lungs were collected to determine Venus and mCherry fluorescence expression using an Ami HT imaging system (A). BF, bright field. Venus and mCherry radiance values were quantified based on the mean values for the regions of interest in mouse lungs (B). Mean values were normalized to the autofluorescence in mock-infected mice at each time point and were used to calculate fold induction. Gross pathological scores in the lungs of mock-infected and rSARS-CoV- 2-infected KI 8 hACE2 transgenic mice were calculated based on the % area of the lungs affected by infection.
FIGS. 11A-11D show viral titers in the lungs, nasal turbinate and brain of KI 8 hACE2 transgenic mice treated with 1212C2 or 1213H7 mAbs and infected with rSARS- CoV-2 WT and rSARS-CoV-2 Beta. Six-to-eight-week-old female K18 hACE2 transgenic mice (n=3) injected (i.p.) with 25 mg/kg of an IgG isotype control, mAb 1212C2, or mAb 1213H7, and infected with 104 PFU of rSARS-CoV-2 Venus (WT) (Fig. 11 A), rSARS-CoV-2 mCherry SA (Beta) (Fig. 1 IB), or both rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) (Fig. 11C). Viral titers in the lungs (top), nasal turbinate (middle) and brain (bottom) at days 2 and 4 pi were determined by plaque assay in Vero E6 cells. Bars indicates the mean and SD of lung virus titers. Dotted lines indicate the limit of detection. Figure 11D shows quantification of rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) in the lungs (top), nasal turbinate (middle) and brain (bottom) from mice co-infected with both rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) at days 2 and 4 pi.
FIG. 12 shows mAb binding to RBD variants. SARS-CoV-2 RBD variants (50nM) were injected over mAbs captured on a biacore chip and their binding levels measured and normalized to the binding response to a reference (WT) Wuhan-1 RBD. Two WT RBD binding experiments were performed at the beginning and end of the experiment to validate the reproducibility of the assay. mAb binding is presented in the matrix as % of WT binding.
FIGS. 13A-13D show mAb epitope mapping. Competition epitope mapping using SPR, combined with the previously described variant analysis were used to define the binding epitopes of the mAbs. FIGS. 13A-13C represent 3 distinct competition mapping experiments performed with hmAbs assigned to different epitope classes Cl, CID, C2, or C4. FIG. 13D shows structural models of the mAb/RBD complexes that explain the epitope mapping and variant binding data. Protein data bank (PDB) entries used to model the mAb/RBD complexes are underlined in the Figure.
FIG. 14 shows MARM analysis of SARS-CoV-2 mAbs. Vero E6 cells were infected with SARS-CoV-2 WA-1 and incubated with decreasing concentration of mAb. Virus that emerged resistant to the indicated mAb was tested for recognition by the mAb using immuno-fluorescence assay (top). The spike of the MARMs was sequenced and mutation noted. Mutations within the RBD are indicated in bold (bottom).
FIG. 15 shows the results of study 2020-20, in particular differential biodistribution and titer of an IgG mAb in serum, bronchial tissue and lung at 0.5, 6, 12 and 24hrs post administration in mouse after administration by IV, IN and IH routes.
FIGS. 16A-16G show representative wells and mAh dose plotted against percent viral infection pre- and post-treatment for a neutralizing antibody assay involving treatment with 1212C2 (A), 1213H7 (B), 1215D1 (C), 1212C2+1213H7 (D), 1213H7+1215D1 (E), 1212C2+1215D1 (F), and 1212C2+1213H7+1215D1 (G).
DETAILED DESCRIPTION
The disclosure provides for broadly neutralizing anti-SARS-CoV-2 monoclonal antibodies and antigen-binding fragments thereof. The disclosure further provides for pharmaceutical compositions comprising an antibody of the disclosure and methods of using the antibodies of the disclosure. The antibodies of the disclosure are shown to be effective in preventing a SARS-CoV- 2 infection as well as in treating a SARS-CoV-2 infection. The antibodies of the disclosure bind epitopes within the spike protein of SARS-CoV-2. In certain embodiments, the antibodies of the disclosure bind an epitope within the receptor binding domain (RBD) of the SARS-CoV-2 spike protein. In certain embodiments, the antibodies of the disclosure bind an epitope within the receptor binding moiety (RBM) of the SARS-CoV-2 spike protein. In certain embodiments, the antibodies of the disclosure bind an epitope within the S 1 region of the SARS-CoV-2 spike protein. In certain embodiments, the antibodies of the disclosure bind an epitope within the S2 region of the SARS-CoV-2 spike protein.
In certain embodiments, the antibodies of the disclosure reduce binding of SARS-CoV-2 to a target cell. In certain embodiments, the antibodies of the disclosure reduce cellular fusion between SARS-CoV-2 and a target cell. In certain embodiments, the antibodies of the disclosure reduce release of infective SARS-CoV-2 from an infected cell. In certain embodiments, the antibodies of the disclosure reduce infection of a target cell by SARS-CoV-2.
As discussed in more detail herein, the antibodies of the disclosure were isolated from a subject with a documented SARS-CoV-2 infection, wherein the subject subsequently recovered from the SARS-CoV-2 infection. As such, the subject mounted an effective immunological response to the SARS-CoV-2 infection. Using single-cell immunoglobulin cloning, neutralizing antibodies to SARS-CoV-2 were isolated from the subject.
Definitions
All patent applications, patents, and printed publications cited herein are incorporated herein by reference in the entireties, except for any definitions, subject matter disclaimers or disavowals, and except to the extent that the incorporated material is inconsistent with the express disclosure herein, in which case the language in this disclosure controls.
As used herein, the term “about” refers to within 10%, preferably within 5%, and more preferably within 1% of a given value or range. Alternatively, the term "about" refers to within an acceptable standard error of the mean, when considered by one of ordinary skill in the art.
As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of an antibody or other molecule and its binding partner (such as, but not limited to, an antigen). Unless indicated otherwise, the term “binding affinity” refers to intrinsic binding affinity which reflects a 1 : 1 interaction between an antibody and an antigen or between members of a binding pair. Affinity is generally be represented by the dissociation constant (KD).
As used herein, the term “antibody” includes whole antibodies and any antigen binding fragment thereof. Examples of an antibody include, but are not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (for example, bispecific antibodies) formed from at least two antibodies or antigen binding fragments thereof, chimeric antibodies, anti -idiotypic (anti-Id) antibodies, intrabodies, and antigen binding fragments of any of the foregoing, Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable (VH) region and a heavy chain constant (CH) region. The CH region is comprised of three to four domains, CHI, CH2, CH3, and CH4. Each light chain is comprised of a light chain variable (VL) region and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Each VH and VL region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy -terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The VH and VL regions form a binding domain that interacts with an antigen in an antigen-specific manner. The CH and CL regions mediate binding of the antibody to host tissues, cells or factors, including various cells of the immune system (e.g, effector cells) and the first component (Clq) of the classical complement system. Antibodies may be of any type, including IgG, IgE, IgM, IgD, IgA and IgY and of any class, including, class IgGl, IgG2, IgG3, IgG4, IgAl and IgA2 or subclass.
As used herein, the terms “antigen-binding fragment” or “antigen-binding portion” refer to one or more fragments derived from an antibody described herein (a parent antibody) that retain the ability to specifically bind to the same antigen as the parent antibody. Examples of binding fragments include, but are not limited to, a Fab fragment (a monovalent fragment consisting of the VL, VH, CL and CHI domains), a F(ab)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region), a Fab' fragment (an Fab fragment comprising a portion of the hinge region), a F(ab’)2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region and containing a portion of the hinge region) a Fd fragment (a monovalent fragment consisting of the VH and CHI domains), a Fv fragment (a monovalent fragment consisting of the VL and VH domains of a single arm of an antibody), a disulfide-linked Fv (sdFv), a dAb fragment (a monomeric fragment consisting of a VH domain), an isolated CDR, a nanobody or single domain antibody (a monomeric fragment consisting of a single variable antibody domain), a portion of the VH region containing a single CDR and two FRs, a portion of the VL region containing a single CDR and two FRs, a diabody (an antibody fragments with two antigen-binding sites; such diabody may be bivalent or bispecific), a triabody (an antibody fragments with three antigen-binding sites; such triabody may be trivalent or trispecific), and a tetrabody (an antibody fragments with four antigen-binding sites; such tetrabody may be tetravalent or tetraspecific). The terms also include single chain Fv (scFv) which are created by recombinantly joining the VH and VL genes by a synthetic linker and expressed as a single polypeptide. Examples of scFv include, but are not limited to, scFv-FC, scFv- CH, scFab, and scFv-zipper. An antigen-binding fragment as described herein may be obtained using conventional methods known in the art and tested for binding as is done with conventional whole antibodies. Suitable antigen-binding fragments are described in Pluckthun (The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994)), Hudson et al., Nat. Med. 9: 129-134 (2003), WO 93/16185, U.S. Pat. Nos. 5,571,894 and 5,587,458. As used herein, the term “antibody of the disclosure” means an antibody disclosed herein, and includes pharmaceutically acceptable forms thereof, such as, but not limited to, pharmaceutically acceptable salts, hydrates and/or solvates. In certain embodiments, an antibody of the disclosure is an antigen-binding fragment.
As used herein, the term “antibody variant” refers to any modified form of an antibody described herein, such as, but not limited to, an antibody having one or more substitutions, deletions or insertions relative to a parental antibody and an antibody linked to a protein or nonprotein moiety.
As used herein, the terms “binds to an epitope” or “recognizes an epitope” with reference to an antibody refers to the epitope bound by the antibody. The term does not require the antibody to directly contact every amino acid within the epitope.
As used herein, the term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same or overlapping amino acids, whether such amino acids are continuous or discontinuous segments. The term does not require the antibodies bind to or contact exactly the same amino acids. The precise amino acids which the antibodies contact can differ. In one example, a first antibody can bind to a group of amino acids that is completely encompassed by the group of amino acids bound by a second antibody. In another example, a first antibody can bind to a group of amino acids that overlap with a group of amino acids bound by a second antibody.
As used herein, the term “chimeric antibody” refers to an antibody in which at least a portion of the variable region sequences (including CDR and FR sequences or just CDR sequences) are derived from one species (for example, a rat) and the constant region sequences are derived from another species (for example, a human). The term also includes an antibody in which its variable region sequence or CDR(s) is derived from one source (e.g., an IgAl antibody) and the constant region sequence or Fc is derived from a different source (e.g., a different antibody, such as an IgG, IgA2, IgD, IgE or IgM antibody). Chimeric antibodies are described in U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)).
As used herein, the term “detectable label” refers to a molecule capable of being detected in a subject or an assay, including, but not limited to, radioactive isotopes, fluorescent compounds, chemiluminescent compounds, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands, intercalating dyes and the like.
As used herein, the term “epitope” refers to an antigenic determinant that interacts with (is bound by) a specific antigen binding site in the variable region of an antibody molecule (the paratope). A single antigen (such as, but not limited to, a polypeptide) may have more than one epitope. Thus, different antibodies may bind to different epitopes on an antigen and may have different biological effects depending on which epitope is bound. The term “epitope” also refers to a site on an antigen to which B and/or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as a structural epitope (the portion of the antigenic determinant that is contacted by the CDR loops of an antibody) or a functional epitope (a subset of a structural epitope comprising those energetic residues centrally located in the structural epitope and directly contribute to the affinity of the antibody-epitope interaction). Epitopes may become immunologically available after fragmentation or denaturation of an antigen (a cryptotope). Epitopes may be linear or conformational (composed of non-linear amino acids brought together in a folded three-dimensional structure). Epitopes may include residues that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope typically includes at least 3 to 15 amino acids.
As used herein, the term “epitope mapping” refers to the process of identification of an epitope for antibody-antigen recognition.
As used herein, the term “functional equivalent” with reference to an antibody disclosed herein refers to an antibody variant of a parent antibody that retains one or more characteristics (such as, but not limited to, binding to the same epitope) of the parent antibody as disclosed herein. A functional equivalent may optionally differ in one or more characteristics of the parent antibody (such as, but not limited to, binding affinity, ADCC, and/or CDC).
As used herein, the term “human antibody” refers to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. Human antibodies can include amino acid residues not encoded by human germline immunoglobulin sequences (such as mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). As used herein, the term human antibody is not intended to include antibodies in which CDR sequences are derived from the germline of another mammalian species, such as a mouse, that have been grafted onto human framework sequence and/or constant regions.
As used herein, the term “human monoclonal antibody” refers to a monoclonal antibody that is obtained from a human.
As used herein, the term “immune response” refers to a biological response within a vertebrate against a foreign agent, which response protects the vertebrate, at least partially, against the foreign agent and diseases caused by the foreign agent. An immune response is mediated by the action of a cell of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from the vertebrate’s body of invading foreign agent, cells or tissues infected with the foreign agent. In preferred embodiments, as used herein, the term “immune response” does not include a response to a self-antigen.
As used herein, the term “isolated antibody” refer to an antibody that is substantially free of other antibodies having different antigenic specificities. An isolated antibody can optionally be substantially further free of other cellular material and/or reagents.
As used herein, the term “isotype” refers to the antibody class (IgG, including IgGl-IgG4, IgM, and IgA. Including IgAl and IgA2, IgD and IgE) that is encoded by the heavy chain constant region genes.
As used herein, the terms “Kassoc” and “Ka” refer to the association rate of a particular antibody-antigen interaction.
As used herein, the terms “Kdis” or “Kd” refer to the dissociation rate of a particular antibody-antigen interaction.
As used herein, the term “KD” refers to the dissociation constant, which is obtained from the ratio of Kd to Ka and is expressed as a molar concentration (M).
As used herein, the term “monoclonal antibody” refers to antibody molecules of a single molecular composition such that each of the antibody molecules displays a single binding specificity and binding affinity for a given epitope. As used herein, the term “pharmaceutically acceptable” refers to a compound that is compatible with an antibody of the disclosure or other ingredients of a composition and not deleterious to the subject receiving the antibody of the disclosure or composition. In some embodiments, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
As used herein, the term “pharmaceutically acceptable carrier or excipient” refers to a carrier medium or an excipient which does not interfere with the effectiveness of an antibody of the disclosure or other active ingredient of the composition and which is not toxic to the subject at the concentrations at which it is administered. The term includes, but is not limited to, a solvent, a stabilizer, a solubilizer, a tonicity enhancing agent, a structure-forming agent, a suspending agent, a dispersing agent, a chelating agent, an emulsifying agent, an anti-foaming agent, an ointment base, an emollient, a skin protecting agent, a gel-forming agent, a thickening agent, a pH adjusting agent, a preservative, a penetration enhancer, a complexing agent, a lubricant, a demulcent, a viscosity enhancer, a bio-adhesive polymer, or a combination thereof. The use of such agents for the formulation of pharmaceutically active substances is well known in the art (see, for example, “Remington's Pharmaceutical Sciences”, E. W. Martin, 18th Ed., 1990, Mack Publishing Co.: Easton, Pa.).
As used herein, the term “pharmaceutically acceptable salt” refers to salts derived from inorganic or organic acids including, for example hydrochloric, hydrobromic, sulfuric, nitric, perchloric, phosphoric, formic, acetic, lactic, maleic, fumaric, succinic, tartaric, glycolic, salicylic, citric, methanesulfonic, benzenesulfonic, benzoic, malonic, trifluoroacetic, trichloroacetic, naphthalene-2 sulfonic and other acids. Pharmaceutically acceptable salt forms may also include forms wherein the ratio of molecules comprising the salt is not 1: 1. For example, the salt may comprise more than one inorganic or organic acid molecule per molecule of antibody, such as two hydrochloric acid molecules per molecule of antibody. As another example, the salt may comprise less than one inorganic or organic acid molecule per molecule of antibody, such as two molecules of compound of antibody per molecule of tartaric acid. Salts may also exist as solvates or hydrates.
A “pharmaceutical composition” refers to a mixture of one or more of the antibodies of the disclosure, with other components, such as, but not limited to, pharmaceutically acceptable carriers and/or excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound of disclosure.
As used herein, the term “recombinant antibody” includes all antibodies that are prepared, expressed, created or isolated by recombinant means, such as, but not limited to, antibodies isolated from an animal that is transgenic or transchromosomal or a hybridoma prepared therefrom, antibodies isolated from a host cell transformed to express the antibody (a transfectoma), antibodies isolated from a recombinant or combinatorial antibody library, and antibodies prepared or created by any means involving the splicing of immunoglobulin gene sequences to other DNA sequences. Such recombinant antibodies may be human recombinant antibodies. Such recombinant antibodies can be subjected to in vitro mutagenesis or in vivo somatic mutagenesis.
As used herein, the term “subject” refers to an animal. Preferably, the animal is a mammal. A subject also refers to for example, primates (e.g, humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds and the like. In a preferred embodiment, the subject is a human.
As used herein, the term “therapeutically effective amount” refers to an amount of an antibody of the disclosure that is sufficient to achieve a beneficial or desired result, including a clinical result. As such, the “therapeutically effective amount” may be sufficient, for example, to reduce or ameliorate the severity and/or duration of a SARS-CoV-2 infection, or one or more symptoms thereof, prevent the recurrence, development, or onset of one or more symptoms associated with a SARS-CoV-2 infection, prevent or reduce the replication or multiplication of SARS-CoV, prevent or reduce the production and/or release of a SARS-CoV-2 particle, or enhance or otherwise improve the prophylactic or therapeutic effect(s) of another therapy used in treating a SARS-CoV-2 infection. In certain embodiments, a “therapeutically effective amount” is an amount of the antibody of the disclosure that avoids or substantially attenuates undesirable side effects.
In certain embodiments, the “therapeutically effective amount” in the context of a SARS- CoV-2 infection is an amount sufficient to reduce one or more of the following steps of a the life cycle of SARS-CoV-2: the docking of the virus particle to a cell, the introduction of viral genetic information into a cell, the expression of viral proteins, the translation of viral RNA, the transcription of viral RNA, the replication of viral RNA, the synthesis of new viral RNA, the production of new virus particles and the release of virus particles from a cell. Such a reduction in any of the foregoing may be by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, the “therapeutically effective amount” in the context of a SARS- CoV-2 infection reduces the replication, multiplication or spread of the virus by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In some embodiments, the “therapeutically effective amount” in the context of a SARS-CoV-2 infection increases the survival rate of infected subjects by at least 5%, preferably at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100%. In each of the foregoing, when a reduction of increase is specified, such reduction of increase may be determined with respect to a subject that has not been treated with an antibody of the disclosure and that has a diagnosed SARS-CoV-2 infection.
As used herein, the terms “treating” or “treatment” mean an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results in the context of a SARS-CoV-2 infection include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, a diminution of extent of disease, a stabilized (z.e., not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state and remission (whether partial or total), whether detectable or undetectable. “Treatment” or “treating” can also mean prolonging survival as compared to expected survival if not receiving treatment.
As used herein, the terms “a”, “an”, “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. Recitation of ranges of values herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range. Unless otherwise indicated herein, each individual value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g. “such as”) provided herein is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the invention.
Human Neutralizing Antibodies The disclosure provides the following neutralizing SARS-CoV-2 antibodies that specifically binds to the spike protein of SARS-CoV-2: 1212C2, 1212F5, 1213H7, 1212F2, 1206D1, 1212D5, 1207B4, 1213F2, 1212D4, 1206A5, 1212D6, 1206D12, 1207F10, 1206G12, 1212C8, 1212E9, 1214E9, and 1215D1. In certain embodiments, an antigen binding fragment of the antibodies 1212C2, 1212F5, 1213H7, 1212F2, 1206D1, 1212D5, 1207B4, 1213F2, 1212D4, 1206A5, 1212D6, 1206D12, 1207F10, 1206G12, 1212C8, 1212E9, 1214E9, and 1215D1 is provided. In certain embodiments, the antibodies are human monoclonal antibodies and/or the antigen binding fragments are derived from human monoclonal antibodies. Table 1 provides the SEQ ID NOS: for the nucleotide sequence (NT) of the heavy chain variable regions and light chain variable regions, the amino acid (AA) sequence of the heavy and light chain variable regions, the AA sequence of the heavy chain CDR1, CDR2, and CDR3, and the AA sequence of the light chain
CDR1, CDR2, and CDR3.
