WO2022074621A1 - Covid-19-binding immunoglobulins and methods for their use - Google Patents

Covid-19-binding immunoglobulins and methods for their use Download PDF

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WO2022074621A1
WO2022074621A1 PCT/IB2021/059246 IB2021059246W WO2022074621A1 WO 2022074621 A1 WO2022074621 A1 WO 2022074621A1 IB 2021059246 W IB2021059246 W IB 2021059246W WO 2022074621 A1 WO2022074621 A1 WO 2022074621A1
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antibody
cov
sars
binding
sample
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French (fr)
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WO2022074621A8 (en
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Sachdev Sidhu
Mart Ustav
Shane MIERSCH
Zhijie Li
James Michael RINI
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The Governing Council Of The University Of Toronto
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56983—Viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
    • G01N2333/08—RNA viruses
    • G01N2333/165—Coronaviridae, e.g. avian infectious bronchitis virus
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/10—Detection of antigens from microorganism in sample from host

Definitions

  • SARS-CoV-2 the cause of the ongoing pandemic of Coronavirus Disease 2019 (COVID-19)
  • coronavirus disease 2019 2019 (COVID-19)
  • coronavirus disease 2019 2019 (COVID-19)
  • coronavirus disease 2019 2019 (COVID-19)
  • coronavirus disease 2019 2019 (COVID-19)
  • coronavirus disease 2019 2019 (COVID-19)
  • coronavirus disease 2019 2019 (COVID-19)
  • coronavirus disease 2019 COVID-19
  • coronavirus disease 2019 COVID-19
  • the SARS-CoV-2 genome shows a high level of homology with SARS-like coronavirus isolated from bats (RaTG13 in particular) and a strain isolated from pangolins (99% identity), and the virus may have originated from recombination between CoVs that infect pangolins and bats (Xiao, K., et al. (2020) Isolation of SARSCoV-2-related coronavirus from Malayan pangolins. Nature).
  • SARS-CoV-2 utilizes an extensively glycosylated spike (S) protein that protrudes from the viral membrane, to mediate host-cell entry (Yan, R., et al. (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444-1448.)
  • S protein contains 1,282 amino acids organized into 2 subunits (SI and S2) and forms a homotrimer on the virus surface.
  • the receptor binding domain located in the SI subunit, recognizes the host cell receptor, angiotensin converting enzyme 2 (ACE2), the binding of which facilitates cleavage of the S2 subunit into S2' and S2" by cell-surface proteases, which in turn enables fusion and internalization of the virus (Hoffmann, et al.
  • ACE2 angiotensin converting enzyme 2
  • antibodies and binding fragments thereof that bind to the spike protein of SARS-CoV-2 (also referred to interchangeably herein as COVID-19), and inhibit or compete for ACE2 binding.
  • the antibodies described herein exhibit neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WAl in Vero E6 cells, and also bind to the receptor binding domain (RBD) of SARS-CoV-2.
  • the antibodies and binding fragments described herein may be engineered to reduce or eliminate effector function.
  • An embodiment of this invention is a full-length SARS-CoV-2-binding antibody, (for example, a monoclonal antibody or a chimeric antibody), or a monovalent binding fragment thereof, (for example, a Fab, a F(ab)2, a F(ab’)2, a Fv, a disulfide linked Fv, a scFv, a disulfide linked scFv), and/or a single chain domain antibody, (for example, a VHH, or a scFab), or a diabody (see e.g., Figure 4).
  • a monovalent binding fragment thereof for example, a Fab, a F(ab)2, a F(ab’)2, a Fv, a disulfide linked Fv, a scFv, a disulfide linked scFv
  • a single chain domain antibody for example, a VHH, or a scFab
  • the antibody and binding fragments thereof comprise the heavy chain complementarity determining regions (CDRs) and/or light chain CDRs of an antibody identified as 15031, 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049.
  • CDRs heavy chain complementarity determining regions
  • Table 2 and Table 3 present the amino acid and nucleotide sequences of the heavy chain and light chain complementarity determining regions (CDR1, CDR2 and CDR3) of the antibodies identified as 15031, 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049.
  • the antibodies described herein may be interchangeably referenced throughout this specification and accompanying figures by a 5- digit nomenclature (for example, 15031, 15032, 15033, etc.) and/or may be identified by the last two to four digits (for example, 31, 32, 33-7, 33-A2, etc.).
  • An embodiment of this invention is a method for detecting SARS-CoV-2, a SARS- CoV-2 S polypeptide or fragment thereof in a sample, e.g., a biological sample from a subject, or a sample not from a subject, e.g., wastewater or a body of water receiving wastewater, using an antibody of this invention.
  • An embodiment of this invention is a method for inhibiting SARS-CoV-2 infection of a cell having a receptor for SARS-CoV-2, e.g., ACE2, by contacting the cell with an effective amount of an antibody or binding fragment described herein.
  • a receptor for SARS-CoV-2 e.g., ACE2
  • An embodiment of this invention is a method for inhibiting SARS-COV-2 S protein binding to an ACE2 receptor by administering an antibody, or an antibody binding fragment, of this invention to a subject in need thereof.
  • An embodiment of this invention is a method for treating as subject infected with SARS-COV-2 with an effective amount of an antibody or binding fragments and pharmaceutical compositions comprising the antibodies and/or binding fragments described herein.
  • Another embodiment includes a nucleic acid encoding an antibody and/or binding fragments described herein.
  • a further embodiment is a vector comprising a nucleic acid of this invention.
  • Another embodiment includes a recombinant cell producing an antibody, binding fragments, nucleic acid, or vector of this invention.
  • Another embodiment is an immunoassay comprising or using an antibody and/or binding fragments described herein.
  • the immunoassay is an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • kits comprising an antibody and/or binding fragments described herein and the nucleic acid molecules and/or vectors comprising the nucleic acid molecules that encode the antibodies and/or binding fragments described herein.
  • the kit may include additional reagents, controls, and instructions for how to use the kit.
  • FIG 1A depicts binding of unique Fab-phage clones to immobilized RBD blocked by solution-phase ACE2. Signal was normalized to the signal in the absence of ACE2 (control).
  • FIG IB depicts serial dilutions of IgGs binding to immobilized SARS-CoV-2 S protein. The ECso values for each curve are shown in parentheses (in qg/mL and qM).
  • FIG 1C depicts binding of Fab-phage to immobilized RBD or negative controls, as indicated.
  • FIG ID depicts binding of ACE2 to immobilized S protein the presence (white bars) or absence (black bars) of 100 qM IgG.
  • FIG. 2 Binding kinetics for IgGs and SARS-CoV-2 S protein.
  • Bio-layer interferometry (BLI) was used to evaluate antibody binding kinetics to immobilized S protein over a range of IgG concentrations (67 - 0.8 qM).
  • Evolved sensor signals were fit and values for binding kinetics were extracted, and are shown in the table at the bottom (Table 1), along with ECso values derived from ELISA curves in Figure IB.
  • the antibodies herein have dissociation constant (KD) for the SARS-COV-2 or a SARS-COV-2 S polypeptide of about or less than 0.8 ⁇ 0.1 qM, about or less than 0.4 ⁇ 0.1 qM or about or less than 0.2 ⁇ 0.1 qM.
  • KD dissociation constant
  • FIG. 3 Neutralization of virus by IgGs. Microneutralization assays were performed in St Louis (Top) or Rome (Bottom). IgG-mediated neutralization of virus was assessed in a focal reduction neutralization assay and expressed as % relative infection, and infection curves were fitted to extract IC50 values, which are shown in parentheses (in rg/mL and r
  • Figure 4 depicts various formats of the antibody, and binding fragments of this invention.
  • Black lines linkers.
  • Spheres represent the cysteines of an intermolecular disulphide bond.
  • FIG. 5 Figure 5 - Characterization of IgG 15031.