Table 1
Figure imgf000016_0001
Figure imgf000017_0001
The neutralization titer 50 (NT50) of the antibodies of the disclosure ranges from less than 0.050 pg/ml to 1.61 gg/ml. Preferably, the NT50 value if the antibodies of the disclosure is less than or equal to 10 pg/ml, less than or equal to 8 pg/ml, less than or equal to 6 pg/ml, less than or equal to 4 pg/ml, less than or equal to 3 pg/ml, less than or equal to 2 gg/ml, or less than or equal to 1 gg/ml. More preferably, the NT50 value if the antibodies of the disclosure is less than or equal to 0.5 pg/ml, less than or equal to 0.25 pg/ml, less than or equal to 0.1 pg/ml, less than or equal to 0.075 pg/ml, less than or equal to 0.05 pg/ml, less than or equal to 0.025 pg/ml, or less than or equal to 0.01 pg/ml. For the purpose of the disclosure, NT50 values are determined as described in Example 4 of the disclosure.
In a first embodiment, the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising: 1. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 38-40, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42-44, respectively (1212C2);
2. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 46-48, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50-52, respectively (1215F5);
3. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 54-56, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 58-60, respectively (1213H7);
4. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 62-64, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 66-68, respectively (1212F2);
5. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 70-72, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 74-76, respectively (1206D1);
6. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 78-80, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 82-84, respectively (1212D5);
7. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 86-88, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 90-92, respectively (1207B4);
8. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 94-96, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 98-100, respectively (1213F2);
9. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 102-104, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 106-108, respectively (1212D4);
10. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 110-112, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 114-116, respectively (1206A5);
11. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 118-120, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 122-124, respectively (1212D6);
12. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 126-128, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 130-132, respectively (1206D12);
13. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 134-136, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 138-140, respectively (1207F10); and
14. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 142-144, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 146-148, respectively (1206G12), or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
In a second embodiment, the disclosure provides an isolated antibody, or an antigenbinding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising: 1. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 37, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 41, or an amino acid sequence at least 80% homologous thereto (1212C2);
2. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 45, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 49, or an amino acid sequence at least 80% homologous thereto (1215F5);
3. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 53, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 57, or an amino acid sequence at least 80% homologous thereto (1213H7);
4. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 61, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 65, or an amino acid sequence at least 80% homologous thereto (1212F2);
5. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 69, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 73, or an amino acid sequence at least 80% homologous thereto (1206D1);
6. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 77, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 81, or an amino acid sequence at least 80% homologous thereto (1212D5);
7. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 85, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 89, or an amino acid sequence at least 80% homologous thereto (1207B4);
8. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 93, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 97, or an amino acid sequence at least 80% homologous thereto (1213F2); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 101, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 105, or an amino acid sequence at least 80% homologous thereto (1212D4); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 109, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 113, or an amino acid sequence at least 80% homologous thereto (1206A5); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 117, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 121, or an amino acid sequence at least 80% homologous thereto (1212D6); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 125, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 129, or an amino acid sequence at least 80% homologous thereto (1206D12); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 133, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 137, or an amino acid sequence at least 80% homologous thereto (1207F10); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 141, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 145, or an amino acid sequence at least 80% homologous thereto (1206G12); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 149, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 150, or an amino acid sequence at least 80% homologous thereto (1215D1); 16. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 151 , or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 152, or an amino acid sequence at least 80% homologous thereto (1214E9);
17. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 153, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 154, or an amino acid sequence at least 80% homologous thereto (1212E9); and
18. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 155, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 156 (1212C8), or an amino acid sequence at least 80% homologous thereto, or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
In a third embodiment, the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising:
1. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 158, 39 and 159, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42, 43, and 161, respectively (1212 variant), or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
In one aspect of the third embodiment, the amino acid at position 3 of SEQ ID NO: 158 is Thr (T) or He (I), the amino acid at position 1 of SEQ ID NO: 159 is Thr (T) or Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid, the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser
(S), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V) or Phe (F).
In one aspect of the third embodiment, the amino acid at position 3 of SEQ ID NO: 158 is Thr
(T), the amino acid at position 1 of SEQ ID NO: 159 is Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid (preferably Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), or Met (M)), the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser (S), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
In one aspect of the third embodiment, the amino acid at position 3 of SEQ ID NO: 158 is Thr (T) or He (I), the amino acid at position 1 of SEQ ID NO: 159 is Thr (T) or Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Leu (L), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid, the amino acid at position 6 of SEQ ID NO: 161 is Asn (N), and the amino acid at position 10 of SEQ ID NO: 161 is Phe (F) or Vai (V).
In one aspect of the third embodiment, the amino acid at position 3 of SEQ ID NO: 158 is Thr (T), the amino acid at position 1 of SEQ ID NO: 159 is Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Leu (L), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid (preferably Ala (A), Vai (V), Leu (L), lie (I), Pro (P), Phe (F), or Met (M)), the amino acid at position 6 of SEQ ID NO: 161 is Asn (N), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
In one aspect of the third embodiment, the amino acid at position 3 of SEQ ID NO: 158 is He (I), the amino acid at position 1 of SEQ ID NO: 159 is Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Phe (F), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid, the amino acid at position 6 of SEQ ID NO: 161 is Ser (S), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
In one aspect of the third embodiment, the amino acid at position 3 of SEQ ID NO: 158 is He (I), the amino acid at position 1 of SEQ ID NO: 159 is Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Phe (F), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid(preferably Ala (A), Vai (V), Leu (L), He (I), Pro (P), Phe (F), or Met (M)), the amino acid at position 6 of SEQ ID NO: 161 is Ser (S), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
In a fourth embodiment, the disclosure provides an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, the isolated antibody, or the antigen-binding fragment thereof, comprising:
1. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 157, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 160, or an amino acid sequence at least 80% homologous thereto (1212 variant); or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
In one aspect of the fourth embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2A below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2B below.
Figure imgf000024_0001
In one aspect of the fourth embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2C below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2D below.
Figure imgf000024_0002
Figure imgf000025_0001
In one aspect of the fourth embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2E below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2F below.
Figure imgf000025_0002
In one aspect of the fourth embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2G below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2H below.
Figure imgf000025_0003
Figure imgf000026_0001
In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is provided as a pharmaceutically acceptable salt.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies bind an epitope within the RBD of the SARS-CoV-2 spike protein. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies bind an epitope within the RBM of the SARS-CoV-2 spike protein. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies bind an epitope within the SI region of the SARS-CoV-2 spike protein. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies bind an epitope within the S2 region of the SARS-CoV-2 spike protein.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies reduce binding of SARS-CoV-2 to a target cell. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies reduce cellular fusion between SARS-CoV-2 and a target cell. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies reduce release of infective SARS-CoV-2 from an infected cell. In one aspect of any of the antibodies of the first to fourth embodiments, the antibodies reduce infection of a target cell by SARS-CoV-2.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antibody variant. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antibody variant comprising one or more substitutions, deletions, and/or insertions relative to the parental antibody. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antibody variant comprising one or more substitutions relative to the parental antibody.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antibody variant that has a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein a protein moiety or non-protein moiety is linked to the Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein a water soluble polymer is linked to the Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein a polyethylene glycol polymer is linked to the Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein a polyoxazoline polymer is linked to the Fc constant region. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody comprises a variant Fc constant region, wherein the variant Fc constant region provides a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is a human antibody. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is a chimeric antibody. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is a cl ass- switched antibody.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is linked to a therapeutic agent. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is linked to a detectable label. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is linked to an enzyme. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is linked to an enzyme inhibitor.
In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antigen-binding fragment. In one aspect of any of the antibodies of the first to fourth embodiments, the antibody is an antigen-binding fragment selected from the groups consisting of: a Fab fragment, a F(ab)2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a disulfide-linked Fv (sdFv), a dAb fragment, an isolated CDR, a nanobody or single domain antibody, a portion of the VH region containing a single variable domain and two constant domains, a diabody, a triabody, a tetrabody, scFv, scFv-FC, scFv-CH, scFab, and scFv-zipper.
In one aspect of any of the antibodies of the second or fourth embodiments, the amino acid sequence has at least 85% homology to the reference sequence, at least 90% homology to the reference sequence, at least 95% homology to the reference sequence, at least 96% homology to the reference sequence, at least 97% homology to the reference sequence, at least 98% homology to the reference sequence, or at least 99% homology to the reference sequence.
In one aspect of any of the antibodies of the second or fourth embodiment, the antibody is 100% homologous to the reference sequence across the CDRs and the amino acid sequence has at least 85% homology to the reference sequence across the FRs, the amino acid sequence has at least 85% homology to the reference sequence across the FRs, at least 90% homology to the reference sequence across the FRs, at least 95% homology to the reference sequence across the FRs, at least 96% homology to the reference sequence across the FRs, at least 97% homology to the reference sequence across the FRs, at least 98% homology to the reference sequence across the FRs, or at least 99% homology to the reference sequence across the FRs.
Antibody Fragments
In certain embodiments, an antibody of the disclosure is an antigen-binding fragment. Antigen-binding fragment include, but are not limited to, Fab, Fab', Fab'-SH, F(ab)2, F(ab’)2, Fv, Fd, sdFv, dAb scFv fragments (including, but not limited to, scFv-FC, scFv-CH, scFab, and scFv- zipper), diabodies, triabodies tetrabodies, nanobodies, and other fragments described here. For discussion of Fab and F(ab) fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.
Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells as described herein or as known in the art.
Chimeric Antibodies
In certain embodiments, an antibody of the disclosure is a chimeric antibody. In one embodiment, a chimeric antibody comprises a non-human variable region (for example, a variable region derived from a mouse, rat, hamster, rabbit, or a monkey or other non-human primate) and a human constant region. In another embodiment, a chimeric antibody is a “class switched” antibody in which the class or subclass of the antibody has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, a chimeric antibody is a humanized chimeric antibody.
Human Antibodies
In certain embodiments, an antibody of the disclosure is a human antibody. Human antibodies can be produced using various techniques known in the art or using techniques described herein. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008). Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals see: Lonberg, Nat. Biotech. 23:1117-1125 (2005); U.S. Pat. Nos. 6,075,181 and 6,150,584; U.S. Pat. No. 5,770,429; U.S. Pat. No. 7,041,870; and U.S. Patent Application Publication No. US 2007/0061900. Human variable regions from intact antibodies generated by such animals may be further modified, such as, but not limited to, by combining with a different human constant region.
Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies are known in the art (Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies generated via human B-cell hybridoma technology are also known in the art (Li et al., Proc. Natl. Acad. Sci. USA, 103:3557- 3562 (2006)). Additional methods include those described in U.S. Pat. No. 7,189,826; Ni, Xiandai Mianyixue, 26(4):265-268 (2006); Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005); and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005). Human antibodies may also be generated by 1 isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.
Antibody Variants
The disclosure also provides for variants of the antibodies disclosed. In certain embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Such variants may be used to improve the binding affinity of an antibody, to improve a biological property of an antibody (such as, but not limited to, half-life), or a combination of the foregoing. Amino acid variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis directly incorporating such amino acid change. Antibody variants include, but are not limited to, fusion proteins including an antibody of the disclosure, substitutions of one or more amino acids, deletion of one or more amino acids, insertion of one or more amino acids, and any combination of the foregoing. In certain embodiments, the antibody variant retains the ability to bind to the same epitope.
In certain embodiments, antibody variants having one or more amino acid substitutions are provided. When a substitution is introduced, the substitution may be made at any desired location. In certain embodiments, when a substitution is introduced, the substitution occurs in the Sites of interest for substitutional mutagenesis include the VH region, the VL region, the heavy chain CDRs, the light chain CDRs, and/or the FR region. Such substitutions may be conservative or nonconservative. Conservative substitutions are defined herein.
In certain embodiments, an antibody of the disclosure comprises a conservative amino acid substitution. Suitably, such conservative amino acid substitution may be made in the VH region, the VL region, the heavy chain CDRs, the light chain CDRs, and/or the FR region. In certain embodiments, the disclosure provides for an antibody variant of one or more of the polypeptides of SEQ ID NOS: 37 to 156. In certain embodiments, the disclosure provides for an antibody variant comprising one or more substitutions, including conservative substitutions, of one or more of the polypeptides of SEQ ID NOS: 37 to 156. In certain embodiments, the disclosure provides for an antibody variant comprising one or more substitutions, including conservative substitutions, of the heavy chain variable region (including CDRs 1-3) and/or light chain variable region (including CDRs 1-3), of one or more of the polypeptides of SEQ ID NOS: 37 to 156. In one embodiment of any of the foregoing, an antibody variant has an amino acid sequence that is at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a parent antibody.
For the purposes of the disclosure, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (% homology = (number of identical positions/total number of positions) x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The percent identity between two amino acid sequences is preferably determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
In one embodiment, an antibody variant includes a conservative modification. As used herein, the term “conservative modifications” refers to an amino acid modification that does not significantly affect or alter the binding characteristics of the antibody containing the conservative modification. Such conservative modifications include amino acid substitutions, additions, and deletions.
In one embodiment, an antibody variant contains a conservative amino acid substitution. Conservative amino acid substitutions are ones in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: amino acids with basic side chains (lysine, arginine, and histidine), acidic side chains (aspartic acid and glutamic acid), uncharged polar side chains (glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan), nonpolar side chains (alanine, valine, leucine, isoleucine, proline, phenylalanine, and methionine), beta-branched side chains (threonine, valine, and isoleucine), and aromatic side chains (tyrosine, phenylalanine, tryptophan, and histidine). Non-conservative substitutions will entail exchanging a member of one of these classes for another class.
For example, a conservative amino acid substitution may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity, steric bulk, charge, hydrophobicity and/or hydrophilicity of the amino acid residue at that position. Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues which are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties. It will be appreciated by those of skill in the art that polypeptide described herein may be chemically synthesized as well as produced by recombinant means.
In making an amino acid substitution as described herein, the hydropathic index of an amino acid may be considered. Each amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics. Hydropathic index values are resented by: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cy stine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is understood in the art (Kyte et al., J. Mol. Biol. 157: 105-131, 1982). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In one embodiment, making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within +/- 1; in an alternate embodiment, the hydropathic indices are within +/- 0.5; in yet another alternate embodiment, the hydropathic indices are within +/- 0.25.
In making an amino acid substitution as described herein, the hydrophilicity may also be considered. In certain embodiments, the greatest local average hydrophilicity of a polypeptide as governed by the hydrophilicity of its adjacent amino acids, correlates with a biological property of the protein. The following hydrophilic index values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0.+-.1); glutamate (+3.0.+-.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5.+-.1); alanine (- 0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (- 1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).
In one embodiment, in making changes based upon similar hydrophilicity values, the substitution of amino acids whose hydrophilicity values are within +/- 1; in an alternate embodiment, the hydrophilicity values are within +/- 0.5; in yet another alternate embodiment, the hydrophilicity values are within +/- 0.25. A skilled artisan will be able to determine suitable substitutions, insertions and deletions, including combinations thereof, of a polypeptide as set forth in any of SEQ ID NOS: 37 to 156 using techniques known in the art. For identifying suitable areas of a polypeptide that may be changed without destroying activity, one skilled in the art may target areas not believed to be important for activity. For example, when homologous polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a polypeptide described herein to such homologous polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides. It will be appreciated that changes in areas of a polypeptide described herein that are not conserved relative to such homologous polypeptide would be less likely to adversely affect the biological activity and/or structure of a polypeptide described herein. One skilled in the art would also know that, even in relatively conserved regions, one may substitute chemically similar amino acids for the naturally occurring residues while retaining activity (for example, conservative amino acid substitutions). Therefore, even areas that may be important for biological activity or for structure may be subject to such amino acid substitutions without destroying the biological activity or without adversely affecting the polypeptide structure.
The deletions, insertions, and substitutions can be selected, as would be known to one of ordinary skill in the art, to generate a desired polypeptide variants. For example, it is not expected that deletions, insertions, and substitutions in a non-functional region of a polypeptide would alter activity. Likewise conservative amino acid substitutions and/or substitution of amino acids with similar hydrophilic and/or hydropathic index values is expected to be tolerated in a conserved region and a polypeptide activity may be conserved with such substitutions.
An exemplary substitution variant is an affinity matured antibody, which may be conveniently generated using phage display-based affinity maturation techniques such as those described in Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001)). Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intra-sequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody. An antibody variant may be screened for a desired activity, including, but not limited to, retained epitope binding, improved epitope binding, decreased immunogenicity, improved antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) or any combination of the foregoing.
An antibody of the disclosure may be modified to contain additional protein moieties and additional moieties that are not protein based. In one embodiment, a non-protein based moiety is a polymer, preferable a water soluble polymer. Suitable water soluble polymers include, but are not limited to, polyethylene glycol (PEG), polyoxazoline (POZ), carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either as a homopolymer or a copolymer), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propylene glycol, poly oxy ethylated polyols (such as glycerol), polyvinyl alcohol, copolymers of ethylene glycol and propylene glycol, copolymers of propylene oxide and ethylene oxide, and mixtures of any of the foregoing. When the water soluble polymer is a copolymer, the copolymer may be present as a block copolymer or a random copolymer. An amino acid substitution may be used to introduce a site for attachment of a water soluble polymer to the antibody.
The water soluble polymer may be of any molecular weight, and may be dendrimers, branched or unbranched. The number of polymers attached to an antibody may vary. In one embodiment, the number average molecular weight of the water soluble polymer is from 2,500 to 75,000 Da, from 5,000 to 50,000 Da, from 7,500 to 40,000 Da, or from 10,000 to 30,000 Da. In one embodiment, 1 to 10 water soluble polymer chains are attached. When more than 1 water soluble polymer is attached, the water soluble polymers may be the same or different and may be of the same of different number average molecular weight. In one embodiment, the water soluble polymers increases the half-life of an antibody of the disclosure and/or decrease immunogenicity of an antibody of the disclosure. Water soluble polymers may be linked to an antibody of the disclosure using conventional reactive groups on the polymer and the antibody. For example, water soluble polymers, such as. But not limited to, POZ and PEG polymers, may be linked to an antibody using acylation alkylation reactions. Water soluble polymers may be linked to the antibody in a site specific manner or randomly (for example, the e-amino group of a lysine residue or the thiol group of cysteine residue may be used in conjugation reactions with an appropriate functionality on the water soluble polymer). In certain embodiments, a non-natural amino acid may be introduced into the antibody and used to link a water soluble polymer to the antibody. For example, a selenocysteine residue may be introduced into the antibody for reaction with a water soluble polymer containing an appropriate functionality (for example, a maleimide group or an iodoacetimide group). In certain embodiments, the water soluble polymer may be linked to a therapeutic agent, a detectable label, an enzyme, or an enzyme inhibitor as described herein.
Methods for linking water soluble polymers to proteins are known in the art and may be applied directly to the antibodies of the disclosure (EP 0154316).
In another embodiment, a non-protein based moiety is a therapeutic agent, for example a therapeutic agent useful in treating a SARS-CoV-2 infection as described herein or a cytotoxic agent. In another embodiment, a non-protein based moiety is a detectable label. In another embodiment, a non-protein based moiety is an enzyme. In another embodiment, a non-protein based moiety is an enzyme inhibitor, for example a serine protease inhibitor, such as, but not limited to, a TMPRSS2 inhibitor.
Glycosylation Variants
In certain embodiments, an antibody of the disclosure is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed. For example, an aglycoslated antibody can be made. Glycosylation can be altered to for a variety of purposes, including, but not limited to, to increase the affinity of the antibody for an antigen. In one embodiment, one or more amino acid substitutions are made that result in elimination of one or more FR glycosylation sites, which may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861. Glycosylation sites in the constant region may also be eliminated. For example, in IgGl, N297 in the Fc portion may be substituted with another residue (for example, alanine) and/or by mutating an adjacent amino acid to thereby reduce glycosylation on N297.
Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished, for example, by expressing the antibody in a host cell with altered glycosylation machinery (such cells are described in EP 1,176,195; PCT Publication WO 03/035835; Shields, R. L. et al. (2002), J. Biol. Chem. 277:26733-26740; PCT Publication WO 99/54342; Umana et al. (1999), Nat. Biotech. 17: 176-180).
Fc Region Variants
The numbering of residues in the Fc region as discussed herein is that of the EU index of Kabat (PCT Publication WO 2000/42072). The variable regions of the antibody described herein can be linked to an Fc region (such as, but not limited to, an IgGl, IgG2, IgG3 or IgG4 Fc), which may be of any allotype or isoallotype (including for IgGl : Glm, Glml(a), Glm2(x), Glm3(f), Glml7(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(gl), G3m28(g5), G3ml l(b0), G3m5(bl), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); for IgA: A2M, A2ml, and A2m2; and for K: Km, Kml, Km2, Km3 (Jefferies et al. (2009) mAbs 1(4), 323-338). In certain embodiments, the variable regions of the antibodies described herein are linked to an Fc that binds to one or more activating Fc receptors (FcR), and thereby stimulate ADCC. In certain embodiments, the variable regions of the antibodies described herein are linked to an Fc region optimized to engage a wider range of Fc receptors. Fc receptors for isotypes other than gamma exist on particular leukocytes. By creating an Fc region that can interact with multiple Fc receptors, such as FcyRI and FcaRI, an antibody with expanded, novel abilities to engage effector cells may be created. Neutrophils are the most abundant leukocyte in the body and engage Fc of IgA antibodies via the FcaRI.
In certain embodiments, the antibody variable regions described herein may be linked to an Fc comprising one or more modification, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, ADCC, and/or CDC.
The Fc region encompasses domains derived from the constant region of an antibody. Suitable immunoglobulins include IgG 1, IgG2, IgG3, IgG4, IgAl, IgA2, and other classes such as IgD, IgE and IgM, The constant region of an antibody is defined as a naturally-occurring or synthetically-produced polypeptide homologous to the antibody C-terminal region, and can include a CHI domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in combination. In some embodiments, an antibody of the disclosure has an Fc region other than that of a wild type IgAl. An antibody of the disclosure may have an Fc region from that of IgG (e.g., IgGl, IgG2, IgG3, and IgG4) or other classes such as IgA2, IgD, IgE and IgM. An antibody of the disclosure may have an Fc region that is contains a substitution, deletion, or insertion of wild-type IgAl.
The Fc of an antibody is responsible for many important functions including FcR binding and complement fixation. There are five major classes of heavy chain constant regions, classified as IgA, IgG, IgD, IgE, and IgM, each with characteristic effector functions designated by isotype. The serum half-life of an antibody is influenced by the ability of that antibody to bind to an FcR.
Antibody molecules interact with multiple classes of cellular receptors. IgG molecules interact with three classes of FcyR specific for the IgG class of antibody, namely FcyRI, FcyRIIa, FcyRIIb, FcyRIIIa, and FcyRIIIb. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains.
In certain embodiments, the Fc region is a variant Fc region (an Fc sequence that has been modified such as by amino acid substitution, deletion and/or insertion) relative to a parent Fc sequence to provide desirable structural features and/or biological activity, including, but not limited to, (i) increased or decreased ADCC; (ii) increased or decreased CDC; (iii) increased or decreased affinity for Clq; and/or (iv) increased or decreased affinity for a FcR (each of the foregoing relative to the parent Fc). Such Fc region variants will generally comprise at least one amino acid substitution, deletion, and/or insertion in the Fc region. In certain embodiments, an Fc region contains from 1 to 6 amino acid substitutions, deletions and/or insertions (preferably substitutions).
A variant Fc region may also comprise a sequence modification wherein an amino acid involved in disulfide bond formation are removed or replaced with another amino acid. In other embodiments, the Fc region may be modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc region, which may be recognized by a digestive enzyme in E. coli such as proline iminopeptidase. In other embodiments, one or more glycosylation sites within the Fc domain are removed. Residues that are typically glycosylated (e.g., asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues e.g., alanine). In other embodiments, sites involved in interaction with complement, such as the Clq binding site, may be removed from the Fc region. For example, one may delete or substitute the EKK sequence of human IgGl. In certain embodiments, sites that affect binding to Fc receptors may be removed, preferably sites other than salvage receptor binding sites. In other embodiments, an Fc region may be modified to remove an ADCC site known in the art. Specific examples of variant Fc domains are disclosed for example, in WO 1997/34631 and WO 1996/32478.
In one embodiment, the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is increased or decreased (U.S. Pat. No. 5,677,425) to, for example, facilitate assembly of the light and heavy chains and/or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc-hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding (U.S. Pat. No. 6,165,745).
In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter an effector function of the antibody. For example, in an IgG antibody one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an FcR or the CI component of complement (U.S. Pat. Nos. 5,624,821 and 5,648,260). In another example, in an IgG antibody one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and/or reduced or abolished CDC (U.S. Pat. No. 6,194). In another example, in an IgG antibody one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement (PCT Publication WO 1994/29351). All references to Fc region amino acid numbering is made according to the EU index of Kabat (Kabat et al., (1983) "Sequences of Proteins of Immunological Interest", US Dept. Health and Human Services).
In yet another example, the Fc region may be modified to increase ADCC and/or to increase the affinity for an FcyR by modifying one or more amino acids at the following positions: 234, 235, 236, 238, 239, 240, 241, 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262, 263,
264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295,
296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 330,
331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D/332E, 236A/332E, 236A/239D/332E, 268F/324T, 267E/268F, 267E/324T, and 267E/268F7324T. Other modifications for enhancing FcyR and complement interactions include but are not limited to substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are described in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.
Fc modifications that increase binding to an FcyR include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region.
Other Fc modifications that can be made are those for reducing or ablating binding to FcyR and/or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, ADCP, and CDC. Exemplary modifications include but are not limited substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index of Kabat. Exemplary substitutions include but are not limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index of Kabat. An Fc variant may comprise 236R/328R. Other modifications for reducing FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 33 IS, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.
Optionally, the Fc region may comprise a non-naturally occurring amino acid residue at additional and/or alternative positions known to one skilled in the art (U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; PCT Publication Nos: WO 2000/42072; WO 2001/58957; WO 2002/06919; WO 2004/016750; WO 2004/029207; WO 2004/035752; WO 2004/074455; WO 2004/099249; WO 2004/063351; WO 2005/070963; WO 2005/040217, WO 2005/092925 and WO 2006/020114). Fc variants that enhance affinity for an inhibitory receptor FcyRIIb may also be used. Such variants may provide an Fc fusion protein with immune-modulatory activities related to FcyRIIb cells, including for example B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRIIb relative to one or more activating receptors. Modifications for altering binding to FcyRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index of Kabat. Exemplary substitutions for enhancing FcyRIIb affinity include but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcyRIIb include 235Y/267E, 236D/267E, 239D/268D, 239D/267E, 267E/268D, 267E/268E, and 267E/328F.
The affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art including but not limited to, equilibrium methods (enzyme-linked immune-absorbent assay, radioimmunoassay, or kinetics, and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer, gel electrophoresis and chromatography). These and other methods may utilize a label on one or more of the components being examined and/or employ a variety of detection methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody -immunogen interactions.
In certain embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this may be done by increasing the binding affinity of the Fc region for FcRn. For example, one or more of following residues can be mutated: 252, 254, 256, 433, 435, and 436, as described in U.S. Pat. No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and/or T256F. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022. Other exemplary variants that increase binding to FcRn and/or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H. 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q/428L (Hinton et al. 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton et al. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 31 1A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 IS, 433R, 433S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y/254T/256E, 433K/434F/436H, 308T/309P/311 S (Dall’Acqua et al. Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281 :23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671.
In certain embodiments, hybrid IgG isotypes with particular biological characteristics may be used. For example, an IgGl/IgG3 hybrid variant may be constructed by substituting IgGl positions in the CH2 and/or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. Thus a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g., 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other embodiments described herein, an IgGl/IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and/or CH3 region with amino acids from IgGl at positions where the two isotypes differ. Thus a hybrid variant IgG antibody may be constructed chat comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to an insertion of a glycine at position 236), and 321 h.
Moreover, the binding sites on human IgGl for FcyRI, FcyRI I, FcyRIII and FcRn have been mapped and variants with improved binding have been described (see Shields, R. L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to improve binding to FcyRIII Additionally, the following combination mutants were shown to improve FcyRIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A, which has been shown to exhibit enhanced FcyRIIIa binding and ADCC activity (Shields et al., 2001). Other IgGl variants with strongly enhanced binding to FcyRIIIa have been identified, including variants with S239D/I332E and S239D/I332E/A330L mutations which showed the greatest increase in affinity for FcyRIIIa, a decrease in FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of the triple mutations into antibodies such as alemtuzumab (CD52-specific), trastuzumab (HER2/neu-specific), rituximab (CD20-specific), and cetuximab (EGFR-specific) translated into greatly enhanced ADCC activity in vitro, and the S239D/I332E variant showed an enhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgGl mutants containing L235V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcyRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that may be used include, but are not limited to: S298A/E333A/L334A, S239D/I332E, S239D/I332E/A330L,
L235V/F243L/R292P/Y300L/P396L, and M428L/N434S.
In certain embodiments, an Fc is chosen that has reduced binding to FcyRs. An exemplary Fc, e.g., IgGl Fc, with reduced FcyR binding comprises the following three amino acid substitutions: L234A, L235E and G237A.
In certain embodiments, an Fc is chosen that has reduced complement fixation. An exemplary Fc, e.g., IgGl Fc, with reduced complement fixation has the following two amino acid substitutions: A33OS and P331S.
In certain embodiments, an Fc is chosen that has essentially no effector function, i.e., it has reduced binding to FcyRs and reduced complement fixation. An exemplary Fc, IgGl Fc that is effectorless comprises the following five mutations: L234A, L235E, G237A, A33OS and P331S. When using an IgG4 constant domain, it is usually preferable to include the substitution S228P, which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules.
Nucleic Acids. Vectors, and Host Cells
The disclosure also provides for nucleic acid sequences encoding the antibodies of the disclosure. The nucleic acid sequences may code for an antigen-binding fragment. In some embodiments, the nucleic acids are codon optimized based on how the antibodies are produced. In one embodiment, the nucleic acid sequence comprises one or more of SEQ ID NOS: 1 to 36. In one aspect of this embodiment, the nucleic acid sequence has at least 75% homology, at least 80% homology, at least 85% homology, at least 90% homology, at least 95% homology, or greater than 95% homology with one or more of SEQ ID NOS: 1 to 36.
The disclosure also provides a vector comprising a nucleic acid sequence coding for an antibody of the disclosure. In certain embodiments, a vector comprises a nucleic acid sequence coding for a variable heavy chain region and a nucleic acid sequence coding for a variable light chain region. Any nucleic acid sequence of the disclosure (SEQ ID NOS: 1-36) may be combined with a vector as described herein. In some embodiments, a nucleic acid sequence coding for a variable heavy chain is on the same vector as a nucleic acid sequence coding for a variable light chain. In some embodiments, a nucleic acid sequence coding for a variable heavy chain region is on a different vector than a nucleic acid sequence coding for a variable light chain region. In some embodiments, the vector is a plasmid. In some embodiments, the vector is a phage vector, such as, but not limited to, X-phage. In some embodiments, the vector is a viral vector, such as, but not limited to, non-replicating adenoviral vector, lentiviral vector, pSV, pCMV, and retroviral vectors. In some embodiments, the vector is a cosmid. In some embodiments, the vector is a recombinant chromosome. In some embodiments, the combinations of the foregoing vectors are employed. The expression of different nucleic acid sequences may occur at the same time generally or be temporally separated. The expression of one or more nucleic acid sequences may be inducible. The vector may comprise a nucleic acid coding for an intact antibody or an antigen binding fragment, particularly those antigen-binding fragments disclosed herein. In some embodiments, the vector comprises a nucleic acid sequence encoding an immunoglobulin constant region, such as, but not limited to, an IgG (e.g. IgGl, IgG2, IgG3, and IgG4) constant region. In exemplary embodiments, the immunoglobulin constant region comprises an IgGl constant region.
In one embodiment, the disclosure provides for a vector comprising nucleotide sequences that code for the heavy and/or light chain variable regions of an antibody selected from the groups consisting of 1212C2, 1212F5, 1213H7, 1212F2, 1206D1, 1212D5, 1207B4, 1213F2, 1212D4, 1206A5, 1212D6, 1206D12, 1207F10, 1206G12, 1212C8, 1212E9, 1214E9, and 1215D1. The vector may be a mammalian expression vectors, such that the vector may be transfected into mammalian cells and the DNA may be integrated into the genome by homologous recombination in the case of stable transfection, or alternatively the cells may be transiently transfected. Common to most engineered vectors are origin of replications, multicloning sites, and selectable markers. Common promoters for mammalian expression vectors include CMV and SV40 promoters, and non-viral promoters such as, but not limited to, EF-1 promoters. In certain embodiments, the disclosure provides a vector comprising one or more nucleic acid sequences encoding one or more CDRs of one or more heavy and/or light chains of one or more of the antibodies of the disclosure (for example, a nucleic acid encoding an amino acid sequences of SEQ ID NOS: 38-40, 42-44, 46- 48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98- 100102-104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-135, 138- 140, 142-144, and 146-148, or an amino acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% homology with SEQ ID NOS: 38-40, 42-44, 46-48, 50-52, 54-56, 58-60, 62-64, 66-68, 70-72, 74-76, 78-80, 82-84, 86-88, 90-92, 94-96, 98-100102-
104, 106-108, 110-112, 114-116, 118-120, 122-124, 126-128, 130-132, 134-135, 138-140, 142- 144, and 146-148). In certain embodiments, the disclosure provides a vector comprising one or more nucleic acid sequences encoding one or more heavy and/or light chain variable regions of one or more of the antibodies of the disclosure (for example, a nucleic acid encoding an amino acid sequences of SEQ ID NOS: 37, 42, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101,
105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, and 149-156 or an amino acid having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or greater than 95% homology with SEQ ID NOS: 37, 42, 45, 49, 53, 57, 61, 65, 69, 73, 77, 81, 85, 89, 93, 97, 101, 105, 109, 113, 117, 121, 125, 129, 133, 137, 141, 145, and 149-156).
The nucleic acid sequences may encode for an antibody variant as described herein, including an antibody containing a conservative substitution. A vector that codes for one or both variable region(s) of an antibody of the disclosure may contain one of or both of SEQ ID NOS: required to generate such antibody, or a nucleotide sequence that shares a degree minimum of homology with such SEQ ID NOS: (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% homology). For example, a vector that encodes the heavy and light chain variable region of antibody 1212C2 may comprise SEQ ID NOS: 1 and 2, or a nucleotide sequence that shares a degree minimum of homology with such SEQ ID NOS: 1 and 2 (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or more than 95% homology).
The disclosure also provides for a cell transformed with a vector described herein. One of ordinary skill in the art will appreciate that different cell types may lead to different antibody products and may possibly impact the therapeutic efficacy of the antibody products, e.g. through having distinct variations in glycosylation patterns, especially N-linked glycosylation patterns. Such discussions may be found in Liu L, J Pharm Sci. 2015 June; 104(6): 1866-84; Rosenlocher et al., J Proteomics. 2016 Feb. 16; 134:85-92; Mimura et al., J Immunol Methods. 2016 January; 428:30-6; and Croset et al., Journal of Biotechnology, 161(3), Oct. 31, 2012. In some embodiments, the cell is a bacterial cell, a yeast cell, a plant cell, or a mammalian cell. In some embodiments, the mammalian cell is one of a Chinese hamster ovary (CHO) cell, including DUXB11, DG44 and CHOK1 lineages, a NSO murine myeloma cell, aPER.C6 cell, and a human embryonic kidney (HEK) cell, including HEK293 lineages. Other less common host cells, include plant cells, for example, those based on the tumor-inducing (Ti) plasmid of Agrobacterium tumefaciens. Cell-free expression systems also exist, for example, based on E. coli cell lysate, containing cellular components necessary for transcript! on/translati on. Eukaryotic and mammalian cell-free systems are also known in the art, for example wheat germ cell-free expression system. Some recombinant antibody production systems express the recombinant antibodies on the surface of the host cell before harvesting, others simply release the antibodies into a medium for collection. Such variations are intended to be within the scope of the disclosure.
The disclosure also provides for a method of making a recombinant antibody of the disclosure. In some embodiments, the antibody is an antigen binding fragment. The host cell comprising a vector described herein is induced to produce the recombinant antibodies and the host cell assembles the antibodies from heavy/light chains in the host cell and then transport the antibodies out of the cell, or the antibodies may self-assemble outside the host cell and be exported as heavy/light chains. An overview of cell culture processes for recombinant monoclonal antibody production may be found in Li et al., Mabs. 2010 September-October; 2(5): 466-477. In one embodiment, the disclosure provides for a method of making a recombinant antibody, or antigenbinding fragment thereof, that specifically binds to SARS-CoV-2 spike protein, the method comprising providing a cell comprising a vector comprising a nucleic acid sequence encoding a heavy chain variable region and/or a light chain variable region of any one of SEQ ID NOS: 1-36, expressing at least one nucleic acid sequence in the vector to create at least one of a heavy chain, a light chain, or combinations thereof, and collecting a formed antibody or the antigen-binding fragment, thereof.
Identification and Isolation of Antibodies
Antibodies of the disclosure may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics (Hoogenboom et al. in Methods in Molecular Biology 178: 1- 37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001); McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).
In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage. Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e. ., from human) to provide a single source of antibodies to a wide range of non-self without any immunization (Griffiths et al., EMBO J, 12: 725-734 (1993)). Finally, naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro (Hoogenboom and Winter, I. Mol. Biol., 227: 381-388 (1992)). Antibodies, including antigen-binding fragments, isolated from human antibody libraries are considered human antibodies.
Methods of Treatment and Use
In a first aspect, the disclosure provides a method for treating a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition. In a preferred embodiment, a combination of 1212C2 and 1213H7 mAbs are used.
In a second aspect, the disclosure provides a method for suppressing a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
In a third aspect, the disclosure provides a method for preventing a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
In a fourth aspect, the disclosure provides a method for treating, suppressing and/or preventing a disease or condition relating to a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition. In certain embodiments of the fourth aspect, the disease or condition is Guillain- Barre Syndrome. In certain embodiments of the fourth aspect, the disease or condition is multisystem inflammatory syndrome, particularly when the subject is under the age of 25 years of age. In certain embodiments of the fourth aspect, the disease or condition is organ injury, such as, but not limited to, lung injury, liver injury, and/or heart injury. In certain embodiments of the fourth aspect, the disease or condition is acute respiratory distress syndrome. In certain embodiments of the fourth aspect, the disease or condition is increased inflammation resulting from an imbalance in the renin-angiotensin system (such as, but not limited to, excess production of angiotensin II and/or the decreased production of angiotensin 1-7).
In a fifth aspect, the disclosure provides a method of reducing or preventing cellular entry of SARS-CoV-2 in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, or a combination of the foregoing, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
In a sixth aspect, the disclosure provides a method of reducing or preventing binding of SARS-CoV-2 to a cellular ACE2 in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
In a seventh aspect, the disclosure provides a method for reducing viral titer of a SARS- CoV-2 in a bodily fluid, tissue or cell of a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition. In certain embodiments, the transmission of the SARS-CoV-2 (for example, from a subject infected with SARS-CoV-2 to a subject that is not yet infected) is reduced as a result of a reduced viral titer.
In an eighth aspect, the disclosure provides a method for reducing or preventing the transmission of a SARS-CoV-2 infection from a first subject to a second subject, the method comprising administering to said first subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition. In certain embodiments of the eighth aspect, such reduction or prevention is obtained, at least in part, by reducing the cellular entry of a SARS-CoV-2 in the first subject. In certain embodiments, administration to the first subject occurs before the first subject has been infected with SARS-CoV-2, after the first subject has been infected with the SARS-CoV-2, or after the first subject has been infected with the SARS-CoV-2 and before the SARS-CoV-2 infection can be detected.