  • A Expressed and Protein A-purified human IgGl antibody derived from phage clone 15031 was assessed for binding to SARS CoV-2 S-protein by ELISA versus control capture protein (neutravidin) over a range of IgG concentrations to estimate EC50 value (shown inset)
  • B Binding kinetics of the interaction between IgG 15031 and SARS CoV-2 S-protein (left panel) was also assessed by biolayer interferometry to estimate the equilibrium dissociation constant (KD) (shown insert) in comparison to a non-specific control protein (right panel)
  • C Analytical SEC of IgG 15031 and the control clinical benchmark IgG (trastuzumab)
  • D Antibody-mediated neutralization was assessed in a focus reduction neutralization assay following incubation of serial dilutions of antibody with the Washington strain of SARS CoV-2 (2019 n-CoV/USA_WAl/2020
  • FIG. 1 Figure 6 - Characterization of IgG 15032.
  • A Expressed and Protein A-purified human IgGl antibody derived from phage clone 15032 was assessed for binding to SARS CoV-2 S-protein by ELISA versus control capture protein (neutravidin) over a range of IgG concentrations to estimate EC50 value (shown inset)
  • B Binding kinetics of the interaction between IgG 15032 and SARS CoV-2 S-protein (left panel) was also assessed by biolayer interferometry to estimate the equilibrium dissociation constant (KD) (shown inset) in comparison to a non-specific control protein (right panel)
  • C Analytical SEC of IgG 15032 and the control clinical benchmark IgG (trastuzumab)
  • D Antibody-mediated neutralization was assessed in a focus reduction neutralization assay following incubation of serial dilutions of antibody with the Washington strain of SARS CoV-2 (2019 n-CoV/USA_WAl/
  • FIG. 7 Figure 7 - Characterization of IgG 33
  • A Expressed and Protein A-purified human IgGl antibody derived from phage clone 15033 was assessed for binding to SARS CoV-2 S- protein by ELISA versus control capture protein (neutravidin) over a range of IgG concentrations to estimate ECso value (shown inset)
  • B Binding kinetics of the interaction between IgG 15033 and SARS CoV-2 S-protein (left panel) was also assessed by biolayer interferometry to estimate the equilibrium dissociation constant (KD) (shown inset) in comparison to a non-specific control protein (right panel)
  • C Analytical SEC of IgG 15033 and the control clinical benchmark IgG (trastuzumab)
  • D Antibody-mediated neutralization was assessed in a focus reduction neutralization assay following incubation of serial dilutions of antibody with the Washington strain of SARS CoV-2 (2019 n- CoV/USA_WAl
  • FIG. 8 Figure 8 - Characterization of IgG 15033-7.
  • Affinity matured IgG 15033-7 obtained following optimization of IgG 15033, was expressed and Protein A-purified then assessed for binding to Fc-tagged SARS CoV-2 RBD protein ELISA versus control capture protein (neutravidin) over a range of IgG concentrations
  • FIG. 9 Figure 9 - Characterization of IgG 15033A2.
  • FIG. 10 Comparing the potency of optimized IgG 15033A2 to IgG 15033-7 by pseudovirus neutralization.
  • IgG 15033-7 and the optimized variant obtained by maturation of IgG 15033-7 (IgG 33 A2) were assessed by pseudovirus neutralization assay against a variant (MB61) toward which IgG 15033-7 showed partially compromised potency.
  • Serial dilutions of the two antibodies were pre-incubated with equivalent concentrations of VLPs in triplicate in parallel to confirm enhanced neutralization.
  • FIG. 11 Figure 11 - Characterization of IgG 36.
  • A Expressed and Protein A-purified human IgGl derived from phage clone 15036 was assessed for binding to SARS CoV-2 S-protein by ELISA versus negative control protein (BSA) over a range of IgG concentrations to estimate the EC50 value (shown inset)
  • BSA negative control protein
  • BSA negative control protein
  • the binding of IgGs 15036 versus Fc-tagged SARS CoV-2 RBD protein was assessed by biolayer interferometry and sensor traces shown for serial dilutions of IgG.
  • C Analytical SEC of IgG 15036 and the control clinical benchmark IgG (trastuzumab)
  • D IgG 15036 was evaluated in a pseudoviral infection assay that employs VLPs pseudotyped with S-protein bearing RBD mutations corresponding to clinically-observed variants of concern to estimate their potency and breadth of neutralization against the indicated variants. Samples were run in triplicate, and the infection signal normalized to a no antibody control. Estimated IC50 values for the neutralizing antibody obtained from the fit curves are shown inset.
  • FIG. 12 Figure 12 - Characterization of IgG 15046.
  • A Expressed and Protein A-purified human IgGl derived from phage clone 15046 was assessed for binding to SARS CoV-2 S- protein by ELISA versus negative control protein (BSA) over a range of IgG concentrations
  • B The binding of IgG 46 versus Fc-tagged SARS CoV-2 RBD protein was assessed by biolayer interferometry and sensor traces shown for serial dilutions of IgG. The equilibrium dissociation constant and associated binding rate constants for each is reported in table format below (C) Analytical SEC of IgG 15046 and the control clinical benchmark IgG (trastuzumab).
  • FIG. 13 Figure 13 - Characterization of IgG 15046A1.
  • Affinity matured IgG 15046A1 obtained following optimization of IgG 15046, was expressed and Protein A-purified then assessed for binding to SARS CoV-2 S-protein by ELISA versus negative control protein (BSA) over a range of IgG concentrations to estimate ECso value (shown inset)
  • BSA negative control protein
  • the binding of IgG 15046A1 versus SARS CoV-2 RBD protein or a biotinylated negative control protein was assessed by biolayer interferometry and sensor traces shown for serial dilutions of IgG.
  • C Analytical SEC of IgG 15046A1 and the control clinical benchmark IgG (trastuzumab)
  • D IgG 15046A1 and the parental IgG 46 was evaluated in a pseudoviral infection assay that employs VLPs pseudotyped with wild-type S-protein corresponding to S-protein from the original Wuhan strain of virus. Samples were run in triplicate, and the infection signal normalized relative to VLP infection in the absence of antibody.
  • FIG. 14 Epitope mapping of IgG 15036 and 15046.
  • A IgG obtained from selections against SARS CoV2 S-protein were assessed for binding to both SARS and SARS CoV-2 RBD protein and a negative control protein by biolayer interferometry. The estimated equilibrium dissociation constants are reported in table format
  • B A phage blocking ELISA was used to map the relative epitopes of antibodies 15033, 15036 and 15046 and previously published antibody (Yuan, Science 2020). Plate immobilized S-protein was incubated with a saturating concentration of the indicated blocking IgG (legend right).
  • Fab-phage supernatant (x-axis) was added to S-protein-coated wells in the presence or absence of the indicated blocking antibody and binding evaluated by development with phage-binding anti -Ml 3 antibodies. Phage binding signals were normalized to the isotype control antibody well and plotted. In each case, Fab-phage clone binding was inhibited by its corresponding blocking IgG, expected as a positive control for blockade.
  • FIG. 15 Biophysical properties of IgG 15036 and 15046
  • A Poly-reactivity of IgGs 15036 and 15046 were assessed by ELISA against a panel of proteins known to be ‘sticky’ and the binding to which is predictive of poor pharmacokinetics. Following exposure of 50 nM IgG to the indicated protein (legend right), binding was detected by development with an anti-human Fc antibody. Binding signals were plotted for the two test samples as well as a positive (non-sticky) IgG control (trastuzumab) and a negative (sticky) control IgG (IgG 6606) for comparison.
  • B Analytical SEC of the indicated IgGs were conducted to assess monodispersity and elution times relative to the clinical benchmark IgG (trastuzumab).
  • FIG. 16 Neutralization of SARS CoV2 variants by pseudovirus infection assay - comparison with clinically authorized antibodies.
  • Neutralization assays were conducted using of a panel of VLPs pseudotyped with SARS-CoV2 S-protein bearing RBD mutations found in clinically isolated variants.
  • Inhibition profiles for were generated for (A) IgG 15033A2, and two clinically authorized antibodies (B) bamlanivimab (Eli-Lilly) and (C) casirivimab (Regeneron) to estimate their potency and breadth of neutralization against the indicated variants. Samples were run in triplicate, normalized to samples run in the absence of antibody and error bars plotted as the standard deviation from the average of triplicate readings.
  • FIG. 17 Neutralization of authentic SARS CoV-2 virus by IgG 33-7 versus clinically authorized antibodies.