In a ninth aspect, the disclosure provides a method for reducing or preventing the transmission of a SARS-CoV-2 infection from a first subject to a second subject, the method comprising administering to the second subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV- 2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition. In certain embodiments of the ninth aspect, the second subject may be at risk for SARS-CoV-2 infection. In certain embodiments of the ninth aspect, such reduction or prevention is obtained, at least in part, by preventing or reducing SARS-CoV-2 cellular entry in the second subject. In certain embodiments of the ninth aspect, such reduction or prevention is obtained, at least in part, by preventing or suppressing a SARS-CoV-2 infection in the second subject. In certain embodiments of the ninth aspect, if a SARS-CoV-2 infection occurs in the second subject, it can be eliminated physiologically (for example, by the immune system) by the second subject, either with or without the administration of additional therapeutic compounds. In certain embodiments of the ninth aspect, administration to the second subject before the second subject has been infected with the SARS-CoV-2, after the second subject has been infected with the SARS-CoV-2, or after the second subject has been infected with the SARS-CoV-2 and before the SARS-CoV-2 infection can be detected. In a tenth aspect, the disclosure provides a method of neutralizing a SARS-CoV-2 in a subject, the method comprising administering to said subject an effective amount of an isolated antibody, or an antigen-binding fragment thereof, that specifically binds to the spike protein of SARS-CoV-2, either alone, combined with one or more other antibodies, and/or as a part of a pharmaceutical composition.
In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds to SARS-CoV-2 viral particles before they are able to interact with cellular ACE2, thereby reducing or preventing SARS-CoV-2 viral particles from entering the cell. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces or prevents SARS-CoV-2 viral particles from binding to cellular ACE2. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces cleavage of the SARS-CoV-2 spike protein. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduce binding of SARS-CoV-2 to a target cell. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces cellular fusion between SARS-CoV-2 and a target cell. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces release of infective SARS-CoV-2 from an infected cell. In certain embodiments of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduce infection of a target cell by SARS-CoV-2.
The methods of the first to tenth aspects may further comprise one or more of the steps: i) identifying a subject in need or treatment, prevention, suppression, reduction, or inhibition; and (ii) providing an antibody, or an antigen-binding fragment thereof, of the disclosure or a pharmaceutical composition comprising the foregoing.
In certain embodiments of the first to tenth aspects, the antibody or antibodies, or an antigen-binding fragment(s) thereof, is any one or more antibodies or antigen binding fragment(s) described herein, or a pharmaceutically acceptable form thereof.
In certain embodiments of the first to tenth aspects, the antibody or antibodies, or an antigen-binding fragment(s) thereof, comprises:
1. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 38-40, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42-44, respectively (1212C2); (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 46-48, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50-52, respectively (1215F5); (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 54-56, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 58-60, respectively (1213H7); (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 62-64, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 66-68, respectively (1212F2); (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 70-72, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 74-76, respectively (1206D1); (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 78-80, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 82-84, respectively (1212D5); (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 86-88, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 90-92, respectively (1207B4); (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 94-96, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 98-100, respectively (1213F2); 9. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 102-104, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 106-108, respectively (1212D4);
10. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 110-112, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 114-116, respectively (1206A5);
11. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 118-120, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 122-124, respectively (1212D6);
12. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 126-128, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 130-132, respectively (1206D12);
13. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 134-136, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 138-140, respectively (1207F10); and
14. (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 142-144, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 146-148, respectively (1206G12), or a pharmaceutically acceptable form of any of the foregoing.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises:
1. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 37, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 41, or an amino acid sequence at least 80% homologous thereto (1212C2); 2. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 45, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 49, or an amino acid sequence at least 80% homologous thereto (1215F5);
3. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 53, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 57, or an amino acid sequence at least 80% homologous thereto (1213H7);
4. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 61, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 65, or an amino acid sequence at least 80% homologous thereto (1212F2);
5. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 69, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 73, or an amino acid sequence at least 80% homologous thereto (1206D1);
6. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 77, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 81, or an amino acid sequence at least 80% homologous thereto (1212D5);
7. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 85, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 89, or an amino acid sequence at least 80% homologous thereto (1207B4);
8. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 93, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 97, or an amino acid sequence at least 80% homologous thereto (1213F2);
9. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 101, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 105, or an amino acid sequence at least 80% homologous thereto (1212D4); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 109, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 113, or an amino acid sequence at least 80% homologous thereto (1206 A5); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 117, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 121, or an amino acid sequence at least 80% homologous thereto (1212D6); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 125, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 129, or an amino acid sequence at least 80% homologous thereto (1206D12); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 133, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 137, or an amino acid sequence at least 80% homologous thereto (1207F10); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 141, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 145, or an amino acid sequence at least 80% homologous thereto (1206G12); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 149, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 150, or an amino acid sequence at least 80% homologous thereto (1215D1); (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 151 , or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 152, or an amino acid sequence at least 80% homologous thereto (1214E9); 17. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 153, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 154, or an amino acid sequence at least 80% homologous thereto (1212E9); and
18. (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 155, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 156 (1212C8), or an amino acid sequence at least 80% homologous thereto, or a pharmaceutically acceptable form of any of the foregoing.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 158, 39 and 159, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42, 43, and 161, respectively (1212 variant), or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
In one aspect of this embodiment, the amino acid at position 1 of SEQ ID NO: 158 is Thr (T) or He (I), the amino acid at position 1 of SEQ ID NO: 159 is Thr (T) or Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid, the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser
(S), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V) or Phe (F).
In one aspect of this embodiment, the amino acid at position 1 of SEQ ID NO: 158 is Thr
(T) or He (I), the amino acid at position 1 of SEQ ID NO: 159 is Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid (preferably Ala (A)), the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser (S), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V).
In one aspect of this embodiment, the amino acid at position 1 of SEQ ID NO: 158 is Thr (T), the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Thr (T) and Leu (L), respectively, and the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid, Asn (N), and Phe (F), respectively. In one aspect of this embodiment, the amino acid at position 1 of SEQ ID NO: 158 is Thr (T), the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Ala (A) and Leu (L), respectively, and the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid (preferably Ala (A)), Asn (N), and Vai (V), respectively.
In one aspect of this embodiment, the amino acid at position 1 of SEQ ID NO: 158 is lie (I), the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Ala (A) and Phe (F), respectively, and the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid, Ser (S), and Vai (V), respectively.
In one aspect of this embodiment, the amino acid at position 1 of SEQ ID NO: 158 is lie (I), the amino acids at positions 1 and 9 of SEQ ID NO: 159 are Ala (A) and Phe (F), respectively, and the amino acids at positions 1, 6, and 10 of SEQ ID NO: 161 are any amino acid (preferably Ala (A)), Ser (S), and Vai (V), respectively.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 157, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 160, or an amino acid sequence at least 80% homologous thereto (1212 variant); or a pharmaceutically acceptable form of any of the foregoing, such as, but not limited to a pharmaceutically acceptable salt, solvate and/or hydrate.
In one aspect of this embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2A and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2B.
In one aspect of this embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2C and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2D.
In one aspect of this embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2E and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2F.
In one aspect of this embodiment, the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2G below and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2H below. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a mixture of any of the foregoing.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is present as a pharmaceutically acceptable salt.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds an epitope within the RBD of the SARS-CoV-2 spike protein. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds an epitope within the RBM of the SARS-CoV-2 spike protein. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds an epitope within the S 1 region of the SARS-CoV-2 spike protein. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, binds an epitope within the S2 region of the SARS-CoV-2 spike protein.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces binding of SARS-CoV-2 to a target cell. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces cellular fusion between SARS-CoV-2 and a target cell. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, reduces release of infective SARS-CoV-2 from an infected cell. In certain embodiments of the first to tenth aspects, the antibody, or an antigenbinding fragment thereof, reduces infection of a target cell by SARS-CoV-2.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antibody variant. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antibody variant comprising one or more substitutions, deletions, and/or insertions relative to the parental antibody. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antibody variant comprising one or more substitutions relative to the parental antibody.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antibody variant that has a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region, wherein a protein moiety or non-protein moiety is linked to the Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region, wherein a water soluble polymer is linked to the Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigenbinding fragment thereof, comprises a variant Fc constant region, wherein a polyethylene glycol polymer is linked to the Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region, wherein a polyoxazoline polymer is linked to the Fc constant region. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, comprises a variant Fc constant region, wherein the variant Fc constant region provides a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is a human antibody. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is a chimeric antibody. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is a class- switched antibody.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is linked to a therapeutic agent. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is linked to a detectable label. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is linked to an enzyme. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is linked to an enzyme inhibitor.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antigen-binding fragment. In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is an antigen-binding fragment selected from the groups consisting of: a Fab fragment, a F(ab)2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a disulfide-linked Fv (sdFv), a dAb fragment, an isolated CDR, a nanobody or single domain antibody, a portion of the VH region containing a single variable domain and two constant domains, a diabody, a triabody, a tetrabody, scFv, scFv-FC, scFv-CH, scFab, and scFv-zipper.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, the amino acid sequence of the antibody, or an antigen-binding fragment thereof, has at least 85% homology to the reference sequence, at least 90% homology to the reference sequence, at least 95% homology to the reference sequence, at least 96% homology to the reference sequence, at least 97% homology to the reference sequence, at least 98% homology to the reference sequence, or at least 99% homology to the reference sequence.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, the amino acid sequence of the antibody, or an antigen-binding fragment thereof, is 100% homologous to the reference sequence across the CDRs and has at least 85% homology to the reference sequence across the FRs, at least 85% homology to the reference sequence across the FRs, at least 90% homology to the reference sequence across the FRs, at least 95% homology to the reference sequence across the FRs, at least 96% homology to the reference sequence across the FRs, at least 97% homology to the reference sequence across the FRs, at least 98% homology to the reference sequence across the FRs, or at least 99% homology to the reference sequence across the FRs.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is used in combination with other anti-viral agents as described herein, such as inhibitors of viral RNA polymerase activity and/or other serine and non-serine protease inhibitors.
In certain embodiments of the first to tenth aspects, a subject is infected with SARs-CoV- 2 and by one or more additional viruses.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is administered in an effective amount. Suitable effective amounts are described in more detail herein. In certain embodiments of the first to tenth aspects, the administering step may comprise administering a single dose of an antibody, or an antigen-binding fragment thereof, according to a course of treatment (where the dose may contain an effective amount). In certain embodiments of the first to tenth aspects, the administering step may comprise administering more than one dose of the antibody, or an antigen-binding fragment thereof, according to a course of treatment (where one or more doses may contain an effective amount). In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, in each dose administered during a course of treatment is not required to be the same. For example, in any of the methods of the first to tenth aspects, the administering step may comprise administering at least one loading dose and at least one maintenance dose during a course of treatment.
In certain embodiments of the first to tenth aspects, the administering step comprises administering a single dose or a plurality of doses comprising the antibody, or an antigen-binding fragment thereof, according to a course of treatment. In certain embodiments of the first to tenth aspects, the administering step comprises administering a dose or a plurality of doses comprising the antibody, or an antigen-binding fragment thereof, by intravenous administration according to a course of treatment. In certain embodiments of the first to tenth aspects, the administering step comprises administering a dose or a plurality of doses comprising the antibody, or an antigenbinding fragment thereof, by intranasal administration according to a course of treatment. In certain embodiments of the first to tenth aspects, the administering step comprises administering a dose or a plurality of doses comprising the antibody, or an antigen-binding fragment thereof, by pulmonary administration according to a course of treatment.
In certain embodiments of the first to tenth aspect, the subject is suffering from or suspected of suffering from a SARS-CoV-2 infection.
In certain embodiments of the first to tenth aspects, a pharmaceutical composition and/or medicaments comprising the antibody, or an antigen-binding fragment thereof, may be administered according to the methods described herein.
In certain embodiments of the first to tenth aspects, the subject is a mammal. In certain embodiments of the first to tenth aspects, the subject is a human.
In certain embodiments of the first to tenth aspects, the administering step occurs before the subject has been infected with SARS-CoV-2 (/.«, the subject is at risk for infection), after the subject has been infected with SARS-CoV-2 (but before an infection can be detected), or after a subject has been infected with SARS-CoV-2 and the infection can be detected.
In certain embodiments of the first to tenth aspects, the antibody, the subject is a healthcare worker, a first responder (for example, a policeman or a fireman), or a member of the military as such individuals may be required to undertake activities that place them at a higher risk of SARS-CoV-2 infection. In certain embodiments of the first to tenth aspects, the subject has travelled to a region where SARS-CoV-2 infections have been documented, the subject has had contact with a person who has travelled to a region where SARS-CoV-2 infections have been documented, the has had contact with a person who has a SARS-CoV-2 infection (including a SARS-CoV-2 infection that has not been detected) or is suspected of having a SARS-CoV-2 infection, the subject is a family member or acquaintance of a person who has a SARS-CoV-2 infection (including a CoV infection that has not been detected) or is at risk of having a SARS- CoV-2 infection, the subj ect is an infant or child (for example, a subject under the age of 18 years) who has a caregiver or parent who has a SARS-CoV-2 infection or is at risk of having a SARS- CoV-2 infection.
In certain embodiments of the first to tenth aspects, the subject may be suffering from pulmonary disease, cardiovascular disease, diabetes mellitus, bacterial superinfection, sepsis syndrome, hypertension, chronic lung disease (inclusive of asthma, chronic obstructive pulmonary disease, and emphysema), chronic renal disease, chronic liver disease, immunodeficiency, an immunocompromised condition, neurologic disorder, neurodevelopmental, or intellectual disability.
In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is administered parenterally, such as by intravenous administration, intramuscular administration, or subcutaneous administration, orally, or via the respiratory tract (for example, by pulmonary or intranasal administration). In certain embodiments of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is administered intravenously. In certain aspects of the methods of the first to tenth aspects, the antibody, or an antigen-binding fragment thereof, is administered intramuscularly. In certain aspects of the methods of the first to tenth aspects, the antibody, or an anti gen -binding fragment thereof, is administered intranasally.
Additional Therapeutic Agents
In addition, the pharmaceutical compositions or methods described herein may further comprise one or more additional anti-viral agents in combination with an antibody of the disclosure. Examples of such anti-viral agents include, but are not limited to, those agent that inhibit replication of SARS-CoV-2, such by inhibition of a RNA polymerase activity of SARS- CoV-2, and protease inhibitors, including but not limited to, serine protease inhibitors (for example, inhibitors of TMPRSS2) and cysteine protease inhibitors. Representative agents include, but are not limited to, galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscarnet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir. Other agents and synergistic combinations thereof are described in Bobrowski et al., bioRxiv (June 2020, https://doi.org/10.1101/2020.06.29.178889) and Ko, et al., bioRxiv (May 2020, https://doi.org/10.1101/2020.05.12.090035).
Compounds that relate to inhibition of influenza polymerase are described, for example, in U.S. Pat. Nos. 7,388,002; 7,560,434; and in U.S. patent application Ser. Nos. 12/440,697 (published as U.S. Patent Publication No. 20100129317); and 12/398,866 (published as U.S. Patent Publication No. 20090227524).
Dosage and Administration
In accordance with the methods of the disclosure, the polypeptides of the disclosure are administered to the subject (or are contacted with cells of the subject) in an effective amount. In some embodiments, an effective amount decreases the viral titer of SARS-CoV-2 in the subject and/or limits or prevents an increase in the viral titer of SARS-CoV-2 viral particles in the subj ect. In some embodiments, an effective amount decreases viral entry of SARS-CoV-2 subject. In some embodiments, an effective amount reduces binding of SARS-CoV-2 to a target cell. In some embodiments, an effective amount reduces cellular fusion between SARS-CoV-2 and a target cell. In some embodiments, an effective amount reduces release of infective SARS-CoV-2 from an infected cell of the subject. In some embodiments, an effective amount reduces infection of a target cell by SARS-CoV-2.
In certain embodiments, the effective amount of an antibody of the disclosure ranges from about 0.01 mg/kg to about 100 mg/kg. For example, doses of about 0.05 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 4 mg/kg, about 0.05 mg/kg to about 2 mg/kg, or about 0.05 mg/kg to about 1 mg/kg.
In certain embodiments, the effective amount of an antibody of the disclosure ranges from: i) about 1 mg/kg to about 5 mg/kg; ii) about 1 mg/kg to about 4 mg/kg; iii) about 1 mg/kg to about 4 mg/kg; or iv) about 1 mg/kg to about 4 mg/kg.
In certain embodiments, the effective amount described herein are administered every day, every other day, every three days, every 4 days, every 5 days, every six days or every seven days. In certain embodiments, the effective amount described herein are administered in weekly intervals (such as every week, every 2 weeks, every three weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer). In certain embodiments, the effective amount described herein are administered in monthly intervals (such as every month, every 2 months, every three months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or longer).
In one embodiment, an effective amounts is administered every day during a course of treatment. The effective amount per day may be administered in a single dose or in more than 1 dose per day (such as two to three doses per day). In certain embodiments, the effective amount per day is administered as a single dose per day. In certain embodiments, the effective amount per day is administered in two doses each day (/.c., b i d ), wherein the amount of the antibody in each dose need not be the same.
In one embodiment, the effective amount described above is administered every other day, every three days, every 4 days, every 5 days, every six days, or every seven days during a course of treatment. The effective amount in the dosing schedules may be administered in a single dose or in more than 1 dose (such as two to three doses). In certain embodiments, the effective amount in the dosing schedules is administered as a single dose on the day of administration. In certain embodiments, the effective amount in the dosing schedules is administered in two doses on the day of administration, wherein the amount of the antibody in each dose need not be the same.
In one embodiment, the effective amounts described above are administered in weekly intervals (such as every week, every 2 weeks, every three weeks, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer), during a course of treatment. The effective amount per weekly interval may be administered in a single dose or in more than 1 dose. In certain embodiments, the effective amount per week is administered as a single dose on the day of administration. In certain embodiments, the effective amount per week is administered in two doses on the day of administration, wherein the amount of the antibody in each dose need not be the same.
In one embodiment, the effective amounts described above are administered in monthly intervals (such as every month, every 2 months, every three months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, every 12 months, or longer), during a course of treatment. The effective amount per monthly interval may be administered in a single dose or in more than 1 dose. In certain embodiments, the effective amount per month is administered as a single dose on the day of administration. In certain embodiments, the effective amount per month is administered in two doses on the day of administration, wherein the amount of the antibody in each dose need not be the same.
As used herein, the term “dose” refers to an amount of an antibody of the disclosure administered at a given time point. For example, if a course of treatment for an antibody of the disclosure is b.i.d (2 times/admini strati ons per day) for 7 days, each administrations on each of days 1-7 would comprise administering a dose (for 2 doses each day).
When 2 or more doses are administered on a given day, each dose administered may contain the same amount of an antibody of the disclosure or one or more of doses administered may contain a greater or lesser amount of an antibody of the disclosure as compared to another dose administered on that day. For example, if a course of treatment for an antibody of the disclosure calls for monthly administration, with the effective amount administered b.i.d at each monthly administration, the first dose administered may contain a first amount (z.e., 10 mg/kg) and the second dose administered may contain a second amount (i.e., 5 mg/kg). As another example, if a course of treatment for an antibody of the disclosure is b.i.d for 7 days, the first dose administered on day 1 may contain a first amount (i.e., 10 mg/kg), the second dose administered on day 1 may contain a second amount i.e., 5 mg/kg), the two doses administered on each of days 2-4 may contain the second amount, and the two doses administered on each of days 5-7 may contain a third amount (i.e., 2 mg/kg).
Furthermore, for a given course of treatment, the administration schedule for the effective amount is not required to be the same. For example, a course of treatment may call for an effective amount to be delivered monthly (either as a single dose or multiple doses) for the first 6 months, and then every three months (either as a single dose or multiple doses) for the next 12 months.
Still further, for a given course of treatment, the effective amount delivered at each administration during a course of treatment is not required to be the same. For example, a course of treatment may call for an effective amount to be delivered monthly (either as a single dose or multiple doses) for the first 6 months, and then every two months (either as a single dose or multiple doses) for the next 6 months, wherein the effective amount for the first monthly administration is 10 mg/kg and the effective amount for the remaining monthly administration and the bi-monthly administrations is 2.5 mg/kg.
Any given dose may be delivered in a single unit dose form or more than one unit dose form. For example, a dose when given by IV administration may be provided as a single IV infusion (z.e., a single 20 mg/kg IV infusion) or as two or more IV infusions administered one after the other and be considered a single dose (z.e., two 10 mg/kg IV infusions). A dose may be further divided into a sub-dose. A sub-dose might be, for example, a number of discrete loosely spaced administrations, such as multiple inhalations from an inhaler, by application of a plurality of drops into the eye, or multiple tablets for oral administration.
In certain embodiments, only one dose of an antibody of the disclosure is administered during a course of treatment and no further doses are administered. Therefore, in the methods described herein the methods may comprise the administration of a single dose of an effective amount of an antibody of the disclosure during the entire course of treatment. In certain embodiments, more than one dose of an antibody of the disclosure is administered during a course of treatment. Therefore, the methods may comprise the administration of multiple doses of an antibody of the disclosure during the course of treatment. In certain embodiments, the course of treatment may range from 2 days to 1 month, from 2 days to 3 weeks, from 2 days to 2 weeks, or from 2 days to 1 week. In certain embodiments, the course of treatment may range from 1 month to 6 months, from 1 month to 12 months, from 1 month to 24 months, or from 1 month to 36 months. In certain embodiments, the course of treatment may range from 2 days to 6 days, from 2 days to 5 days, from 2 days to 4 days, or from 2 days to 3 days. In certain embodiments, a dose is delivered at least 1 time per day (z.e., 1 to 3 times) during the course of treatment. In certain embodiments, the course of treatment is continuous. In certain embodiments, a dose is not administered every day during the course of treatment (for example, a dose is be administered at least 1 time per day every other day during the course of treatment). Furthermore, the amount of an antibody of the disclosure in each dose need not be the same as discussed above. In certain embodiments, of the foregoing, one or more doses, preferably all of the doses, contain an effective amount of an antibody of the disclosure.
In certain embodiments, a course of treatment may comprise administering at least one dose as a loading dose and at least one dose as a maintenance dose, wherein the loading dose contains a greater amount of an antibody of the disclosure as compared to the maintenance dose (such as, but not limited to, 2 to 10 times higher). In one aspect of this embodiment, the loading dose is administered initially, followed by administration of one or more maintenance doses through the remaining course of treatment. For example, for a course of treatment that is b.i.d. for 7 days, a loading dose of 10 mg/kg may be administered as the first dose on day 1, followed by maintenance doses of 2 mg/kg for the remainder of the course of treatment. As a further example, for a course of treatment that is b.i.d. for 7 days, a loading dose of 20 mg/kg may be administered as the first dose on day 1, followed by maintenance doses of 5 mg/kg as the second dose on day 1 and each dose on days 2-4, followed by maintenance doses of 2 mg/kg for the remainder of the course of treatment.
Furthermore, a loading dose may be given as a dose that is not the first dose administered during a course of treatment. For example, a loading dose may be administered as the first dose on day 1 and as a dose on one or more additional days (for example, day 4). For example, for a course of treatment that is b.i.d. for 7 days, a loading dose of 10 mg/kg may be administered as the first dose on day 1, followed by a maintenance dose of 2 mg/kg as the second dose on day 1 and each dose on days 2-3, followed by a loading dose of 10 mg/kg as the first dose on day 4, followed by a maintenance dose of 2 mg/kg for the remainder of the course of treatment. When more than one loading dose is administered during a course of treatment, the loading dose may be the same (z.e., 10 mg/kg) or different (z. e. , 20 mg/kg for the first loading dose and 10 mg/kg for each other loading dose).
In certain embodiments, the loading dose comprises 2 to 15 times more of an antibody of the disclosure as compared to a maintenance dose administered during the same course of treatment. In certain embodiments, the loading dose comprises 2 to 10 times more of an antibody of the disclosure as compared to a maintenance dose administered during the same course of treatment. In certain embodiments, the loading dose comprises 2 to 5 times more of an antibody of the disclosure as compared to a maintenance dose administered during the same course of treatment.
In certain embodiments of loading and maintenance doses as discussed above, one or more of the loading and maintenance doses, preferably all of the loading and maintenance doses, contain an effective amount of an antibody of the disclosure. Furthermore, in certain embodiments of the loading and maintenance doses discussed above, administration of one or more loading and/or maintenance doses may comprise administering one or more sub-doses and/or administering one or more unit dose forms.
In certain embodiments, the course of treatment is initiated (i.e., the first dose administered) after a subject has been infected with SARS-CoV-2. In certain embodiments, the course of treatment is initiated any time after a subject has been infected with SARS-CoV-2. In certain embodiments, the course of treatment is initiated any time after a subject has been infected with SARS-CoV-2 and before an active SARS-CoV-2 infection can be detected (i.e., by laboratory diagnosis or other methods). In certain embodiments, the course of treatment is initiated any time during which a subject has an active SARS-CoV-2 infection (i.e., by laboratory diagnosis or other methods). In certain embodiments, the course of treatment is initiated 1-5 days after a subject has been infected with SARS-CoV-2. In certain embodiments, the course of treatment is initiated 5- 10 days after a subject has been infected with SARS-CoV-2.
In certain embodiments, the course of treatment is initiated before a subject is infected with SARS-CoV-2 (i.e., a prophylactic administration). For example, if a subject is planning to travel to a region where SARS-CoV-2 infection has been reported or believes he/ she may be exposed to SARS-CoV-2, the subject may undergo a course of treatment with an antibody of the disclosure prior to travel to the region or prior to potential exposure. Furthermore, a subject may be someone that is not initially exposed to SARS-CoV-2 infection from a non-human vector source. For example, the spouse or partner of someone who has been exposed to SARS-CoV-2 or who is at risk for exposure to SARS-CoV-2 (for example, by traveling to an area where SARS-CoV-2 infection has been reported) may undergo a course of treatment with an antibody of the disclosure as well. Such a prophylactic use of the antibodies of the disclosure are beneficial not only to protect the subject that is administered an antibody of the disclosure, but also in protecting those the subject comes into contact with (for example, family members and co-workers).
In any of the foregoing embodiments, the dose may comprise an antibody of the disclosure alone or an antibody of the disclosure in a pharmaceutical composition. In any of the foregoing embodiments, each dose is delivered by parenteral administration. In any of the foregoing embodiments, each dose is delivered by IV administration. In any of the foregoing embodiments, each dose is delivered by IM administration. In any of the foregoing embodiments, each dose is delivered by subcutaneous administration. In any of the foregoing embodiments, each dose is delivered by via the pulmonary route. In any of the foregoing embodiments, each dose is delivered by pulmonary administration. In any of the foregoing embodiments, each dose is delivered by intranasal administration.
In any of the foregoing embodiments, each dose contains an amount of an antibody of the disclosure in a pharmaceutically acceptable form, such as a pharmaceutically acceptable salt. Pharmaceutical Compositions
The antibodies of the disclosure may be formulated into pharmaceutical compositions for administration to subjects in a pharmaceutically acceptable form suitable for administration in vivo. The disclosure provides a pharmaceutical composition comprising antibodies of the disclosure in combination with a pharmaceutically acceptable carrier and/or excipient. Often, the pharmaceutically acceptable carrier and/or excipient is chemically inert toward the active compounds and is non-toxic under the conditions of use. The pharmaceutically-acceptable carrier and/or excipient employed herein may be selected from various organic or inorganic materials that are used as materials for pharmaceutical compositions and which are incorporated as analgesic agents, buffers, wetting agents, emulsifying agents, pH adjusting agents, binders, disintegrants, diluents, emulsifiers, extenders, glidants, solubilizers, stabilizers, suspending agents, tonicity agents, vehicles, viscosity-increasing agents, antioxidants, colorants, flavor-improving agents, preservatives, and sweeteners.
In one embodiment, the disclosure provides a pharmaceutical composition comprising an antibody of the disclosure, wherein the pharmaceutical composition comprises 1 to 6,000 mg of the antibody. For example, the pharmaceutical composition may contain 1 to 2,000 mg, 1 to 600 mg, 1 to 500 mg, 1 to 400 mg, 1 to 300 mg, 1 to 200 mg, or 1 to 100 mg of the antibody. In one embodiment, the antigen of the disclosure is present in the pharmaceutical composition at a concentration of at least 1 mg/mL, 5 mg/mL, 10 mg/mL, 50 mg/mL, 100 mg/mL, 150 mg/mL, 200 mg/mL, 300 mg/mL, 400 mg/ml„ or 500 mg/ml (with the concentration in each of the foregoing being less than 750 mg/ml).
The amount of an antibody of the disclosure in a pharmaceutical composition may very as is known in the art. Generally, the amount of an antibody of the disclosure will range from about 0.01% to about 99% by total weight of the pharmaceutical composition, preferably from about 0.1% to about 70%, from about 0.5% to 50%, or from about 1% to about 30%.
Examples of pharmaceutically acceptable carriers may include, for example, water or saline solution, polymers such as polyethylene glycol, carbohydrates and derivatives thereof, oils, fatty acids, or alcohols. In some embodiments, the pharmaceutically acceptable carrier is saline or water. In some embodiments, the pharmaceutically acceptable carrier is saline. In some embodiments, the pharmaceutically acceptable carrier is water or a saline solution.
Surfactants such as, but not limited to, detergents, are also suitable for use in the formulations. Specific examples of surfactants include polyvinylpyrrolidone, polyvinyl alcohols, copolymers of vinyl acetate and of vinylpyrrolidone, polyethylene glycols, benzyl alcohol, mannitol, glycerol, sorbitol or polyoxyethylenated esters of sorbitan; lecithin or sodium carboxymethylcellulose; or acrylic derivatives, such as methacrylates and others, anionic surfactants, such as alkaline stearates, in particular sodium, potassium or ammonium stearate; calcium stearate or triethanolamine stearate; alkyl sulfates, in particular sodium lauryl sufate and sodium cetyl sulfate; sodium dodecylbenzenesulphonate or sodium dioctyl sulphosuccinate; or fatty acids, in particular those derived from coconut oil, cationic surfactants, such as water-soluble quaternary ammonium salts of formula N+R'R"R'"R""Y‘, in which the R radicals are identical or different optionally hydroxylated hydrocarbon radicals and Y' is an anion of a strong acid, such as halide, sulfate and sulfonate anions; cetyltrimethylammonium bromide is one of the cationic surfactants which can be used, amine salts of formula N+R'R"R"', in which the R radicals are identical or different optionally hydroxylated hydrocarbon radicals; octadecyl amine hydrochloride is one of the cationic surfactants which can be used, non-ionic surfactants, such as optionally polyoxyethylenated esters of sorbitan, in particular Polysorbate 80, or polyoxyethylenated alkyl ethers; polyethylene glycol stearate, polyoxyethylenated derivatives of castor oil, polyglycerol esters, polyoxyethylenated fatty alcohols, polyoxyethylenated fatty acids or copolymers of ethylene oxide and of propylene oxide, amphoteric surfactants, such as substituted lauryl compounds of betaine,
When administered to a subject, the pharmaceutical composition is preferably sterile. The pharmaceutical compositions of the disclosure are prepared by methods well-known in the pharmaceutical arts. For example, the antibodies of the disclosure are brought into association with a carrier and/or excipient, as a suspension or solution. Optionally, one or more accessory ingredients (e.g, buffers, flavoring agents, surface active agents, and the like) also are added. The choice of carrier and/or excipient is determined by the solubility and chemical nature of the antibodies, chosen route of administration and standard pharmaceutical practice. In some embodiments, the pharmaceutical composition comprises an antibody of the disclosure and water. In some embodiments, the formulation comprises an antibody of the disclosure and saline.
For oral administration, a pharmaceutical composition of the disclosure may be presented as capsules, tablets, powders, granules, or as a suspension or solution. Capsule formulations may be gelatin, soft-gel or solid. Tablets and capsule formulations may further contain one or more adjuvants, binders, diluents, disintegrants, excipients, fillers, or lubricants, each of which are known in the art. Examples of such include carbohydrates such as lactose or sucrose, dibasic calcium phosphate anhydrous, corn starch, mannitol, xylitol, cellulose or derivatives thereof, microcrystalline cellulose, gelatin, stearates, silicon dioxide, talc, sodium starch glycolate, acacia, flavoring agents, preservatives, buffering agents, disintegrants, and colorants. Orally administered pharmaceutical compositions may contain one or more optional agents such as, but not limited to, sweetening agents such as fructose, aspartame or saccharin; flavoring agents such as peppermint, oil of wintergreen, or cherry; coloring agents; and preservative agents, to provide a pharmaceutically palatable preparation.
For parenteral administration the antibodies of the disclosure may be combined with a sterile aqueous solution that is isotonic with the blood of the subject. Such a formulation may be prepared by dissolving a solid active ingredient in water containing physiologically-compatible substances, such as sodium chloride, glycine and the like, and having a buffered pH compatible with physiological conditions, so as to produce an aqueous solution, then rendering said solution sterile. The formulation may be presented in unit dose form, such as sealed ampules or vials. The formulation may be delivered by any mode of injection, including, without limitation, epifascial, intracapsular, intracranial, intracutaneous, intrathecal, intramuscular, intraorbital, intraperitoneal, intraspinal, intrasternal, intravascular, intravenous, inhalation, intranasal, parenchymatous, subcutaneous, or sublingual or by way of catheter into the subject's body. A preferred mode of administration is intravenous, intramuscular, or intranasal.
Parenteral administration includes aqueous and non-aqueous based solutions. Examples of which include, for example, water, saline, aqueous sugar or sugar alcohol solutions, alcoholic (such as ethyl alcohol, isopropanol, glycols), ethers, oils, glycerides, fatty acids, and fatty acid esters. In some embodiments, water is used for parenteral administration. In some embodiments, saline is used for parenteral administration. Oils for parenteral injection include animal, vegetable, synthetic or petroleum based oils. Examples of sugars for solution include sucrose, lactose, dextrose, mannose, and the like. Examples of oils include mineral oil, petrolatum, soybean, corn, cottonseed, peanut, and the like. Examples of fatty acids and esters include oleic acid, myristic acid, stearic acid, isostearic acid, and esters thereof. In some embodiments, water is the excipient and/or carrier when the antibody of the disclosure is administered intravenously. In some embodiments, the excipient and/or carrier is a saline solution when the antibody of the disclosure is administered intravenously. In some embodiments, the excipient and/or carrier is a lactated Ringer’s solution when the antibody of the disclosure is administered intravenously. Aqueous dextrose and glycerol solutions may also be employed as an excipient and/or carrier when the antibody of the disclosure is administered intravenously.
The antibodies of the disclosure can be formulated into aerosol formulations to be administered via the respiratory tract (for example, pulmonary or nasal administration). These aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, and nitrogen. Such aerosol formulations may be administered by metered dose inhalers. They also may be formulated as pharmaceuticals for non-pressured preparations, such as in a nebulizer or an atomizer.
The antibodies of the disclosure, alone or in combination with suitable excipients and/or carriers, may be administered in an aqueous solution as a nasal or pulmonary spray and may be dispensed in spray form by a variety of methods known to those skilled in the art. The formulations may be presented in multi-dose containers, for example in the sealed dispensing system disclosed in U.S. Pat. No. 4,511,069. Additional aerosol delivery forms may include, e.g., compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, which deliver the active agent dissolved or suspended in a pharmaceutical solvent, e.g., water, ethanol, or a mixture thereof.
Nasal and pulmonary solutions of the disclosure typically comprise the drug or drug to be delivered, optionally formulated with a surface-active agent, such as a nonionic surfactant (e.g., polysorbate-80), and one or more buffers. In some embodiments of the present invention, the nasal spray solution further comprises a propellant. The pH of the nasal spray solution is optionally between about pH 3.0 and 6.0, preferably 4.5+-0.5. Suitable buffers for use within these compositions are as described above or as otherwise known in the art. Other components may be added to enhance or maintain chemical stability, including preservatives, surfactants, dispersants, or gases. Suitable preservatives include, but are not limited to, phenol, methyl paraben, paraben, m-cresol, thimerosal, chlorobutanol, benzylalkonimum chloride, and the like. Suitable surfactants include, but are not limited to, oleic acid, sorbitan trioleate, polysorbates, lecithin, phosphatidyl cholines, and various long chain diglycerides and phospholipids. Suitable dispersants include, but are not limited to, ethylenediaminetetraacetic acid, and the like. Suitable gases include, but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs), hydrofluorocarbons (HFCs), carbon dioxide, air, and the like.
Within alternate embodiments, nasal and pulmonary formulations are administered as dry powder formulations comprising the active agent in a dry, usually lyophilized, form of an appropriate particle size, or within an appropriate particle size range, for intranasal delivery. Minimum particle size appropriate for deposition within the nasal or pulmonary passages is often about 0.5 pm. mass median equivalent aerodynamic diameter (MMEAD), commonly about 1 pm MMEAD, and more typically about 2 pm MMEAD. Maximum particle size appropriate for deposition within the nasal passages is often about 10 pm MMEAD, commonly about 8 pm MMEAD, and more typically about 4 pm MMEAD. Intranasally and pulmonary respirable powders within these size ranges can be produced by a variety of conventional techniques, such as jet milling, spray drying, solvent precipitation, supercritical fluid condensation, and the like. These dry powders of appropriate MMEAD can be administered to a patient via a conventional dry powder inhaler (DPI), which relies on the patient's breath, upon pulmonary or nasal inhalation, to disperse the power into an aerosolized amount. Alternatively, the dry powder may be administered via air-assisted devices that use an external power source to disperse the powder into an aerosolized amount, e.g., a piston pump.
To formulate compositions for nasal or pulmonary delivery, the active agent can be combined with various pharmaceutically acceptable additives, as well as a base or carrier for dispersion of the active agent(s). Desired additives include, but are not limited to, pH control agents, such as arginine, sodium hydroxide, glycine, hydrochloric acid, citric acid, etc. In addition, local anesthetics (e.g, benzyl alcohol), isotonizing agents e.g., sodium chloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80), solubility enhancing agents (e.g., cyclodextrins and derivatives thereof), stabilizers (e.g., serum albumin), and reducing agents (e.g., glutathione) can be included. When the composition for nasal or pulmonary delivery is a liquid, the tonicity of the formulation, as measured with reference to the tonicity of 0.9% (w/v) physiological saline solution taken as unity, is typically adjusted to a value at which no substantial, irreversible tissue damage will be induced in the nasal mucosa at the site of administration. Generally, the tonicity of the solution is adjusted to a value of about 1/3 to 3, more typically 1/2 to 2, and most often 3/4 to 1.7.
The antibodies of the disclosure may be dispersed in a base or vehicle, which may comprise a hydrophilic compound having a capacity to disperse the active agent and any desired additives. The base may be selected from a wide range of suitable carriers, including but not limited to, copolymers of polycarboxylic acids or salts thereof, carboxylic anhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl (meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such as polyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulose derivatives such as hydroxymethylcellulose, hydroxypropylcellulose, etc., and natural polymers such as chitosan, collagen, sodium alginate, gelatin, hyaluronic acid, and nontoxic metal salts thereof. Often, a biodegradable polymer is selected as a base or carrier, for example, polylactic acid, poly(lactic acid-glycolic acid) copolymer, polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid) copolymer and mixtures thereof. Alternatively or additionally, synthetic fatty acid esters such as polyglycerin fatty acid esters, sucrose fatty acid esters, etc. can be employed as carriers. Hydrophilic polymers and other carriers can be used alone or in combination, and enhanced structural integrity can be imparted to the carrier by partial crystallization, ionic bonding, crosslinking and the like. The carrier can be provided in a variety of forms, including, fluid or viscous solutions, gels, pastes, powders, microspheres and films for direct application to the nasal mucosa. The use of a selected carrier in this context may result in promotion of absorption of the active agent.
In some embodiments, the antibodies of the disclosure are in unit dose form such as a tablet, capsule, infusion bag for intravenous administration, or single-dose vial. Suitable unit dose forms may contain and effective amount (including specific examples of an effective amount described herein), The effective amount may be determined and/or modified during clinical trials designed appropriately for each of the conditions for which administration of an antibody of the disclosure is indicated and will, of course, vary depending on the desired clinical endpoint.