  • a focus reduction neutralization assay was conducted using serial 10-fold dilutions of IgG or the indicated bi-specific IgG against an authentic SARS CoV-2 strain isolated in the USA (Washington strain (2019 n-CoV/USA_WAl/2020) and an isogenic version of the SARS-CoV-2 strain isolated in S. Africa (B.1.351) bearing multiple S-protein mutations including in the RBD. Samples were run in triplicate and error bars are expressed as the standard deviation of the average infection signal.
  • amino acid includes all of the naturally occurring amino acids as well as modified amino acids.
  • an "affinity matured” antibody or “maturation of an antibody” refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent or source antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen or to other desired properties of the molecule.
  • amino acid includes all of the naturally occurring amino acids as well as modified amino acids.
  • antibody as used herein is intended to include human antibodies, monoclonal antibodies, polyclonal antibodies, antibody binding fragments, single chain and other chimeric antibodies.
  • the antibody may be from recombinant sources and/or produced in transgenic animals.
  • the basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
  • the amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
  • a “whole antibody” or “full-length” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof.
  • Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3.
  • Each light chain is comprised of a light chain variable region (VL) and a light chain constant region.
  • the light chain constant region is comprised of one domain, CL or CL1.
  • the VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDR complementarity determining regions
  • Each VH and VL 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, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • IMGT IMMLNOGENETICS INFORMATION SYSTEM
  • antibody binding fragment or “binding fragment” or “antigen binding fragment” as used herein is intended to include without limitations Fab, Fab', F(ab')2, scFv, scFab, dsFv, ds-scFv, diabodies and single domain antibodies, e.g., VHH.
  • Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • Fab, Fab' and F(ab')2, scFv, scFab, VHH, dsFv, ds-scFv, diabodies, and other fragments can also be synthesized by recombinant techniques, see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 and Holliger et al. (1993) PNAS. USA 90:6444-6448.
  • An antigen binding site or “binding site” is formed by amino acid residues of the N- terminal variable regions of the heavy chain (VH) and N-terminal variable regions of the light chains (VL).
  • the three CDRs of a light chain and the CDRs of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface.
  • the antigen-binding surface is complementary to the three-dimensional surface of a bound antigen.
  • the binding site is formed by only one heavy variable domain (VH).
  • VHH refers to a human VH that has been engineered to be independent of the light chain (Nilvebrant et al. Curr Pharm Des. (2016) 22(43):6527-6537; Barthelemy et al. Journal of Biological Chemistry (2007) 283:3639-3654. Nilvebrant et al. J Mol Biol. (2021) 433(21):Epub).
  • capture antibody refers to an antibody or binding fragment thereof bound to a solid support and used to capture the target antigen in a sample, for example SARS-COV-2 or a SARS-COV-2 S polypeptide, e.g., an SI and or S2 subunit, optionally soluble SARS-COV-2 S polypeptide by forming a complex with the target antigen.
  • composition or method comprising essentially of', it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention.
  • antibodies or binding fragments “consisting essentially of' a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based upon the sequence from which it was derived, e.g. about 5 residues, 4 residues, 3 residues, 2 residues or about 1 residue less than the recited bounding amino acid residue, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited bounding amino acid residue.
  • compositions, methods, or kit of any element, step, or ingredient not specified in the claim For example, antibodies or binding fragments "consisting of' a disclosed sequence consists only of the disclosed amino acid sequence.
  • a "conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another.
  • Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size; alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, and valine are all typically considered to be small.
  • the “COVID-19 S polypeptide” as used herein is an extensively glycosylated spike (S) protein of SARs-CoV-2 that protrudes from the viral membrane, to mediate host-cell entry.
  • S polypeptide contains 1,282 amino acids organized into 2 subunits (SI and S2) and the receptor binding domain (RBD), is located in the SI subunit.
  • denatured refers to a polypeptide that has lost tertiary and/or secondary structure (e.g. fully unfolded protein), for example when exposed to denaturing conditions in SDS sample loading buffer.
  • detecttable tag or “detectable label” as used herein refers to moieties such as peptide sequences, a radio-opaque or a radioisotope label, such as 3 H, 14 C; 32 P; 35 S; 123 I, 125 I, 133 I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion that can be appended or introduced into recombinant protein.
  • a radio-opaque or a radioisotope label such as 3 H, 14 C; 32 P; 35 S; 123 I, 125 I, 133 I
  • detection antibody refers to an antibody or binding fragment thereof that binds a capture antibody or a target antigen, for example SARS-CoV-2 or a SARS-CoV-2 S polypeptide, or optionally a target antigen already in a complex with a capture antibody.
  • the detection antibody binds the capture antibody: SARS- CoV-2 or a SARS-CoV-2 S polypeptide complex at an epitope on the target antigen that is different than the one recognized by the capture antibody.
  • “Diabody” or “Diabodies” as used herein are dimeric antibody fragments.
  • each antigen-binding site is formed by pairing of the VH and VL of one polypeptide with the VH and VL of the other polypeptide.
  • Diabodies thus have two antigen-binding sites, and can be monospecific or bispecific, (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123; Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5) incorporated herein by reference.
  • the term “effective amount” as used herein refers generally to an amount of the antibodies and binding fragments described herein, or a pharmaceutical composition comprising the antibodies or binding fragments, sufficient at dosages and periods of time necessary to achieve the desired result, e.g., to ameliorate at least one physical adverse effect of SARS-COV-2 infection or proliferation. This might be an amount sufficient to interfere with the binding of SARS-COV-2 to its target, and/or delay onset of, one or more symptoms of the disease.
  • the amount required to be administered will depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered.
  • Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight and from about 2 mg/kg to about 30 mg/kg.
  • Common dosing frequencies may range, for example, from twice daily to once a week.
  • epitopic determinants refers to the site on the antigen that is recognized by the antibodies or binding fragments disclosed herein.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An antibody is said to specifically bind an antigen when the dissociation constant is ⁇ 1 pM; e.g., ⁇ 100 r
  • the term the “Fc domain” refers to the constant region of immunoglobulin molecules and is also called the fragment crystallizable region.
  • the Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains and the Fc domains of IgGs bear a highly conserved N- glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity.
  • heavy chain complementarity determining region refers to regions of hypervariability within the heavy chain variable region of an antibody molecule.
  • the heavy chain variable region has three complementarity determining regions termed heavy chain complementarity determining region 1 (CDR1), heavy chain complementarity determining region 2 (CDR2) and heavy chain complementarity determining region 3 (CDR3) from the amino terminus to carboxy terminus.
  • CDR1 heavy chain complementarity determining region 1
  • CDR2 heavy chain complementarity determining region 2
  • CDR3 heavy chain complementarity determining region 3
  • All CDRs and framework regions (FRs) disclosed herein, amino acid sequences of CDRs and FRs disclosed herein, and CDR- encoding or FR-encoding nucleic acid sequences disclosed herein are intended to be defined in accordance with IMGT numbering (Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Development and Comparative Immunology. 2003
  • heavy chain variable region or “heavy chain variable domain” or “VH” as used herein refers to the variable domain of the heavy chain comprising the heavy chain complementarity determining region 1, heavy chain complementarity determining region 2 and heavy chain complementarity determining region 3.
  • host cell refers to a cell into which a recombinant DNA expression cassette or vector can be introduced to produce a recombinant cell.
  • Host cells include but are not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as S2 drosophila cells and Sf9, or animal cells, including human cells, e.g., fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.
  • inhibitor or “inhibition” or “inhibiting” includes its generally accepted meaning which includes prohibiting, preventing, restraining, alleviating, ameliorating, and slowing, stopping or reversing progression or severity of an infection.
  • the present method includes both therapeutic and/or prophylactic administration, as appropriate.
  • Inhibition of virus pathogenesis or virus infection of the cell is indicated when the antibody inhibits virus pathogenesis, infection and/or proliferation, in vitro or in vivo, by at least 20%, more preferably by at least 30%, even more preferably by at least 40%, at least 50%, at least 60, at least 70%, at least 80% or at least 90%, as compared to a non-specific, control antibody.
  • isolated antibody or binding fragment thereof' or isolated and purified antibody or binding fragment thereof' refers to an antibody or binding fragment thereof that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized and/or other antibodies, for example directed to a different epitope.
  • KD refers to the dissociation constant of a complex for example of a particular antibody-antigen interaction.
  • light chain complementarity determining region refers to regions of hypervariability within the light chain variable region of an antibody molecule.
  • Light chain variable regions have three complementarity determining regions termed light chain complementarity determining region 1 (CDR1), light chain complementarity determining region 2 (CDR2) and light chain complementarity determining region 3 (CDR3) from the amino terminus to the carboxy terminus.
  • CDR1 light chain complementarity determining region 1
  • CDR2 light chain complementarity determining region 2
  • CDR3 light chain complementarity determining region 3
  • light chain variable region or “light chain variable domain” or “VL” as used herein refers to the variable domain of the light chain comprising the light chain complementarity determining region 1, light chain complementarity determining region 2 and light chain complementarity determining region 3.
  • the term "monoclonal antibody” refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product.
  • the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population.
  • MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
  • non-native or “natively folded” as used herein refers to a protein in its native conformation (e.g. 3D conformation) or in a conformation sufficient to confer functionality, including for example partially unfolded protein capable of binding a receptor or ligand.
  • nucleic acid sequence refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof.
  • the nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases.
  • nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand.
  • nucleic acid sequences includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents.
  • isolated nucleic acid refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.
  • paratope includes the antigen binding site in the variable region of an antibody that binds to an epitope.
  • polypeptide refers to a polymer consisting of a large number of amino acid residues bonded together in a chain.
  • the polypeptide can form a part or the whole of a protein.
  • the polypeptide may be arranged in a long, continuous and unbranched peptide chain.
  • the polypeptide may also be arranged in a biologically functional way.
  • the polypeptide may be folded into a specific three-dimensional structure that confers it a defined activity.
  • polypeptide as used herein is used interchangeably with the term "protein”.
  • polypeptide fragment refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence.
  • isolated polypeptide refers to substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
  • the term "reference agent” as used herein refers to an agent that can be used in an assay and that can be for example a standard amount of SARS-CoV-2 or a SARS-CoV-2S polypeptide used as a reference for example for detecting SARS-COV-2 or a SARS-CoV-2 polypeptide in a sample or for screening or for diagnosing a subject having or suspected of having a SARS-CoV-2 infection.
  • sample from a subject refers to any biological fluid, cell or tissue sample from a subject, which can be assayed for SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
  • the sample can comprise lung or nasal secretions, lung lavage, urine, serum, plasma or cerebrospinal fluid.
  • the sample can for example be a "post-treatment” sample wherein the sample is obtained after one or more treatments, or a "base-line sample” which is for example used as a base line for assessing disease progression.
  • the term "sandwich ELISA” as used herein refers to an ELISA comprising a solid support and a capture antibody or binding fragment thereof (specific for the antigen) immobilized onto the solid support.
  • the capture antibody e.g. SARS-CoV-2 or a SARS-CoV-2 S polypeptide comprised in a sample.
  • the bound antigen is detected by a second antibody or binding fragment thereof, i.e. a detection antibody or binding fragment thereof, which recognizes an epitope that is different from the one recognized by the capture antibody.
  • the capture antibody SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex
  • the detection antibody which can be covalently linked to an enzyme or can itself be detected by addition of a secondary antibody which is linked to an enzyme.
  • the capture antibody and/or the detection antibody can comprise CDR regions disclosed herein.
  • SARs-CoV-2 or “COVID-19” as used herein is the novel coronavirus belonging to Coronaviridae family, beta-coronavirus genus and Sarbecovirus subgenus and the cause of the ongoing pandemic of Coronavirus Disease 2019 (COVID-19).
  • Single-chain Fv or “scFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain.
  • the Fv polypeptide (Fv fragment has the antigen-binding site made of the VH and VL regions, but they lack the constant regions of Fab (CHI and CL) regions) further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding.
  • the “COVID-19 S polypeptide” as used herein is an extensively glycosylated spike (S) protein of SARs-CoV-2 that protrudes from the viral membrane, to mediate host-cell entry.
  • S polypeptide contains 1,282 amino acids organized into 2 subunits (SI and S2) and the receptor binding domain (RBD), is located in the SI subunit.
  • subject means a human being, a monkey, an ape, or a dog or cat, for whom diagnosis, treatment, or therapy is desired.
  • treatment means obtaining a desired pharmacologic and/or physiologic effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease.
  • Treatment covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., slowing or arresting its development; or (c) relieving the disease, i.e., causing regression of the disease.
  • the therapeutic agent may be administered before, during or after the onset of disease or injury.
  • the treatment of ongoing disease where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues.
  • the subject therapy may be administered before the symptomatic stage of the disease, during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
  • variant includes one or more amino acid and/or nucleotide modifications in a sequence (polypeptide or nucleic acid respectively) for example, one or more modifications of a light chain or a heavy chain complementarity determining region (CDR) disclosed herein that perform substantially the same function as the light chain and heavy chain CDRs disclosed herein in substantially the same way.
  • CDR complementarity determining region
  • variants of the CDRs disclosed herein have the same function of being able to specifically bind to an epitope on SARS-CoV-2 or a SARS-CoV-2 S polypeptide or in the case of nucleotide modifications, encode CDRs that have same function of being able to specifically bind to an epitope on SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
  • codon optimized and degenerate sequences are included.
  • Variants of CDRs disclosed herein include, without limitation, amino acid substitutions as well as additions and deletions to the CDR sequences disclosed herein.
  • the substitution, addition or deletion can be 1, 2, 3, 4, 5, 6, 7, or 8 amino acids and/or the corresponding number of nucleotides.
  • vector refers to a nucleic acid delivery vehicle or plasmid that can be engineered to contain a nucleic acid molecule, e.g., a nucleic acid sequence encoding the binding molecules described herein.
  • the vector that can express protein when inserted with a polynucleotide is called an expression vector.
  • Vectors can be inserted into the host cell by transformation, transduction, or transfection, so that the carried genetic substances can be expressed in the host cell.
  • Vectors are well known to the technical personnel in the field, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl derived artificial chromosome (PAC); phage such as /.phage or Ml 3 phage and animal viruses etc.
  • Animal viruses may include but not limited to, reverse transcriptase virus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e. g. herpes simplex virus), chicken pox virus, baculovirus, papilloma virus, and papova virus (such as SV40).
  • a vector can contain multiple components that control expression of the binding molecules described herein, including but not limited to, promoters, e.g., viral or eukaryotic promoters, e.g., a CMV promoter, signal peptides, e.g., TRYP2 signal peptide, transcription initiation factor, enhancer, selection element, and reporter gene.
  • promoters e.g., viral or eukaryotic promoters, e.g., a CMV promoter
  • signal peptides e.g., TRYP2 signal peptide
  • transcription initiation factor e.g., enhancer, selection element, and reporter gene.
  • the vector may also contain replication initiation site(s).
  • the term "level" as used herein refers to an amount (e.g. relative amount or concentration) of SARS-CoV-2 or a SARS-CoV-2 S polypeptide that is detectable or measurable in a sample.
  • the SARS-CoV-2 or a SARS-CoV-2 S polypeptide level can be a concentration such as pM or a relative amount such as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and/or 10 times a control level, where for example, the control level is the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a healthy or asymptomatic subject.
  • compositions comprising antibodies or binding fragments are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
  • a composition comprising antibodies or binding fragments is a composition that may comprise other elements in addition to the antibodies or binding fragments, e.g. functional moieties such as polypeptides, small molecules, or nucleic acids bound, e.g.
  • the present disclosure relates to an antibody and/or binding fragment thereof as well as methods of making and use for example for diagnosing and/or prognosing SARS-CoV-2 infection and/or proliferation.
  • Described herein are antibodies and antibody binding fragments that specifically bind SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g., S protein subunits SI and S2.
  • the antibodies described herein exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WAl in Vero E6 cells, and also bound to the receptor binding domain (RBD).
  • an embodiment of this invention is an antibody and/or binding fragment thereof that specifically binds SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g., S protein subunits SI or S2.
  • the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention may be at least 50%, at least 55%, at least 60%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy chain (hlgl) CDRs of an antibody of Table 2.