The disclosure also provides articles of manufacture for treating and preventing disorders, such as a SARS-CoV-2 infection, in a subject. The articles of manufacture comprise an antibody of the disclosure or a pharmaceutical composition comprising an antibody of the disclosure, optionally further containing at least one additional antiviral compound, as described herein. The articles of manufacture may be packaged with indications for various disorders that the pharmaceutical compositions are capable of treating and/or preventing. For example, the articles of manufacture may comprise a unit dose of an antibody of the disclosure that is capable of treating or preventing a certain disorder, and an indication that the unit dose is capable of treating or preventing a certain disorder, for example a SARS-CoV-2 infection.
Detection and Diagnosis
The antibodies of the disclosure may be used for detection and diagnosis of SARS-CoV-2. Detection may occur by any known means in the art, for example, by immunoassay, including ELISA. Other immunoassays include, but are not limited to, enzyme immune assays (EIA), ELISPOT (enzyme-linked immunospot), radioimmunoassays (RIAs), immunofluorescence, and other assays known in the art, including but not limited to Western Blot analysis and/or immunoprecipitation methods.
For example in an ELISA (either direct or sandwich), a buffered solution of an antigen or a sample containing an antigen, is added to a well of a microtiter plate. A solution of non-reacting protein is then added to the well to prevent non-specific binding. An antibody of the disclosure, or antigen-binding fragment thereof, is added. Such antibody or antigen binding fragment may be typically conjugated to a reporter molecule, such as, but not limited to, luciferase, horse-radish peroxidase, alkaline phosphatase, or P-D-galactosidase. If the antibody of the disclosure, or antigen-binding fragment thereof, is not conjugated to a reporter molecule, secondary antibody that recognized the antibody of the disclosure may be added that is conjugated to a reporter molecule. A substrate for the reporter molecule is then added, which leads to a detectable signal. ELISAs may be run in a qualitative or quantitative format. Qualitative results provide a simple positive or negative result (yes or no) for a sample. A competitive ELISA may also be used. In this format, an unlabeled antibody of the disclosure, or antigen-binding fragment thereof, (the primary antibody, which may be conjugated to a reporter molecule or unlabeled) is incubated in the presence of a sample containing the antigen, which mixture is then added to a microtiter plate which is coated with the same antigen (the reference antigen) antigen-coated well. The plate is washed so as to remove unbound antibodies. If the primary antibody is not conjugated to a reporter molecule, a secondary antibody conjugated to a reporter molecule that is specific to the primary antibody is added to generate the detectable signal. Depending on the amount of antigen in the sample, more or less primary antibody will be available to bind the reference antigen. This means the more antigen there is in the sample, the less reference antigen will be detected and the weaker the detectable signal. Labeled antigens rather than labeled antibodies may be used. In this format, the less antigen in the sample, the more labeled antigen is retained and the stronger a detectable signal results.
In RIAs, typically a known quantity of an antigen is linked to a radioactive tracer, such as, but not limited to, 1-125, which is then mixed with a known amount of antibody of the disclosure to bind to an antigen (such as SARS-CoV-2 spike protein). Subsequently, a sample containing unknown quantity of the antigen is added and as the concentration of unlabeled antigen is increased, the binding between the antibodies and the labeled standard is decreased, which is directly measurable by measuring radioactivity.
In one aspect, the present disclosure provides a method for the determination (such as an immunoassay determination) of SARS-CoV-2 in a patient, the method comprising: a) incubating a bodily sample from the patient with at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37 and a detectable label, wherein the detectable label is present on the antibody, or antigen binding fragment thereof, or the detectable label is present on a binding partner for either the SARS-CoV-2 or the antibody, or the antigen binding fragment thereof, to form an immunological complex containing the determinable group; and b) determining the presence of the detectable label in the sample, wherein the presence of the detectable label indicates SARS-CoV-2 is present in the sample.
In one embodiment, such method may further comprise isolating the immunological complex from the sample and determining the presence of the detectable label in either in the isolated immunological complex or in the sample remaining.
In certain embodiments, the bodily sample is a serum sample, a blood sample, a plasma sample, a throat swab sample, a nasopharyngeal swab sample, a sputum sample, a fecal sample, a urine sample, a saliva sample, or a bronchoalveolar lavage fluid sample.
Any assay described herein may be used for diagnostic purposes. Therefore, the disclosure provides for methods of diagnosing SARS-CoV-2 infection in a subject using the antibodies of the disclosure. Such a diagnostic use may be directed to determining if a SARS-CoV-2 infection is present, monitoring recovery from a SARS-CoV-2 infection, evaluating the efficacy of a therapeutic treatment for treating a SARS-CoV-2 infection, and for other purposes known in the art.
Kits The disclosure also provides a kit for use in the methods described herein, the kit comprising an antibody of the disclosure, or a pharmaceutically acceptable form thereof, and at least one of the following: (i) at least one other therapeutic agent; (ii) a buffer; (iii) instructions for administering the antibody of the disclosure, or pharmaceutically acceptable form thereof to a subject to treat a SARS-CoV-2 infection in the subject or detect SARS-CoV-2 in a sample (such as a sample from a subject).
In one embodiment of the kits disclosed, the subject is a human. In one embodiment of the kits disclosed, the antibody is an antibody from Table 1 or Tables 2A-H, an antibody of the first to fourth embodiments, or any of the specific aspects of the first to fourth embodiments described above.
The disclosure is further described by the following non-limiting Examples. EXAMPLES
Example 1 : Identification. Purification. Sequencing, and Recombinant Production of SARS-CoV- 2 Specific Antibodies
Peripheral blood samples were obtained from a single individual at the time of or following verified SARS-CoV-2 infection. Peripheral blood mononuclear cells (PBMC) were isolated by density gradient purification.
To obtain SAR-CoV-2 Spike Receptor Binding Domain (RBD)-specific B cells, PBMC were stained with fluorescently conjugated custom generated recombinant RBD tetramer protein in addition to anti-CD19, anti-CD20, anti-IgD-PE, anti-CD3, and anti-CD14 antibodies at 4° C for 60 min similar to as described in Kobie et al. Monoclon Antib Immunodiagn Immunother. 2015 Apr. 1; 34(2): 65-72. Single CD3- CD14- CD19+ RBD+ cells were sorted using a FACSMelody cell sorter (BD Biosciences) directly into 96-well PCR plates (Bio-Rad, Hercules, Calif.) containing 4 pL/well 0.5><PBS with 10 mM DTT (Invitrogen), and 8 U RiboLock (ThermoFisher) RNAse inhibitor. Plates were sealed with MICROSEAL F FILM (Bio-Rad) and immediately frozen at -80° C until used for RT-PCR. cDNA was synthesized and semi-nested RT-PCR for IgH, IgX, and IgK V gene transcripts was performed as described in Kobie et al. IgGl expression vector cloning and transfection of human HEK293T cells (ATCC, Manassas, Va.) were performed as previously described in Kobie et al. Monoclon Antib Immunodiagn Immunother. 2015 Apr. 1; 34(2): 65-72 and Tiller et al., J Immunol Methods. 2008 Jan. 1; 329(1-2): 112-124. PCR products were sequenced at Genewiz Sequences and analyzed by IgBlast and IMGT/V-QUEST to identify germline V(D)J gene segments with highest identity and to determine sequence properties (shown in Table 3). IgG was purified from culture supernatant using MAGNA PROTEIN G beads (Promega, Madison, Wis.).
Table 3 provides a description of the SARS-CoV-2 hmAbs of the disclosure indicating the germline V(D)J for the heavy chain and light chain variable regions for each antibody as well as the original isotype of the antibody.
Table 3
Figure imgf000076_0001
Example 2: The Antibodies of the Disclosure Bind to SARS-CoV-2 Proteins To determine if the antibodies of the disclosure bound to SARS-CoV-2 spike protein, binding experiments were performed. ELISA plates were coated with the following recombinant SARS-CoV-2 polypeptides: i) SARS-CoV-2 receptor binding domain (RBD) polypeptide (SEQ ID NO: 162); ii) SARS-CoV-2 SI domain polypeptide (containing the RBD) (SEQ ID NO: 163); iii) SARS-CoV-2 SI and S2 domain polypeptide (SEQ ID NO: 164); and iv) SARS-CoV-2 nucleoprotein (N; negative control) (SEQ ID NO: 165). The CR3022 antibody (recombinant human anti-SARS-CoV monoclonal antibody that specifically binds to spike protein) served as a positive control while wells incubated with PBS only (no recombinant SARS-CoV-2 polypeptide) also served as a negative control. Recombinant SARS-CoV-2 polypeptides were coated at a concentration of 1 pg/ml and plates were blocked with 3% bovine serum albumin (BSA) in PBS. The antibodies of the disclosure were diluted in PBS and tested at 1 and 10 pg/ml. Antibody binding was detected using horseradish peroxidase-conjugated anti-human IgG. The results are shown in FIG. 1, with the values expressed as area under the curve of the OD reading for both the 1 and 10 pg/ml antibody concentrations.
As can be seen from FIG. 1, the antibodies of the disclosure bound recombinant SARS- CoV-2 Spike protein polypeptides specifically, with no significant binding to the negative control SARS-CoV-2 N polypeptide. FIG. 1 also shows the antibodies of the disclosure showed more favorable binding to the recombinant SARS-CoV-2 RBD polypeptide as compared to the SARS- CoV-2 SI polypeptide and S1+S2 polypeptide. Antibodies 1213H7, 1206D1, 1212D4, 1206D12, 1207F10, and 1206G12 demonstrated 2-fold or greater binding to recombinant SARS-CoV-2 RBD polypeptide as compared to recombinant SARS-CoV-2 SI polypeptide, suggesting that at least a portion of the epitope may be masked in the full length SARS-CoV-2 Spike protein. In contrast, antibodies 1212C2, 1212F2, 1212D5, 1206A5, 1212D6, and 1206D12 showed the least different in binding to recombinant SARS-CoV-2 RBD polypeptide as compared to recombinant SARS- CoV-2 SI polypeptide and S1+S2 polypeptide.
Example 3: The Antibodies of the Disclosure Bind to SARS-CoV-2 Infected Cells
To determine if the antibodies of the disclosure bound to SARS-CoV-2 infected cells, cell binding experiments were performed. Vero E6 cells were infected with SARS-CoV-2 virus (USA- WA1/2020 (Gen Bank: Accession No. MN985325.1) obtained from BEI Resources (NR-52281)) or mock infected and incubated for different times post-infection. Cells were then fixed with 4% neutral formaldehyde for 30 minutes followed by permeabilization using 0.5% triton X-100 for 15 minutes. Cells were then blocked with 2.5% Bovine Serum Albumin (BSA) at 37° C for 1 h. Cells were then incubated with indicated antibodies (1 pg/ml) at 37° C for 1 h, followed by incubation with FITC-conjugated anti-human IgG secondary Ab. Images of FITC Ab was taken using fluorescent microscope. The results are shown in FIG. 2. All antibodies tested bound to SARS-CoV-2 infected cells, with no significant binding to the mock infected cells.
Example 4: The Antibodies of the Disclosure Neutralized SARS-CoV-2 Virus by SARS-CoV-2 Antibodies
The ability of the antibodies of the disclosure to neutralize SARS-CoV-2 virus was analyzed using a plaque reduction microneutralization (PRMNT) assay. Since serial dilutions of the Ab containing samples are used, the PRMNT assay measures the neutralizing activity of Abs in a concentration dependent manner. The ability of a neutralizing antibody to inhibit virus infection is manifested in the reduced capacity of the virus to produce visible plaques when compared to virus-only infected control cells. The PRMNT assay is similar to a plaque reduction neutralization assay with the only difference that the PRMNT utilizes 96-well plates.
The ability of the antibodies to neutralize SARS-CoV-2 virus was determined using two different approaches, a pre-treatment approach and a post-treatment approach. In the pre-treatment experiments, antibodies of the disclosure are pre-incubated with SARS-CoV-2 at 37°C for Ih. This enables the antibodies to bind to the Spike protein blocking virus attachment, and subsequently interfering with the process of virus entry. In the post-treatment experiments, virus adsorption is allowed to progress for Ih at 37°C. This gives the virus an opportunity to initiate viral entry by binding to the cell surface receptor (ACE2). Antibodies incubated with the infected cells may interfere with active viral replication partly by blocking later steps of the virus entry into the cell and/or by inhibiting the cell-to-cell spread of virus progeny. In both pre- and post-treatment experiments, the extent of virus neutralization can be visualized upon staining with a cross-reactive anti-SARS-CoV-1 antibody, and the neutralization titer 50 (NT50) is calculated using a sigmoidal dose-response curve. The NT50 of an antibody is the dilution which inhibited viral replication in 50% of the infected cells.
While the pre-treatment PRMNT assay can be described, to some extent, as a prophylactic assessment of an antibody activity, the post-treatment PRMNT assay offers a therapeutic evaluation of the neutralizing activity of Abs against SARS-CoV-2. In addition, while the pretreatment PRMNT assay focuses on identifying antibodies targeting the SARS-CoV-2 Spike protein RBD, the post-treatment PRMNT assay allows the identification of antibodies targeting other regions on the viral Spike protein involved in the proteolytic cleavage step (for example, the SI region), the fusion event (for example, the S2 region), or antibodies affecting other steps in the replication cycle of the virus (for example, virus assembly and/or budding). In general, a potent SARS-CoV-2 neutralizing antibody will show a low NT50 titer whereas a weak neutralizing antibody will give a high NT50 value.
For the pre-treatment experiments, 100 PFU/well of SARS-CoV-2 (USA-WA1/2020 from BEI Resources (NR-52281)) was mixed with decreasing concentrations of the antibodies of the disclosure (2-folds dilutions, starting concentration 25,000 ng/well for antibodies of the disclosure in 100 pl of infection media (DMEM with 1% penicillin-streptomycin-L-glutamate) and incubated at 37° C, 5% CO2 for Ih in infection media. Vero HL cells (96-well plate format, 4 x 104 cells/well, quadruplicate) were infected with virus-antibody mixture and incubated (virus adsorption) for Ih. After Ih incubation, the media was replaced with post-infection media (DMEM containing 2% FBS and 1% Avicel).
For the post-treatment experiments, Vero HL cells (96-well plate format, 4 x 104 cells/well, quadruplicate) in infection media were infected with 100 PFU/well of SARS-CoV-2 (USA- WA1/2020 from BEI Resources (NR-52281))). After 1 h of viral adsorption, the infection media was changed with the 100 pl of post-infection media containing decreasing concentrations of the antibodies of the disclosure (2-folds dilutions, starting 50,000 or 25,000 ng/ml for antibodies of the disclosure.
At 24 h post infection, infected cells (both pre-treatment and post-treatment) were fixed with 10% neutral formalin for 24 h and were immunostained using anti-NP (nucleoprotein) monoclonal 1C7C7 antibody. Virus neutralization was evaluated and quantified using ELISPOT, and the percentage of infectivity calculated using sigmoidal dose response curves. Mock-infected cells and viruses in the absence of the antibodies of the disclosure were used as internal controls.
The results are shown in Table 4 below. The results show the antibodies of the disclosure are neutralizing SARS-CoV-2 antibodies. Nine of the antibodies tested displayed similar activity in the pre-treatment and post-treatment conditions (with NT50 values for the pre-treatment and post-treatment conditions being within 2-fold of one another). Eight of the antibodies tested displayed different activity in the pre-treatment and post-treatment conditions (with NT50 values for the pre-treatment and post-treatment conditions being greater 2-fold of one another).
Table 4
Figure imgf000079_0001
Figure imgf000080_0001
Of note, antibody 1212C2 exhibited potent neutralizing activity in both the pre-treatment and post-treatment conditions, with NT50 values <50 ng/ml in each assay. Antibody 1212C2 exhibited strong binding to recombinant SARS-CoV-2 RBD polypeptide (FIG. 1). Similarly, antibodies 1212F5 and 1213H7 exhibited potent neutralizing activity in both the pre-treatment and post-treatment conditions, with NT 50 values <100 ng/ml in each assay.
Example 5: High affinity and distinct binding profiles of SARS-CoV-2 antibodies to the SARS- CoV-2 Receptor Binding Domain
To ascertain the affinity of the hmAbs for SARS-CoV-2 Spike RBD surface plasmon resonance (SPR) was performed. SPR experiments were performed on a Biacore T200 (Cytiva) at 25°C using a running buffer consisting of lOmM HEPES, 150mM NaCl, 0.0075% P20, and 100 pg/mL BSA. For affinity measurements, antibodies were captured onto CM-5 sensor chips using the human IgG capture kit (Cytiva). All SPR affinity experiments were double referenced (e.g., sensorgram data was subtracted from a control surface and from a buffer blank injection). The control surface for all experiments consisted of the capture antibody. Approximately 100RU of each hmAb was captured onto the chip surface. RBD was injected over the NmAbs at four concentrations (80nM, 20nM, 5nM, and 1.25nM) for 90 s, followed by 300 s dissociation phase. After each injection, the surface was regenerated with a 30 s pulse of 3M MgC12, followed by capture of fresh hmAb. The buffer flowrate for affinity analysis was 50 pL/min. Sensorgrams were globally fit to a 1 :1 model, without a bulk index correction, using Biacore T-200 evaluation software version 1.0. Standard errors for the rate constants were obtained from the fitted sensorgram data. Replicate KD errors were estimated by repeating the experiments for four of NmAb-RBD interactions (1212C2, 1206D1, 1215D1 and CB6/JS016). Standard deviations from these experiments were used to define an average KD error of 11%.
As shown in Figure 3 and in Table 5 below, this analysis demonstrated a variety of high- affinity hmAbs exhibiting equilibrium dissociation constants (KD) ranging from 71 pM to ~10 nM. Several of the hmAbs (1212C2, 1215D1, 1215D5) exhibited ~8-fold or higher affinity to RBD than control hmAbs (REGN10987, REGN10933,20 and CB6/JS01617), predominantly due to their slower off rates (kd). These results indicate that this panel of hmAbs recognizes SARS- CoV-2 RBD specifically, with unique characteristics and with high affinity.
Table 5
Figure imgf000081_0001
Example 6: Broad and distinct neutralization of SARS-CoV-2 variants by SARS-CoV-2 antibodies SARS-CoV-2 has undergone substantial mutation, including within the Spike protein with such mutations impacting the ability of antibodies to recognize these emerging variants. Variants of Concern (VoC) are particularly threatening to human health and have driven multiple waves of the SARS-CoV-2 pandemic. The defining mutations in spike for the VoC are as follows as reported by the US Center for Diseases Control and Prevention and Nexstrain. Beta: D80A, D215G, del241-243, K417N, E484K, N501Y, D614G, A701V. Delta: T19R, dell 56-157 R158G, L452R, T478K, D614G, P681R, D950N. Gamma: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F. Epsilon: S13I, W152C, L452R, D614G.
The in vitro neutralization activity of the SARS-CoV-2 antibodies against the wild-type SARS-CoV-2 USA-WA1/2020 virus, Variants of Concern (WHO Label) Beta, Gamma, Delta, and Epsilon, and recombinant viruses containing single or triple mutations in the RBD was tested. hmAbs were tested for neutralization of SARS-CoV2 (isolate USA-WA1/2020, BEI Resources) as described in Jun-Gyu Park, et al. J Virol Methods. 2021 Jan;287: 113995. 100-200 plaque forming units (PFU)/well of SARS-CoV-2 containing 2-folds dilutions (starting concentration 5 pg/ml) for hmAbs were mixed in 100 pL of media and incubated at 37°C for Ih. Vero E6 cells (96-well plate format, 4 x 104 cells/well, quadruplicate) were infected with virus-hmAb or virusserum (control well) mixture for virus adsorption for Ih, followed by changing media with postinfection media containing 2% FBS, 1% Penicillin-Streptomycin L-glutamine (PSG), and 1% Avicel. At 24 hpi, infected cells were fixed with 10% neutral formalin for 24 h and were immune- stained using anti-NP monoclonal 1C7C7 antibody. Virus neutralization was evaluated and quantified using ELISPOT, and the percentage of infectivity calculated using sigmoidal dose response curves. The formula to calculate percent viral infection for each concentration is given as [(Average # of plaques from each treated wells - average # of plaques from “no virus” wells)/(average # of plaques from “virus only” wells - average # of plaques from “no virus” wells)] x 100. A non-linear regression curve fit analysis over the dilution curve can be performed using GraphPad Prism to calculate NT50. Mock-infected cells and viruses in the absence of hmAb were used as internal controls.