  • the antibodies and antigen-binding fragments thereof contain a heavy chain variable region having a CDR amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% 98%, or at least 99% identical to the amino acid sequence of the heavy chain (hlgl) CDRs of an antibody of Table 2.
  • the amino acid sequence of the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention consist essentially of the amino acid sequence of the heavy chain (hlgl) CDRs of an antibody of Table 2.
  • the light chain CDRs of the antibodies and antigen-binding fragments of this invention may be at least 50%, at least 55%, at least 60%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light chain (hk) CDRs of an antibody of Table 2.
  • the antibodies and antigen-binding fragments thereof contain a light chain variable region having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2.
  • the amino acid sequence of the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention consist essentially of the amino acid sequence of light chain (hk) CDRs of an antibody of Table 2.
  • the antibodies and antigen-binding fragments thereof comprise (a) a VH domain comprising three heavy chain variable region CDRs, the CDRs having an amino acid sequence at least 90%/o, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of three heavy chain (hlgl) CDRs of an antibody of Table 2, and (b) a VL domain comprising three light chain CDRs having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2.
  • the antibodies and antigen-binding fragments thereof comprise (a) a VH domain comprising three heavy chain variable region CDRs, the CDRs having an amino acid sequence of three heavy chain (hlgl) CDRs of an antibody of Table 2, and/or (b) a VL domain comprising three light chain CDRs having an amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2.
  • the antibody comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of a molecule of Table 4.
  • the antibody comprises an amino acid sequence of the molecules of Table 4.
  • the antibody consists essentially of an amino acid sequence of the molecules of Table 4.
  • the amino acid and nucleotide sequences of the molecules in Table 4 comprise the heavy chain and light chain CDRs of antibody 15031
  • embodiments of this invention include the amino acid and nucleotide of the molecules in Table 4 wherein the CDRs of 15031 are replaced with the CDRs of antibodies 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049 set forth in Tables 2 and 3.
  • a further aspect is an antibody and/or binding fragment of this invention, wherein the antibody and/or binding fragment thereof specifically binds to SARS-CoV-2 or a SARS- CoV-2 S polypeptide in an unfixed or mildly fixed sample.
  • the antibody and/or binding fragment thereof comprises a VL domain comprising a CDR3 and a VH domain comprising a CDR1, a CDR2 and a CDR3, wherein the amino acid sequences of the CDRs comprise one or more of the CDR sequences set forth in Table 2.
  • the antibody and/or binding fragment thereof comprises a light chain variable region comprising an hk CDR3 of Table 2 and the heavy chain variable region comprises a hlgl CDR1, a CDR2 and a CDR3 of an antibody set forth in Table 2.
  • the antibody, binding fragment thereof, or CDRs disclosed herein may have one or more amino acid substitutions, including but not limited to conservative amino acid substitutions.
  • the antibody comprising the light chain and heavy chain CDRs set forth in Table 2 can be any class of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; and any isotype, including: IgGl, IgG2, IgG3 and IgG4.
  • Chimeric antibodies can be prepared using routine recombinant techniques.
  • an Fab of an IgG of Table 4 may be reformatted into another full length IgG by subcloning the variable domains of the immunoglobulin’s light and heavy chains into mammalian expression vectors and producing the IgG protein using human embryonic kidney cells (HEK293T).
  • HEK293T human embryonic kidney cells
  • any cell type suitable for expressing an antibody can be used.
  • the antibody may be a full-length immunoglobulin molecule, e.g., a chimeric antibody, a human antibody, a humanized antibody, a polyclonal antibody, or a minibody
  • the antibody binding fragment may be a Fab, a Fab', a F(ab)2, a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a disulfide linked scFv, a single chain domain antibody scFab or VHH, or a diabody, that does not comprise an Fc domain or fragment thereof .
  • the antigen binding fragments e.g., Fab, Fab' and F(ab')2, scFv, scFab, dsFv, ds-scFv, VHH, or diabodies, and other fragments can be synthesized or expressed by recombinant techniques.
  • Antibodies can also be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
  • the antibody is a synthetic monoclonal antibody or a chimeric antibody comprising one or more CDRs selected from the CDRs of Table 2.
  • the antibody is a chimeric antibody comprising a VL domain comprising the hk CDR1, CDR2 and CDR3 from an antibody of Table 2 and/or a VH domain comprising the hlgl CDR1, CDR2 and CDR3 from an antibody of Table 2.
  • the SARS-CoV-2 binding molecule is a VHH fragment, a scFv, an Fab or a diabody that comprises the hlgl CDR1, CDR2 and CDR3 and/or the hk CDR1, CDR2 or CDR3 from an antibody of Table 2.
  • the antibodies and antibody fragments described herein may be isolated antibodies and antibody fragments.
  • the Fc domain of the antibody is engineered such that it does not target the cell that binds the antibody for ADCC or CDC-dependent death.
  • the Fc domain is dimerized via a knob-in-hole configuration.
  • Methods are well known in the art for mitigating antibody effector function, including for example amino acid substitutions in the Fc regions, e.g., the N297G (NG) and D265A, N297G (DANG) variants or L234A, L235A, P329G (LALA-PG) substitutions, see e.g., Lo et al. “Effector Attenuating Substitutions that Maintain Antibody Stability and Reduce Toxicity in Mice. The Journal of Biological Chemistry Vol. 292, No. 9, pp. 3900 -3908, March 3, 2017, incorporated herein by reference.
  • the antibody and/or binding fragment thereof is linked, e.g., conjugated or recombinantly fused, to a therapeutic moiety, e.g., a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioactive metal ion.
  • a therapeutic moiety e.g., a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioactive metal ion.
  • the antibody and/or binding fragment is labelled and/or conjugated or fused to a tag, for example to produce a detection tool or a diagnostic agent.
  • the detectable tag can be a purification tag such as a His-tag, a HA-tag, a GST- tag, biotin or a FLAG-tag.
  • the label is preferably capable of producing, either directly or indirectly, a detectable signal.
  • the label may be radio-opaque or a radioisotope, such as 3 H, 14 C; 32 P; 35 S; 123 I, 125 I, 131 I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion.
  • a radioisotope such as 3 H, 14 C; 32 P; 35 S; 123 I, 125 I, 131 I
  • a fluorescent (fluorophore) or chemiluminescent (chromophore) compound such as fluorescein isothiocyanate, rhodamine or luciferin
  • Another aspect of the disclosure relates to an antibody complex comprising the antibody and/or binding fragment described herein and SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
  • nucleic acid encoding an antibody and/or binding fragment described herein.
  • the nucleic acid encodes an antibody and/or binding fragment comprising a light chain variable region comprising a CDR1, CDR2 and CDR3 and a heavy chain variable region comprising a CDR1 CDR2 and CDR3, wherein one or more of the CDRs are the light chain and heavy chain CDRs set forth in Table 2.
  • nucleic acids that encode the same amino acid sequence. Accordingly, also included are nucleotide sequences that encode an antibody disclosed herein that specifically binds SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
  • nucleic acid sequences are at least 70%, at least 75%, at least 80%, at least 90% and at least 95% identical to nucleic acid sequences of Table 3 or Table 4 encoding the molecules set forth in Table 2 or Table 4.
  • the nucleic acid is an isolated nucleic acid.
  • Another aspect of this invention is a vector comprising the nucleic acid herein disclosed.
  • the vector is an isolated vector.
  • the vector can be any vector suitable for producing an antibody and/or binding fragment thereof, including for example vectors described herein.
  • Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses).
  • a further aspect of this invention is a recombinant cell producing the antibody and/or binding fragment thereof herein disclosed or the vector herein disclosed.
  • the recombinant cell can be generated using any cell suitable for producing a polypeptide, for example suitable for producing an antibody and/or binding fragment thereof.
  • Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells.
  • the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.
  • bacterial host cells suitable for producing recombinant antibody producing cells include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genus Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art.
  • Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication.
  • Representative promoters include the R-lactamase (penicillinase) and lactose promoter system, the trp promoter and the tac promoter.
  • selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes.
  • Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322, the pUC plasmids pUC18, pUC19, pUC118, pUC119, and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).
  • Suitable yeast and fungi host cells include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus.