The SARS-CoV-2 antibodies demonstrate unique neutralization profiles against the SARS- CoV-2 variants. 1212C2 demonstrates potent neutralization with NT50 <50 ng/ml for WAI, Delta, Epsilon, and rK417. Neutralization by 1212C2 is somewhat diminished for rN501 and is not evident for Beta, Gamma, rE484K, or the rK417N E484K N501Y triple mutation variant. This pattern is distinct from CB6/JS016, RGN10933, and RGN10987. 1213H7 is able to neutralize all the viruses tested which is in contrast to CB6/JS016 and RGN10933. 1213H7 has superior neutralizing activity against WAI and the rK417N E484K N501Y triple mutation variant compared to RGN1087.
Table 6
Figure imgf000083_0001
Table 6 above shows in vitro neutralization of indicated live SARS-CoV-2 Variants of Concern by indicated hmAb. Example 7: Improved neutralization of SARS-CoV-2 by modifying 1212C2 heavy chain variable residues
In an effort to improve the activity of the SARS-CoV-2 antibody 1212C2, a series of mAbs were synthesized wherein amino acids in the variable region of the heavy chain were altered (Fig. 4A). Particularly amino acids within and in close proximity to complementarity determining regions were modified. These 1212C2 variant mAbs were tested for neutralization activity.
1212C2-V1 (Fig. 4C) was created by introducing the I28T, I34M, H93Y, T96A, S114Y changes in to 1212C2 and resulted in 33% increase in neutralization potency. 1212C2-V2 (Fig. 4D) was created by introducing the Q1E, Q6E, I28T, D31A, I34L, H93Y, T96A, SI 14Y and resulted in at least a 44% increase in neutralization potency. These results indicate modifications of 1212C2 (Fig. 4A) that can improve its activity against SARS-CoV-2.
Example 8: SARS-CoV-2 antibodies have prophylactic and therapeutic activity in hamsters
The in vivo activity of SARS-CoV-2 antibodies 1212C2 and 1206D1 was evaluated in the golden Syrian hamster model of SARS-CoV-2 infection. To test for prophylactic activity, 10 mg/kg hmAb was administered by intraperitoneal (i.p.) injection 6 h before intranasal (i.n.) challenge with SARS-CoV-2. At 2 days post-infection (dpi), all PBS control and isotype control hamsters had detectable live virus as measured by plaque assay in their nasal turbinates and lungs. In contrast, at 2 dpi, hamsters that received 1212C2 already started to exhibit meaningful viral load reduction in their nasal turbinate and lungs (Fig. 5A). At 4 dpi, virus was detected in the nasal turbinates and lungs of all of the hamsters in the PBS and isotype control groups, although an overall decrease compared to 2 dpi, consistent with the viral dynamics of SARS-CoV-2 infection in hamsters. In comparison to the control groups at 4 dpi, prophylactically treated 1212C2 hamsters exhibited the eradication of viral loads in the nasal turbinates and lungs in 3 of 4 animals. Consistent with the viral load reduction, 1212C2 treated animals exhibited significantly less lung pathology compared to the PBS-treated group at 2 dpi (p = 0.0334) and 4 dpi (p = 0.0004), with this reduction also reaching significance compared to the isotype control group at 4 dpi (p < 0.0001) (Fig. 5C). There was -80% reduction in lung pathology at 4 dpi when 1212C2 was given prophylactically. 1206D1 hmAb exhibited modest activity, noted by 50% of hamsters having no detectable virus in the nasal turbinates and all 1206D1 hamsters having detectable virus in the lungs, although trending to lower titers compared to PBS and isotype control groups. These in vivo results are consistent with the lower in vitro viral neutralization activity of 1206D1 in comparison to 1212C2. Overall, 1212C2 demonstrated substantial prophylactic activity as seen in sterilizing protection in 63% of the hamsters.
Therapeutic activity was tested by treatment with 25 mg/kg of 1212C2 6 h following i.n. infection of hamsters with SARS-CoV-2 and evaluating viral burden at 4 dpi only due to the limited availability of hmAb. SARS-CoV-2 was detected in the nasal turbinates in 3 of 4 animals in the PBS and isotype control groups. No virus was detected in the nasal turbinates of the 1212C2 treated hamsters. SARS-CoV-2 was detected in the lungs of all PBS and isotype control treated hamsters, but only in 1 of 4 of the 1212C2 treated hamsters (Fig. 5B). Overall, 1212C2 demonstrated substantial therapeutic activity, reducing virus to undetectable levels in 75% of the treated hamsters. SARS-CoV-2 infection in hamsters results in gross lung lesions, including consolidation, congestion, and atelectasis. Therapeutically, significantly less lung pathology was also observed in the 1212C2 treated hamster group compared to the PBS-treated group (p = 0.0136), and ~58% reduction in lung pathology in the 1212C2 group compared to the control hamsters (Fig. 5D). Importantly, these differences observed in lung lesions are consistent with the viral burden seen in the upper and lower respiratory tract (A+B). Overall, these results demonstrate the ability of 1212C2 to substantially reduce the viral burden and lung pathology of SARS-CoV- 2 infection when used either prophylactically or therapeutically.
Example 9: Delivery of inhaled SARS-CoV-2 antibody
Therapeutic mAbs are typically delivered systemically through intravenous infusion or intramuscular injection. Such delivery typically results in <0.1% of the injected dose in the lung epithelial lining fluid at Cmax, thus highly inefficient in achieving optimal concentrations in the respiratory tract for the protection or treatment of respiratory infections, such as SARS-CoV-2.
To evaluate the efficiency of lung-delivered dosing and clearance of an IgGl hmAb when delivered as an i.p. bolus dose as compared to an inhaled nebulized aerosol, hamsters were administered the non-specific IgG used in the challenge studies and the serum and bronchoalveolar lavage (BAL) concentrations were analyzed at 30 min and 42 h post-dosing. Intraperitoneal administration of ~25 mg/kg hmAb resulted in high serum concentrations but little to no detectable hmAb in the BAL at 30 min post-treatment. However, 42 h later, the hmAb was detectable in the BAL, suggesting that i.p. -injected hmAb gradually penetrates the lungs from the serum compartment. Inhalation (IH) administration of the hmAb using liquid aerosols delivered whole body to the animals from a commercially available nebulizer (Aerogen Aeroneb Solo nebulizer) showed that ~1.7% of the inhaled dose was deposited in the BAL. The low percentage of the inhaled dose that is deposited in the lungs of hamsters is in line with prior lung uptake studies for the ~4-pm diameter droplets produced by the nebulizer used here (volume mean diameter measured by laser diffraction 4.1 pm), and is a result of a higher degree of inertial impaction of liquid aerosols in the upper airways of small animals. Nevertheless, these lung-deposited (BAL) doses are still substantially higher at both time points than the BAL concentration achieved with the i.p. route. A higher lung-deposited dose afforded by the inhaled route demonstrates higher delivery efficiency to the lungs than the i.p. route. At 42 h post-inhaled dose, most of the mAb is cleared from the BAL (-85% <30 min post-IH), which appears to be faster clearance rate than an expected lung half-life of -8 days reported in other studies.
Table 7
Figure imgf000086_0001
Example 10: Inhaled delivery of 1212C2 provides more efficient therapeutic activity against SARS-CoV-2 infection in hamsters as compared to intraperitoneal delivery
The crystallizable fragment (Fc) of 1212C2 was modified with the LALA mutation (z.e., a double mutation of Leu234Ala together with Leu235Ala) to reduce Fc receptor (FcR) binding and subsequent Fc-mediated effector functions, and further modified to increase half-life (referred to as 1212C2-HLE-LALA with the mutations M252Y/S254T/T256E (YTE)). To determine the efficacy of inhaled 1212C2 and evaluate the therapeutic dose-response, hamsters were infected i.n. with 2 x 105 plaque-forming units (PFU) SARS-CoV-2 and treated with a single dose of 1212C2 hmAb or isotype control hmAb 12 h later using inhaled (IH) or i.p. routes. As measures of efficacy, body weight was recorded, pulmonary lung lesions were measured, and viral titers quantitated from nasal turbinates and lungs on days 2 and 4 following infection. At 2 dpi, the virus was detected in the nasal turbinates and lungs of all infected control hamsters (PBS and isotype control hmAb) (Fig. 6A). Virus was not detectable in the lungs of any hamsters treated via IH with 1212C2-HLE-LALA at 16.3 mg/kg, and virus was lower in their nasal turbinates compared to controls. In contrast, virus was detectable at 2 dpi in the lungs of 50% of the hamsters treated i.p. with 1212C2-HLE-LALA at 25 mg/kg, and not significantly decreased in nasal turbinates compared to controls. At 2 dpi, virus was only detectable in the lungs of 25% and 50% of the hamsters treated via IH with lung-delivered doses of 3.2 mg/kg and 0.6 mg/kg 1212C2-HLE- LALA, respectively. At 2 dpi, 16.3 and 3.2 mg/kg inhaled 1212C2-HLE-LALA was superior in reducing viral titer in the lungs compared to i.p. administration. At 4 dpi, virus was detected in the nasal turbinates and lungs of all infected control hamsters (saline and isotype hmAb), with a trend toward modest non-specific reduction in viral titer in nasal turbinates only with isotype control hmAb (Fig. 6B). Viral titers were only sporadically detected in the nasal turbinates of the 1212C2 treated groups, with the exception of detectable virus in all of the 0.6 mg/kg inhaled 1212C2-HLE- LALA group. At 4 dpi, there was no detectable viral titer in the lungs of any 1212C2 treated hamsters, including the 0.6 mg/kg IH 1212C2-HLE-LALA group, representing at least a 2-log reduction compared to the control groups. Control PBS-treated hamsters exhibited an ~10% maximum weight loss; the 1212C2 treated animals did not have appreciable weight loss. Lung pathology was evident at 4 dpi, particularly in the control groups; however, overall, relative to non-infected hamsters, no lung pathology was evident in 9/24 (37.5%) of the 1212C2 treated hamsters, compared with lung pathology evident in 11/12 (91.7%) of the control treated infected hamsters (Fig. 6C). At 4 dpi, lung lesions were significantly decreased in hamsters that were treated with 3.2 mg/kg (p = 0.0028) of inhaled 1212C2 hmAb compared to PBS-treated hamsters. With regard to effector function removal brought about by the addition of the LALA mutation, animals treated with 1212C2-HLE-LALA appeared to achieve comparable viral reduction as the unmodified 1212C2 mAb. Overall, these results confirm the therapeutic activity of 1212C2 hmAb against SARS-CoV-2 infection and suggest the increased efficacy of inhaled 1212C2 hmAb, with the elimination of viral lung burden with a single inhaled dose of only 0.6 mg/kg. Further, inhaled therapeutic treatment of SARS-CoV-2 infected hamsters was shown to preserve body weight (Fig. 7).
Example 11 : SARS-CoV-2 antibodies prevent SARS-CoV-2 in mice
The efficacy of 1212C2 mAb was tested in K18 human angiotensin converting enzyme 2 (hACE2) transgenic mice. KI 8 hACE2 transgenic mice were prophylactically treated with 1212C2, and then 12 h later infected them (105 PFU) with recombinant SARS-CoV-2 that expresses Nano luciferase (rSARS-CoV-2/Nluc-2A). Non-invasive longitudinal imaging of the mice revealed that 1212C2 treated mice dramatically restricted rSARS-CoV-2/Nluc-2A multiplication since no significant Nine signal was detected at any time point examined (Figs. 8A and 8B). These results were also supported by the observation of reduced lung surface lesions (Figs. 8C and 8D), Nluc expression (Fig. 8E) and virus titers (Fig. 8F) in the nasal turbinate, lungs, and brains of 1212C2-treated mice. Moreover, 1212C2 was able to protect mice from clinical symptoms of rSARS-CoV-2/Nluc-2A infection as determined by changes in body weight (Fig. 8G) and survival rate (Fig. 8H).
Example 12: SARS-CoV-2 antibodies prevent SARS-CoV-2 variant infection in mice
Also assessed was the ability of 1212C2 and 1213H7 mAbs to neutralize rSARS-CoV-2 wild-type virus expressing Venus flourescent protein and rSARS-CoV-2 engineered with the RBD mutations of the Beta varaint and expressing mCherry flourscent protein, alone or in combination, in the K18 hACE2 transgenic mouse model of SARS-CoV-2 infection. Mice were treated intraperitoneally (i.p.) with 25 mg/kg of 1212C2, 1213H7, or an IgG isotype control 24 h prior to challenge with 104 PFU of rSARS-CoV-2 Venus, rSARS-CoV-2 mCherry Beta, or both rSARS- CoV-2 Venus and rSARS-CoV-2 mCherry Beta together. Body weight (Fig. 9A) and survival (Fig. 9B) were evaluated for 12 days post-infection (pi). IgG isotype control-treated mice infected with rSARS-CoV-2 Venus, rSARS-CoV-2 mCherry Beta, or both rSARS-CoV-2 Venus and rSARS- CoV-2 mCherry Beta together, exhibited weight loss starting on day 4 pi (Fig. 9A) and succumbed to viral infection between days 6 to 8 pi (Fig. 9B). However, all mice treated with 1212C2 or 1213H7 survived challenge with rSARS-CoV-2 Venus, consistent with efficient neutralization of SARS- CoV-2 WA-1 in vitro by these two mAbs. In contrast, only 1213H7, but not 1212C2, was able to protect mice infected with rSARS-CoV-2 mCherry Beta (Fig. 9A and Fig. 9B), consistent with the inability of 1212C2 to neutralize SARS-CoV-2 Beta in vitro. When mice were co-infected with both rSARS-CoV-2 Venus and rSARS-CoV-2 mCherry Beta, only mice treated with 1213H7 retained their initial body weight and survived infection (Fig. 9A and Fig. 9B), similar to results obtained using individual infections.
1212C2 and 12137H7 similarly limited lung, nasal, and brain viral burden consistent with their activity against SARS-CoV-2 wild-type, or wild-type and Beta, respectively. As shown in Figure 10A and 10B, six-to-eight- week-old female KI 8 hACE2 transgenic mice (n=3) were injected (i.p.) with 25 mg/kg of an IgG isotype control, mAb 1212C2, or mAb 1213H7 and infected with 104 PFU of rSARS-CoV-2 Venus (top), rSARS-CoV-2 mCherry SA (middle), or both, rSARS-CoV-2 Venus and rSARS-CoV-2 mCherry SA (Beta) (bottom). At days 2 and 4 pi, lungs were collected to determine Venus and mCherry fluorescence expression using an Ami HT imaging system (Fig. 10A). BF, bright field. Venus and mCherry radiance values were quantified based on the mean values for the regions of interest in mouse lungs (Fig. 10B). Mean values were normalized to the autofluorescence in mock-infected mice at each time point and were used to calculate fold induction. Gross pathological scores in the lungs of mock-infected and rSARS-CoV- 2-infected KI 8 hACE2 transgenic mice were calculated based on the % area of the lungs affected by infection.
Six-to-eight-week-old female KI 8 hACE2 transgenic mice (n=3) injected (i.p.) with 25 mg/kg of an IgG isotype control, mAb 1212C2, or mAb 1213H7, and infected with 104 PFU of rSARS-CoV-2 Venus (WT) (Fig. 11 A), rSARS-CoV-2 mCherry SA (Beta) (Fig. 11B), or both rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) (Fig. 11C). Viral titers in the lungs (top), nasal turbinate (middle) and brain (bottom) at days 2 and 4 pi were determined by plaque assay in Vero E6 cells. Bars indicates the mean and SD of lung virus titers. Dotted lines indicate the limit of detection. As shown in Fig. 1 ID, quantification of rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) in the lungs (top), nasal turbinate (middle) and brain (bottom) from mice co-infected with both rSARS-CoV-2 Venus (WT) and rSARS-CoV-2 mCherry SA (Beta) at days 2 and 4 pi.
These results demonstrate that both 1212C2 and 1213H7 are able to prevent SARS-CoV- 2 WT infection and pathology in hACE2 transgenic mice, and further that 1213H7 is also able to prevent SARS-CoV-2 Beta infection.
Example 13:_SARS-CoV-2 antibodies have distinct residue-dependent SARS-CoV-2 Receptor Binding Domain binding
Based on the in silico mAb epitope analysis, nine RBD variants that localize to the Cl (K417T, A475V, N501Y, Y505W), C1D/C2 (G446V, L452R and E484K) and C3 epitopes (T345I, N440Y, and G446V) were produced for binding studies. The structural analysis shown in Figure 12 confirms that the loss of mAb binding to certain SARS-CoV-2 RBD variants can be used to identify mAb classes. Furthermore, the assays can be used to identify mAbs that are not sensitive to RBD variants and thus potentially useful as therapeutics. To address these questions, the unknown mAbs and three controls [CB6, RGN10933 and RGN10987] were tested for their ability to bind to the SARS-CoV-2 RBD variants. The data revealed 6 antibodies (1212C2, 1212D4, 1212F2, 1212F5, 1213H7, and 1215D1) with superior breadth ofbinding to RBD variants for down selection of optimal mAbs for further studies. Notably, 1212C2 and Regeneron 10987 exhibit reduced binding to E484K and G446V mutations, respectively.
Example 14: Classification of SARS-CoV-2 antibodies by competition epitope mapping
Competition-based epitope mapping was performed to aid in determining assignment of the mAbs to the different classes. Overall, there was excellent correlation between the mAb assignments and the epitope analysis. Cl mAbs 1212D5, 1212F2, 1213F2, 1212D4, and 1212C8, allowed simultaneous binding of C2 mAbs 1207B4, 1215D1, and 1207F10. In contrast, the C2 mAb 1212C2 blocked all Cl and C2 mAbs that were assayed (Figs. 13A-13C). To address these observations, structures of the NAb-RBD complexes assigned to the different C2 NAbs were compared (Fig. 13D). Specifically, 1215D1, 1207B4, and 1207F10 mAbs were assigned to the C386-2/RBD complex, while 1212C2 was assigned to the C121/RBD complex. 1212C2 (modeled by C121-RBD) overlaps with Cl (modeled by P2C-1F11/RBD) and C2 NAbs, while 1215D1 (modeled by C386-2/RBD) can simultaneously bind with several Cl (1214D4, 1212C8, 1212D5 etc.) and CID (1213H7) mAbs.
Example 15: SARS-CoV-2 antibody binding dependence on SARS-CoV-2 residues resolved through viral escape
Constant exposure of SARS-CoV-2 to suboptimal levels of mAbs in infected individuals or in vitro can promote the selection of monoclonal antibody resistant mutant (MARM) SARS- CoV-2. Using molecular approaches, MARMs were selected and identified using an in vitro model (Vero E6 cells) of SARS-CoV-2 infection (Fig. 14). Culture of SARS-CoV-2 in the presence of 1212C2, 1213H7, or 1215D1 resulted in the generation of MARM that had developed mutations within the Spike protein, resulting in loss of recognition by the mAb.
The 1212C2 MARM has the E484K mutation in RBD. The 1212C2/RBD model identifies HCDR2 N53 and HCDR3 R103 as residues that interaction with RBD E484 and sterically prevents 1212C2 from binding to RBD E484K, These and additional residues form a pocket optimized for glutamic acid and smaller residues, but occlude lysine and Arginine sidechains at RBD E484.
The 1213H7 MARM has RBD mutations G476D and F490S. Our current model of the 1213H7/RBD complex is of lower resolution than 1212C2 and 1215D1 interactions. Nonetheless, the 1213H7 has been assigned as having a CID epitope that binds on the opposite side of the RBD tip, relative to the 1215D1 epitope. This model is consistent with the competition epitope mapping. F490 sits in the center of the heavy chain binding site of the 1215D1/RBD model, which provides an initial explanation of 1215D 1 ’s sensitivity to the F490S MARM and validates the general location of the 1215D1/RBD model. Hydrophobic packing interactions, contributed from HCDR3 Fi l l is expected to be an important contributor to the 1215D1/RBD interactions. The mutations are described below in Table 8.