  • yeast S. cerevisiae examples include pYepSecl, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for the transformation of yeast and fungi are well known to those of ordinary skill in the art.
  • Suitable mammalian cells include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g, ATCC No. CCL 2), 293 (ATCC No. 1573), NS-1 cells and any derivatives of these lines.
  • the mammalian cells used to produce a recombinant antibody are selected from CHO, HEK293 cells or FREESTYLETM 293-F cells (Life technologies).
  • FREESTYLE 293-F cell line is derived from the 293 cell line and can be used with the FREESTYLETM MAX 293 Expression System, FREESTYLETM 293 Expression System or other expression systems.
  • Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences.
  • a promoter e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40
  • other transcriptional and translational control sequences e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40
  • the vector is designed for production of a light chain or a heavy chain, e.g., an IgGl heavy chain.
  • Suitable insect cells include cells and cell lines from Bombyx or Spodotera species.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series and the pVL series.
  • the recombinant expression vectors may also contain genes which encode a fusion moiety (i.e. a "fusion protein") which provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including for example tags and labels described herein.
  • a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
  • GST glutathione S-transferase
  • maltose E binding protein or protein A, respectively, to the recombinant protein.
  • operably linked is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.
  • Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate regulatory sequences is dependent on the host cell chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
  • expression of the antibody or binding fragment thereof is under the control of an inducible promoter.
  • inducible non-fusion expression vectors include pTrc (28) and pET l id.
  • the recombinant expression vectors may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention.
  • selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase.
  • selectable marker gene Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as - galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest. Other selectable markers include fluorescent proteins such as GFP which may be cotransduced with the nucleic acid of interest.
  • selectable marker protein such as - galactosidase, chloramphenicol acetyltransferase, or firefly lucifer
  • compositions e.g., a pharmaceutical comprising the antibody and/or binding fragment thereof, the nucleic acid herein disclosed or the recombinant cell herein disclosed, optionally in combination with a suitable diluent or carrier or excipient.
  • the pharmaceutical composition may consist or consist essentially of the antibodies or binding fragment or the nucleic acid molecules expression cassettes and vectors encoding the antibodies or binding fragment described herein, and a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers, diluents and excipients, and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa.
  • an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic.
  • the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution.
  • the pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5.
  • Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the agonist, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. Delivery systems for administering therapeutic agents, e.g., antibodies and fragments thereof, are known in the art, see e.g., Wu and Wu, J.
  • polymers used in sustained release formulations include, but are not limited to, polyphydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene- co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, polypvinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides, poly(lactide-co-glycolides) (PLGA), and polyorthoesters.
  • the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of the antibodies or binding fragment being administered.
  • the composition can be a lyophilized powder or aqueous or non-aqueous solution or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes.
  • Other components that may be present in such compositions include, but are not limited to, water, surfactants (pharmaceutically acceptable non-ionic surfactants such as, for example, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), zwitterionic, or ionic surfactants), alcohols, polyols, glycerin and vegetable oils.
  • surfactants pharmaceutically acceptable non-ionic surfactants such as, for example, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), zwitterionic, or ionic surfactants
  • alcohols polyols, glycerin and vegetable oils.
  • Suitable diluents for nucleic acids include but are not limited to water, saline solutions and ethanol.
  • Suitable diluents for polypeptides, including antibodies or fragments thereof and/or cells include but are not limited to saline solutions, pH buffered solutions and glycerol solutions or other solutions suitable for freezing polypeptides and/or cells.
  • composition can further comprise stabilizing agents, for example reducing agents, hydrophobic additives, and protease inhibitors which are added to physiological buffers.
  • stabilizing agents for example reducing agents, hydrophobic additives, and protease inhibitors which are added to physiological buffers.
  • Additional aspects of the invention are methods for using, producing or isolating an antibody and/or binding fragment thereof described herein with specific binding affinity to SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
  • COVID-19 infections include, e.g., fever, cough, shortness of breath, chills, repeated shaking with chills, muscle pain, headache, sore throat, loss of taste or smell, and can lead to physical complications, e.g., pneumonia and trouble breathing, organ failure in several organs, heart problems, severe lung condition, e.g., acute respiratory distress syndrome, blood clots, acute kidney injury and additional viral and bacterial infections.
  • physical complications e.g., pneumonia and trouble breathing, organ failure in several organs, heart problems, severe lung condition, e.g., acute respiratory distress syndrome, blood clots, acute kidney injury and additional viral and bacterial infections.
  • An embodiment of the invention relates to a method for treating, preventing, mitigating, or delaying the progression of a SARS-CoV-2 infection, or ameliorating a symptom or complication of a CO VID-19 infection, comprising administering an effective amount of an antibody or binding fragment of this invention to a subject having or suspected of having a CO VID-19 infection.
  • a method for treating, preventing, mitigating, or delaying the progression of a SARS- CoV-2 infection includes reducing viral load in a subject in need thereof.
  • the antibody or binding fragment of the present invention may be administered systemically or locally, e.g., by injection (e.g. subcutaneous, intravenous, intraperitoneal, intrathecal, intraocular, etc.), inhalation, implantation, topically, or orally.
  • the antibody or binding fragment may be coated in a material to protect the molecules from the action of acids and other natural conditions which may inactivate the molecules.
  • the antibody or binding fragment described herein may be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier.
  • composition comprising the binding molecule can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc.
  • excipients such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc.
  • An extensive listing of excipients that can be used in such a composition can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000).
  • the antibody or binding fragment of this invention can also be administered in conjunction with other therapeutic regimens or agents for treatment of COVID-19 infections and the associated symptoms or complications, e.g., convalescent serum therapy, small molecules, oleandrin, neutralizing monoclonal antibodies, recombinant antibodies, cytokines, or antivirals, e.g., LY-CoV555 (Eli Lilly), e.g., recombinant anti-CD47/PD-Ll bispecific antibody (IBI322)( Innovent Biologies, Inc.
  • remdesivir Actemra, Oluminant (baricitinib), merimepodib, dexamethaone (glucocorticoid), Aviptadil (RLF-100) , hydroxy cl oropuine, Avigan (favipiravir, Avifavir), Bucillamine , niclosamide, aviptadil, PIKfyve kinase inhibitor, tyrosine kinase inhibitor efineptakin alfa, interferon beta, or SLV213 (Selva Therapeutics).
  • Effective dosages and schedules for administering the antibodies and fragments thereof of this invention and nucleic acids that encode them described herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of such antibodies and fragments thereof that must be administered will vary depending on, for example, the subject that will receive the binding molecule, the route of administration, the particular type of antibody or fragment thereof used and other drugs being administered. Guidance in selecting appropriate doses for antibodies and fragments thereof of this invention is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp.
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the inflammation in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the antibody, fragments thereof, nucleic acid and vector is within the skill of the art.
  • An embodiment of the invention relates to a method for preventing or delaying the cleavage of the S2 subunit, or the fusion of SARS-CoV-2 with the cell and internalization of SARS-CoV-2 by a cell, e.g., a cell having an ACE2 receptor, by contacting the cell with an effective amount of an antibody or antibody binding fragment of this invention.
  • Yet another aspect relates to a method for screening or for diagnosing a subject having or suspected of having a SARS-CoV-2 infection, the method comprising measuring the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample from a subject by contacting the sample with an antibody or binding fragment of this invention, wherein detecting a level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide bound by the antibody or binding fragment is indicative that the subject is infected with SARS-CoV-2.
  • the method may further comprise the step of comparing the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample bound by the antibody or binding fragment with a negative or positive control, wherein an increased level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample bound by the antibody or binding fragment as compared to a negative control, e.g., a value derived from a group of subjects without SARS-CoV-2 infection, or convalescing from a SARS-CoV-2 infection, is indicative that the subject is infected with SARS-CoV-2 or wherein a level of SARS-CoV-2 similar to or exceeding the level of SARS-CoV-2 of the positive control, e.g., a value derived from a group of subjects known to have a SARS-CoV-2 infection, is indicative that the subject is infected with SARS- CoV-2.
  • a negative control e.g., a value derived from a group of subjects without
  • An additional aspect of the invention is a method for prognosing CO VID-19 progression or lack thereof (e.g. recovery or worsening of disease). Accordingly an aspect is a method of prognosing a likelihood of recovery after CO VID-19 infection, the method comprising: a) measuring a level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample from a subject by contacting the sample with the antibodies or antibody fragments described herein; and b) comparing the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide bound by the antibodies or fragments thereof in the sample with a control, for example a control value derived from a group of subjects who recovered from or in whom the COVID-19 disease symptoms regressed, wherein a decreased level of SARS-CoV-2 in the sample bound by the antibodies or binding fragments compared to the control is indicative that the subject has an increased likelihood of recovery after COVID-19 infection.
  • a control for example a control value derived
  • the level of SARS-CoV-2 may be measured by, e.g., measuring the amount of SARS-CoV-2 or a SARS-CoV-2 S polypeptide bound to the antibodies or antibody fragments of this invention by e.g., immunoprecipitation, an immunoassay, e.g. ELISA, immunoblot detection.
  • the antibody-based detection can also be combined with a mass spectrophometric assay, for example as in the case of a particle-based flow cytometric assay.
  • control is a control value derived from a group of subjects recovered from COVID-19 infection and an increased level of SARS-CoV-2 or a SARS- CoV-2 S polypeptide in the sample compared to the control is indicative that the subject has a decreased likelihood of recovery after CO VID-19 infection and/or an increased likelihood of COVID-19 infection and associated adverse effects progressing.
  • a further embodiment of the invention is a method for prognosing the likelihood of progression of a COVID-19 infection and associated adverse effects, the method comprising: a) measuring a level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample from a subject by contacting the sample with the antibodies or antibody fragments described herein; and b) comparing the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample bound to the antibodies or antibody fragments described herein with a control, wherein an increased level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample compared to the control, for example wherein the control is a control value derived from a group of subjects who did not recover or progressed is indicative that the subject has an decreased likelihood of recovering from a SARS-CoV-2 infection.
  • control is a control value derived from a group of subjects who recovered and an increased level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample compared to the control is indicative that the subject has a decreased likelihood of recovery after SARS-CoV-2 and/or an increased likelihood of SARS-CoV-2 infection and associated adverse effects progressing.
  • the sample can for example be taken after the subject has received a treatment and compared for example to a pre-treatment sample.
  • the patient can be monitored after a repeating interval to assess for example if treatment or other intervention is necessary.
  • the test is repeated and plotted to assess the subject's progression.
  • the sample may be a tissue sample, e.g. lung tissue, or a biological fluid such as blood, or a fraction thereof such as plasma or serum, or a lung secretion or a pulmonary lavage.
  • a biological fluid such as blood, or a fraction thereof such as plasma or serum, or a lung secretion or a pulmonary lavage.
  • the biological fluid is saliva or a nasal secretion.
  • the sample is selected from a fresh sample such as a fresh (e.g. not frozen) biological fluid sample or tissue sample, a onetime frozen biological fluid sample or tissue sample (e.g. frozen a single time at the time of obtaining the sample)) or a repeat frozen sample (e.g. frozen and thawed and frozen biological fluid sample or repeat frozen tissue sample).
  • a fresh sample such as a fresh (e.g. not frozen) biological fluid sample or tissue sample, a onetime frozen biological fluid sample or tissue sample (e.g. frozen a single time at the time of obtaining the sample)) or a repeat frozen sample (e.g. frozen and thawed and frozen biological fluid sample or repeat frozen tissue sample).
  • the sample comprises native or natively folded SARS-CoV-2 polypeptide.
  • the sample is a fixed sample such as a mildly fixed sample wherein the fixation induces limited denaturation and/or unfolding.
  • the isolated and purified antibody and/or binding fragment thereof is affinity matured.
  • Affinity maturation can be performed as described for the initial selection, with antigen adsorbed to plastic plates, using a for example a phage library comprising variants of the CDR sequences.
  • a person skilled in the art will appreciate that several methods can be used to isolate and produce antibodies and/or binding fragments thereof with specific binding affinity to SARS-CoV-2 or a SARS-CoV-2 polypeptide, e.g. SARS-CoV-2 S polypeptide.
  • a method that can be used is a phage display method.
  • SARS-CoV-2 or a SARS-CoV-2 S polypeptide is produced in order to isolate and characterize the antibody and/or binding fragment thereof. Phage from a human Fab phage-displayed library are selected following several rounds of panning.
  • Phage with specific binding affinity to the SARS-CoV-2 or a SARS-CoV-2 S polypeptide, as determined by ELISA, are sequenced and cloned into vectors designed for production of light chain or heavy chain.
  • the heavy chain can be for example an IgG, or an IgG isotype such as an IgGl or an IgG4.
  • Antigen binding fragments and IgG polypeptides are then affinity purified by using, for example, Protein A affinity columns.
  • a nucleic acid encoding an antibody or antibody binding fragment described herein is expressed in a host cell to make the antibody and/or binding fragment.
  • the method comprises: a) expressing in a host cell a nucleic acid encoding an antibody and/or binding fragment; b) culturing the host cell to produce the antibody and/or binding fragment; and c) isolating and/or purifying the antibody and/or binding fragment from the host cell.
  • nucleic acid encoding a single chain antibody is expressed.
  • multiple nucleic acids are expressed, for example a nucleic acid encoding an antibody light chain and a nucleic acid encoding an antibody heavy chain.
  • Vectors and nucleic acids encoding an antibody or antibody biding fragment described herein may be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection and other liposome-based transfection agents, electroporation or microinjection.
  • Nucleic acid encoding an antibody or antibody biding fragment described herein may be directly introduced into mammalian cells using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors.
  • the SARS-CoV-2 or SARS-CoV-2 S polypeptide specific antibodies disclosed herein can be used in a variety of assays for binding, detecting and measuring SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample.
  • the antibodies can be used in an ELISA, as well as immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA) (Muhammad S. Alam, Nov 2018) Current Protocols in Immunology Vol 123, Issue 1., mass spectroscopy -based techniques, particle-based flow cytometric assay, fluorescence-activated cell sorting (FACS) and ELISA.
  • Immunodetection methods as described herein generally involve the detection or measuring of antibody :SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g. an SI of S2 subunit, complexes using antibodies and/or binding fragments thereof disclosed herein.
  • the detection of such complexes is well known in the art and may be achieved through different methods, for example by using a detectable label or marker, such as a radioactive, fluorescent or enzymatic tag.
  • Detection of these complexes may also involve the use of a ligand such as a secondary antibody and/or binding fragment thereof specific for SARS-CoV-2 or a SARS- CoV-2 S polypeptide or for the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex.
  • a ligand such as a secondary antibody and/or binding fragment thereof specific for SARS-CoV-2 or a SARS- CoV-2 S polypeptide or for the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex.
  • detection assays for example a sandwich ELISA, as one antibody can be used as a capture reagent to isolate the SARS-CoV-2 or a SARS-CoV-2 S polypeptide while another antibody binding a distinct epitope can be used as a detection reagent.
  • an immunoassay comprising one or more antibodies and/or binding fragments thereof described herein.
  • the immunoassay is an enzyme linked immunosorbent assay (ELISA).
  • ELISA enzyme linked immunosorbent assay
  • Antibodies and/or binding fragments thereof may be used in the context of detection assays such as ELISAs, for example sandwich ELISAs.
  • the antibodies and binding fragments of this invention may be used as capture and detection antibodies for the detection of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in solution.
  • the full-length antibodies or binding fragments of this invention that recognize the S protein e.g., the SI subunit, are immobilized and incubated with SARS-CoV-2 or a SARS-CoV-2 S polypeptide or subunit.
  • the samples are then incubated with biotinylated IgGs recognizing epitopes on the virus, polypeptide, or subunit.
  • the ELISA is a sandwich ELISA comprising a capture antibody and a detection antibody that bind to different epitopes.
  • the capture antibody is an antibody or binding fragment which specifically binds a first epitope of SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g., a SI subunit or the RBD
  • the detection antibody is an antibody or binding fragment which specifically binds a second epitope of the SARS- CoV-2 or a SARS-CoV-2 S polypeptide, e.g., the SI subunit or the RBD, wherein the first and second epitopes are different.