Table 8
Figure imgf000091_0001
Example 16: Inhaled Delivery of IgG mAb in Hamsters
The goals of this study were to compare and contrast the efficiency and kinetics of delivery of a human mAb to the lung tissues following IV, IN and IH routes of delivery. Measurements of delivery were determined by quantitating BAL, lung and serum titer that were evaluated for human IgG using a quantitative ELISA assay. The comparative analysis is shown in Fig. 15 and indicate IgG titer in serum, BAL and lung over a 24 hour of evaluation at 30mins, 6hrs, 12hrs and 24hrs following dosing by IV, IN or IH routes of delivery. It should be noted that the concentration of drug use in IV, IN and IH was different for each route and reflect differences in the efficiency of delivery. With IV delivery, serum titers were high (above limit of detection at 30mins) and remained high over 24 hours. This is in contrast to BAL titer after IV delivery, which was low at 30mins and remained at low levels over the 24 hour period. When the mAb was delivered by inhalation, the BAL titer was highest at 30 mins and was maintained at similar levels over the 24 hour period. Of note is that the BAL titer following inhalation was similar at both concentrations of 4mg/kg and lOmg/kg of mAb tested. Intranasal delivery also resulted in BAL titer that was increased compared to IV delivery. However, although inhalation appeared to be the more efficient route of delivery compared to intranasal route, the dose of mAb used by intranasal delivery was 0.04mg/kg reflecting a 100 fold reduction in dose compared to 4mg/kg and lOmg/kg dose used for inhalation.
These data clearly demonstrate differences in titer and distribution in of the delivered human mAb in BAL and serum at each time point depending on the route of delivery. Differences were clearly observed between BAL and serum. As expected, the titer of delivered mAb to the BAL following inhalation was high from the early time point and was maintained at high levels over the 24 hour period. In contrast, the mAb titer in the BAL was low following IV delivery and increased marginally over the 24hr period. The titer is the lung was more challenging to interpret owing to the presence of both blood and BAL in this tissue. Based on these data, delivery to the BAL was the most efficient, with highest titer and earliest time point observed by the inhalation route of delivery.
Example 17: Neutralization Assay for hmAbs alone or in combination
The 1212C2, 1213H7, and 1215D1 mAbs were assessed both alone and in combination in a neutralizing antibody assay against WAI P7 (1.7x105 pfu/mL). 96-well plates were inoculated with lOOPFU/well (MOI 0.05), except for control wells containing no virus. Wells containing virus were treated with lOOng, 50. Ong, 25ng, 12.5ng, 6.25ng, 3.12ng, 1.6ng, 0.8ng, 0.4ng, 0.2ng or Ong of one or more antibodies for 24 hours in 1% Avicel media and NT50 was assessed pre- and posttreatment. NT50 for 1212C2 was 4.5ng and 3.2ng pre- and post-treatment, respectively. NT50 for 1213H7 was 6.6ng and 4.8ng pre- and post-treatment, respectively. NT 50 for 1215D1 was 22.6ng and 5.9ng pre- and post-treatment, respectively.
Assessed combinations of mAbs included: 1212C2 (Fig. 16A); 1213H7 (Fig. 16B); 1215D1 (Fig. 16C); 1212C2+1213H7 (Fig. 16D); 1213H7+1215D1 (Fig. 16E); 1212C2+1215D1 (Fig. 16F); and 1212C2+1213H7+1215D1 (Fig. 16G).

Claims

What is claimed:
1. At least one isolated antibody, or an antigen-binding fragment thereof, that specifically binds to a spike protein of SARS-CoV-2 selected from the group consisting of: 1212C2, 1212F5, 1213H7, 1212F2, 1206D1, 1212D5, 1207B4, 1213F2, 1212D4, 1206A5, 1212D6, 1206D12, 1207F10, 1206G12, 1212C8, 1212E9, 1214E9, 1215D1 and 1212 variant.
2. The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 38-40, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42-44, respectively (1212C2).
3. The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 46-48, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 50-52, respectively (1215F5).
4. The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 54-56, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 58-60, respectively (1213H7).
5. The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 62-64, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 66-68, respectively (1212F2).
6. The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 70-72, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 74-76, respectively (1206D1). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 78-80, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 82-84, respectively (1212D5). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 86-88, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 90-92, respectively (1207B4). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 94-96, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 98-100, respectively (1213F2). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 102-104, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 106-108, respectively (1212D4). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 110-112, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 114-116, respectively (1206A5). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 118-120, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 122-124, respectively (1212D6). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 126-128, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 130-132, respectively (1206D12). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 134-136, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 138-140, respectively (1207F10). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 142-144, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 146-148, respectively (1206G12). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOS: 158, 39, and 159, respectively, and (ii) a light chain variable region that comprises LCDR1, LCDR2 and LCDR3 comprising the amino acid sequences of SEQ ID NOS: 42, 43, and 161, respectively (1212 variant). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 16, wherein the amino acid at position 1 of SEQ ID NO: 158 is Thr (T) or He (I), the amino acid at position 1 of SEQ ID NO: 159 is Thr (T) or Ala (A), the amino acid at position 9 of SEQ ID NO: 159 is Leu (L) or Phe (F), the amino acid at position 1 of SEQ ID NO: 161 is any amino acid, the amino acid at position 6 of SEQ ID NO: 161 is Asn (N) or Ser (S), and the amino acid at position 10 of SEQ ID NO: 161 is Vai (V) or Phe (F). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 37, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 41, or an amino acid sequence at least 80% homologous thereto (1212C2). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 45, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 49, or an amino acid sequence at least 80% homologous thereto (1215F5). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 53, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 57, or an amino acid sequence at least 80% homologous thereto (1213H7). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 61, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 65, or an amino acid sequence at least 80% homologous thereto (1212F2). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 69, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 73, or an amino acid sequence at least 80% homologous thereto (1206D1). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 77, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 81, or an amino acid sequence at least 80% homologous thereto (1212D5). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 85, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 89, or an amino acid sequence at least 80% homologous thereto (1207B4). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 93, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 97, or an amino acid sequence at least 80% homologous thereto (1213F2). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 101, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 105, or an amino acid sequence at least 80% homologous thereto (1212D4). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 109, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 113, or an amino acid sequence at least 80% homologous thereto (1206A5). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 117, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 121, or an amino acid sequence at least 80% homologous thereto (1212D6). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 125, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 129, or an amino acid sequence at least 80% homologous thereto (1206D12). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 133, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 137, or an amino acid sequence at least 80% homologous thereto (1207F10). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 141, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 145, or an amino acid sequence at least 80% homologous thereto (1206G12). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 149, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 150, or an amino acid sequence at least 80% homologous thereto (1215D1). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 151, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 152, or an amino acid sequence at least 80% homologous thereto (1214E9). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 153, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 154, or an amino acid sequence at least 80% homologous thereto (1212E9). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 155, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 156, or an amino acid sequence at least 80% homologous thereto (1212C8). The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 1, comprising: (i) a heavy chain variable region that comprises the amino acid sequences of SEQ ID NO: 157, or an amino acid sequence at least 80% homologous thereto, and (ii) a light chain variable region that comprises the amino acid sequences of SEQ ID NO: 160, or an amino acid sequence at least 80% homologous thereto (1212 variant).
37. The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 36, wherein the amino acids at positions 13, 23, 28, 59, 65, 77, 79, 94, 97, 105, and 113 of SEQ ID NO: 157 are selected from Table 2A and the amino acids at positions 1, 2, 3, 91, 96, and 100 of SEQ ID NO: 160 are selected from Table 2B.
38. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, wherein the antibody, or antigen binding fragment thereof, is an antibody variant.
39. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, further comprising a variant Fc constant region.
40. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, further comprising a variant Fc constant region, wherein a protein moiety or a non-protein moiety is linked to the Fc constant region.
41. The at least one isolated antibody, or the antigen-binding fragment thereof, of claim 40, wherein the non-protein moiety is a water soluble polymer.
42. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1-37, having a longer half-life in vivo in a subject relative to the parental antibody, decreased immunogenicity in vivo in a subject relative to the parental antibody, or a combination of the foregoing.
43. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, wherein the antibody, or antigen binding fragment thereof, is a chimeric antibody, a class-switched antibody, or a monoclonal antibody.
44. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, wherein the antibody, or antigen binding fragment thereof, is linked to a therapeutic agent, an enzyme, an enzyme inhibitor, or a detectable label.
45. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, wherein the antigen binding fragment is selected from the groups consisting of: a Fab fragment, a F(ab)2 fragment, a Fab' fragment, a Fd fragment, a Fv fragment, a disulfide-linked Fv, a dAb fragment, an isolated CDR, a nanobody, a single domain antibody, a portion of the VH region containing a single variable domain and two constant domains, a diabody, a triabody, a tetrabody, a scFv, a scFv-FC, a scFv-CH, a scFab, and a scFv-zipper.
46. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, wherein the antibody, or antigen binding fragment thereof, binds an epitope within the receptor binding domain of the SARS-CoV-2 spike protein, binds an epitope within the receptor binding motif of the SARS-CoV-2 spike protein, binds an epitope within the SI region of the SARS-CoV-2 spike protein, or binds an epitope within the S2 region of the SARS-CoV-2 spike protein.
47. The at least one isolated antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37, wherein the antibody, or antigen binding fragment thereof, reduces binding of SARS-CoV-2 to a target cell, reduces cellular fusion between SARS-CoV-2 and the target cell, reduces release of infective SARS-CoV-2 from an infected cell, reduces infection of the target cell by SARS-CoV-2, or a combination of the foregoing.
48. An isolated nucleic acid encoding the heavy chain variable region, the light chain variable region, or both the heavy chain variable region and the light chain variable region of the antibody, or antigen binding fragment thereof, of any one of claims 2 to 33.
49. An expression vector comprising the nucleic acid of claim 48.
50. A cultured host cell comprising the expression vector of claim 49.
51. A pharmaceutical composition comprising the antibody, or the antigen-binding fragment thereof, of any one of claims 1 to 37 and at least one of a pharmaceutically acceptable carrier and a pharmaceutically acceptable excipient.
52. The pharmaceutical composition of claim 51 formulated for intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration.
53. A method for treating a SARS-CoV-2 infection in a subject, the method comprising administering to said subject an effective amount of at least one isolated antibody, or antigen-binding fragment thereof, of any one of claims 2 to 37.
54. The method of claim 53, wherein the administering step comprises administering a single dose or a plurality of doses comprising the at least one antibody, or an antigen-binding fragment thereof, according to a course of treatment.
55. The method of claim 53, wherein the administering step occurs before the subject has been infected with SARS-CoV-2, after the subject has been infected with SARS-CoV-2 but before an infection can be detected, or after a subject has been infected with SARS-CoV- 2 and after the infection can be detected.
56. The method of claim 53, wherein the at least one antibody, or an antigen-binding fragment thereof, is administered is by intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration.
57. The method of any one of claims 53 to 56, further comprising administering to the subject an additional therapeutic agent.
58. The method of claim 57, wherein the additional therapeutic agent is a SARS-CoV-2 RNA polymerase inhibitor, a serine protease inhibitor, a cysteine protease inhibitor, or a combination of the foregoing.
59. The method of claim 57, wherein the additional therapeutic agent is selected from the group consisting of: galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscamet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir.
60. A method of reducing cellular entry of SARS-CoV-2 in a subject, the method comprising administering to said subject an effective amount of at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37.
61. The method of claim 60, wherein the administering step comprises administering a single dose or a plurality of doses comprising the at least one antibody, or an antigen-binding fragment thereof, according to a course of treatment.
62. The method of claim 60, wherein the administering step occurs before the subject has been infected with SARS-CoV-2, after the subject has been infected with SARS-CoV-2 but before an infection can be detected, or after a subject has been infected with SARS-CoV- 2 and after the infection can be detected.
63. The method of claim 60, wherein the at least one antibody, or an antigen-binding fragment thereof, is administered is by intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration.
64. The method of any one of claims 60 to 63, further comprising administering to the subject an additional therapeutic agent.
65. The method of claim 64, wherein the additional therapeutic agent is a SARS-CoV-2 RNA polymerase inhibitor, a serine protease inhibitor, a cysteine protease inhibitor, or a combination of the foregoing.
66. The method of claim 64, wherein the additional therapeutic agent is selected from the group consisting of: galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscamet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir.
67. A method of reducing binding of SARS-CoV-2 to a cellular ACE2 in a subject, the method comprising administering to said subject an effective amount of at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37.
68. The method of claim 67, wherein the administering step comprises administering a single dose or a plurality of doses comprising the at least one antibody, or an antigen-binding fragment thereof, according to a course of treatment.
69. The method of claim 67, wherein the administering step occurs before the subject has been infected with SARS-CoV-2, after the subject has been infected with SARS-CoV-2 but before an infection can be detected, or after a subject has been infected with SARS-CoV- 2 and after the infection can be detected.
70. The method of claim 67, wherein the at least one antibody, or an antigen-binding fragment thereof, is administered is by intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration.
71. The method of any one of claims 67 to 70, further comprising administering to the subject an additional therapeutic agent. The method of claim 71, wherein the additional therapeutic agent is a SARS-CoV-2 RNA polymerase inhibitor, a serine protease inhibitor, a cysteine protease inhibitor, or a combination of the foregoing. The method of claim 71 , wherein the additional therapeutic agent is selected from the group consisting of: galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscamet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir. A method for reducing viral titer of a SARS-CoV-2 in a bodily fluid, tissue or cell of a subject, the method comprising administering to said subject an effective amount of at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37. The method of claim 74, wherein the administering step comprises administering a single dose or a plurality of doses comprising the at least one antibody, or an antigen-binding fragment thereof, according to a course of treatment. The method of claim 74, wherein the administering step occurs before the subject has been infected with SARS-CoV-2, after the subject has been infected with SARS-CoV-2 but before an infection can be detected, or after a subject has been infected with SARS-CoV- 2 and after the infection can be detected. The method of claim 74, wherein the at least one antibody, or an antigen-binding fragment thereof, is administered is by intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration. The method of any one of claims 74 to 77, further comprising administering to the subject an additional therapeutic agent. The method of claim 77, wherein the additional therapeutic agent is a SARS-CoV-2 RNA polymerase inhibitor, a serine protease inhibitor, a cysteine protease inhibitor, or a combination of the foregoing.
80. The method of claim 77, wherein the additional therapeutic agent is selected from the group consisting of: galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscamet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir.
81. A method for reducing the transmission of a SARS-CoV-2 infection from a first subject to a second subject, the method comprising administering to said first subject an effective amount of at least one isolated antibody, or an antigen-binding fragment thereof, of any one ofclaims 2 to 37.
82. The method of claim 81, wherein the administering step comprises administering a single dose or a plurality of doses comprising the at least one antibody, or an antigen-binding fragment thereof, according to a course of treatment.
83. The method of claim 81, wherein the administering step occurs before the subject has been infected with SARS-CoV-2, after the subject has been infected with SARS-CoV-2 but before an infection can be detected, or after a subject has been infected with SARS-CoV- 2 and after the infection can be detected.
84. The method of claim 81, wherein the at least one antibody, or an antigen-binding fragment thereof, is administered is by intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration.
85. The method of any one of claims 81 to 84, further comprising administering to the subject an additional therapeutic agent.
86. The method of claim 84, wherein the additional therapeutic agent is a SARS-CoV-2 RNA polymerase inhibitor, a serine protease inhibitor, a cysteine protease inhibitor, or a combination of the foregoing.
87. The method of claim 84, wherein the additional therapeutic agent is selected from the group consisting of: galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin,
102 oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscamet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir.
88. A method for reducing the transmission of a SARS-CoV-2 infection from a first subject to a second subject, the method comprising administering to the second subject an effective amount of at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37.
89. The method of claim 88, wherein the administering step comprises administering a single dose or a plurality of doses comprising the at least one antibody, or an antigen-binding fragment thereof, according to a course of treatment.
90. The method of claim 88, wherein the administering step occurs before the subject has been infected with SARS-CoV-2, after the subject has been infected with SARS-CoV-2 but before an infection can be detected, or after a subject has been infected with SARS-CoV- 2 and after the infection can be detected.
91. The method of claim 88, wherein the at least one antibody, or an antigen-binding fragment thereof, is administered is by intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration.
92. The method of any one of claims 88 to 91, further comprising administering to the subject an additional therapeutic agent.
93. The method of claim 92, wherein the additional therapeutic agent is a SARS-CoV-2 RNA polymerase inhibitor, a serine protease inhibitor, a cysteine protease inhibitor, or a combination of the foregoing.
94. The method of claim 92, wherein the additional therapeutic agent is selected from the group consisting of: galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscamet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir.
103 A method of neutralizing a SARS-CoV-2 in a subject, the method comprising administering to said subject an effective amount of at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37. The method of claim 95, wherein the administering step comprises administering a single dose or a plurality of doses comprising the at least one antibody, or an antigen-binding fragment thereof, according to a course of treatment. The method of claim 95, wherein the administering step occurs before the subject has been infected with SARS-CoV-2, after the subject has been infected with SARS-CoV-2 but before an infection can be detected, or after a subject has been infected with SARS-CoV- 2 and after the infection can be detected. The method of claim 95, wherein the at least one antibody, or an antigen-binding fragment thereof, is administered is by intravenous administration, intramuscular administration, subcutaneous administration, pulmonary administration, or intranasal administration. The method of any one of claims 95 to 98, further comprising administering to the subject an additional therapeutic agent. . The method of claim 99, wherein the additional therapeutic agent is a SARS-CoV- 2 RNA polymerase inhibitor, a serine protease inhibitor, a cysteine protease inhibitor, or a combination of the foregoing. . The method of claim 99, wherein the additional therapeutic agent is selected from the group consisting of galidesivir, remdisivir, hydrochloroquine, chloroquine, irbesartan, toremifene, camphor, equiline, mesalazine, mercaptopurine, nafamostat, paraoxetine, sirolimus, carvedilol, dactinomycin, melatonin, quinacrine, eplerenone, enoclin, oxymethalone, ENU2000, azithromycin, lopinovir/ritonavir, umifenovir, cytovene, ganciclovir, trisodium phosphonoformate, ribavirin, interferon, d4T, ddl, AZT, amantadine, rimantadine, acyclovir, foscamet, laninamivir, oseltamivir, zanamivir, favipiravir, baloxavir marboxil, and peramivir. . A kit for detecting the presence of SARS-CoV-2 in a sample comprising (i) at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37, and at least one of the following: (i) at least one other therapeutic agent; (ii) a buffer; and (iii) instructions for administering the antibody to a subject to treat a SARS-CoV-2 infection in the subject or detect SARS-CoV-2 in a sample.
104
. A method for the immunoassay determination of SARS-CoV-2 in a patient, the method comprising: a. incubating a bodily sample of the patient with at least one isolated antibody, or an antigen-binding fragment thereof, of any one of claims 2 to 37 and a detectable label, wherein the detectable label is present on the antibody, or antigen binding fragment thereof, or the detectable label is present on a binding partner for either the SARS-CoV-2 or the at least one antibody, or the antigen binding fragment thereof, to form an immunological complex containing the determinable group; and b. determining the presence of the detectable label in the sample, wherein the presence of the detectable label indicates SARS-CoV-2 is present in the sample. . The method of claim 103 further comprising isolating the immunological complex from the sample and determining the presence of the detectable label in either in the isolated immunological complex or in the sample remaining. . The method of claim 103 or 104 wherein the bodily sample is a serum sample, a blood sample, a plasma sample, a throat swab sample, a nasopharyngeal swab sample, a sputum sample, a fecal sample, a urine sample, a saliva sample, or a bronchoalveolar lavage fluid sample. . The method of claim 103 or 104 wherein the immunoassay determination is used to determine if SARS-CoV-2 infection is present in the subject, to monitoring recovery of the subject from SARS-CoV-2 infection, or to evaluate the efficacy of a therapeutic treatment for treating a SARS-CoV-2 infection.
105
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