  • one or both of the capture or detection antibodies is an antibody and/or binding fragment thereof herein.
  • the immunoassay is for the detection and/or measuring of SARS- CoV-2 or a SARS-CoV-2 S polypeptide, e.g., the SI or S2 subunit, in a sample
  • the method of making the immunoassay comprises: a) coating a solid support with the capture antibody; b) contacting the capture antibody with the sample under conditions to form a capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex; c) removing unbound sample; d) contacting the capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex with the detection antibody; e) removing unbound detection antibody; and f) detecting and/or measuring the capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex, wherein one or both of the capture or detection antibodies is an antibody and/or binding fragment thereof herein.
  • the ELISA is a competitive ELISA. In an embodiment, the ELISA is a direct ELISA. In an embodiment, the ELISA is an indirect ELISA.
  • solid supports include any material to which SARS-CoV-2 or a SARS-CoV-2 S polypeptide and antibodies and/or binding fragments thereof herein disclosed are capable of binding to.
  • the solid support can include plastic, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses and polyacrylamides.
  • the solid support is a microtiter plate, magnetic beads, latex beads or array surfaces.
  • the sample may be contacted with an antibody and/or binding fragment thereof under appropriate conditions, for example, at a given temperature and for a sufficient period of time, to allow effective binding of SARS-CoV-2 or a SARS-CoV-2 S polypeptide to the antibody or binding fragment, thus forming an antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex, such as a capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex.
  • the contacting step is carried out at room temperature for about 30 minutes, about 60 minutes, about 2 hours or about 4 hours.
  • the contacting step is carried out at about 4°C. overnight.
  • the antibody and/or binding fragment as described herein may be, e.g., complexed with SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a suitable buffer.
  • the buffer has a pH of about 5.0 to about 10.0.
  • the buffer has a pH of 4.5, 6.5 or 7.4.
  • the buffer is a HBS-EP buffer, a KRH buffer or Tris-buffered saline.
  • the buffer comprises BSA and/or TWEENTM20.
  • Any unbound material in a sample may be removed by washing so that only the formed antibody :SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex remains on the solid support.
  • the unbound sample is washed with phosphate-buffered saline, optionally comprising bovine serum albumin (BSA).
  • BSA bovine serum albumin
  • the detection antibody is labelled and/or conjugated to a tag.
  • the detection antibody directly labelled and/or conjugated.
  • the detection antibody is indirectly labelled and/or conjugated.
  • Indirect labels include for example fluorescent or chemiluminescent tags, metals, dyes or radionuclides attached to the antibody.
  • Indirect labels include for example horseradish peroxidase, alkaline phosphatase (AP), beta-galactosidase and urease.
  • HRP can be used with a chromogenic substrate, for example tetramethybenzidine, which produces a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
  • An embodiment of the invention is an assay for detecting and/or measuring level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample, the assay comprising: a) contacting a sample with the antibody and/or binding fragment described herein under conditions to form an antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex; and b) detecting and/or measuring the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex.
  • a further embodiment is an assay for detecting and/or measuring SARS-CoV-2 or a SARS-CoV-2 S polypeptide the method comprising: a) contacting a sample, the sample being a biological fluid, with the antibody and/or binding fragment of the antibody as described herein under conditions to form an antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex; and b) detecting and/or measuring the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex.
  • the assay for detecting and/or measuring the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide is performed under non-denaturing or mildly denaturing conditions.
  • the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex is detected directly for example by using an antibody labeled with a detectable tag or fusion moiety.
  • the complex is detected indirectly using a secondary antibody specific for the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex.
  • the assay for detecting or measuring the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide is an immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA), mass spectroscopy -based techniques and fluorescence-activated cell sorting (FACS), and ELISA.
  • Detecting can be performed using methods that are qualitative or measured using quantitative methods, for example by comparing to a standard or standard curve.
  • the sample used in the methods described herein may be a biological sample from a subject, e.g., a human or other primate, or the sample may be from another source.
  • the biological sample from a subject may be a tissue or a fluid, e.g., blood and its component parts, serum or plasma, saliva, a lung secretion or a lung lavage, or urine etc.
  • Other samples may be, e.g., wastewater or a body of water where wastewater drains, e.g., a river, canal, pond, bay etc.
  • a further aspect of this invention relates to a kit comprising i) an antibody and/or binding fragment described herein, ii) a nucleic acid encoding the an antibody and/or binding fragment described herein, iii) a composition comprising the an antibody and/or binding fragment described herein or the nucleic acid encoding the antibody or fragment, or iv) a recombinant cell described herein, comprised in a vial such as a sterile vial or other housing and optionally a reference agent, and/or instructions for use thereof.
  • the kit comprises components and/or is for use in performing an assay described herein.
  • the kit is an ELISA kit and can comprise a first antibody, e.g. a capture antibody, for example attached to a solid support, and a second antibody, e.g. a detection antibody, that binds to SARS-CoV-2 or a SARS-CoV-2 S polypeptide and/or binds the capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex, and that is conjugated to a detectable label.
  • the first and/or second antibody may be an antibody or antibody fragment of this invention. Any combination of antibodies or antibody fragments described herein can be used.
  • the kit is a diagnostic kit and the instructions are directed to a method described herein.
  • scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art.
  • Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)).
  • the PB-CMV version was used for large- scale transient expression.
  • the PB-TRE version was used for establishing inducible stable cell lines using the piggyBac transposase-based method (Li, Z., et al. (2013) Simple piggyBac transposon-based mammalian cell expression system for inducible protein production. Proc. Natl. Acad. Sci. U.S. A. 110, 5004-5009.).
  • cDNAs encoding the SARS-CoV and SARS-CoV-2 spike proteins were human codon optimized and synthesized by Genscript.
  • cDNA encoding human ACE2 was obtained from MGC clone 47598.
  • the soluble SARS-CoV-2 spike ectodomain trimer included residues 1- 1211, followed by a foldon trimerization motif (Tao et al. (1997) Structure of bacteriophage T4 fibritin: A segmented coiled coil and the role of the C-terminal domain. Structure 5, 789- 798), a 6xHis tag (SEQ ID NO: 1) and an AviTag biotinylation motif (Fairhead, M., and Howarth, M.
  • Residues 682-685 were mutated to SSAS (SEQ ID NO: 101) to remove the furin cleavage site on the SARS-CoV-2 spike protein.
  • Residues 986-987 were mutated to two proline residues to stabilize the pre-fusion form of the spike (Pallesen et al. (2017) Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc. Natl. Acad. Sci. U. S.A. 114, E7348-E7357.).
  • the SARS-CoV-2 receptor binding domain (RBD, residues 328-528), the soluble human ACE2 construct (residues 19-615), and the SARS-CoV RBD (residues 315-514) were eachfollowed by a 6xHis tag (SEQ ID NO: 1) and an AviTag.
  • Freestyle 293-F cells were grown in Freestyle 293 expression medium (Thermo Fisher) in suspension culture. Upon transfection, 300 mL cells were seeded to 1-L shaker flasks at a cell density of 10 6 cells/mL. 15 mL Opti-MEM medium (Thermo Fisher) containing 300 pg of PB-CMV plasmid DNA was mixed with 15 mL Opti-MEM medium containing 400 pL 293fectin reagent (Thermo Fisher). The mixture was incubated for 5 minutes before being added to the shaker flask. Two days post transfection, each 300 mL culture was expanded to 3 shaker flasks containing a total of 900 mL medium, and the expression was continued for another 4 days.
  • Freestyle 293-F cells were grown in Freestyle 293 expression medium (Thermo Fisher) in suspension culture. Upon transfection, 300 mL cells were seeded to 1-L shaker flasks at a cell density of 10
  • Freestyle 293-F cells or a GnTl-knockout Freestyle 293-F cell line was used for generating the stable cell lines.
  • the cells were transfected on 6-well plates. Each well contained 10 6 cells growing in 2 mL Freestyle 293 expression medium.
  • the cell lines were grown as suspension cultures in 1-L shaker flasks. Each flask contained 300 mL Freestyle 293 expression medium supplemented with 1 pg/mL doxycycline and 1 pg/mL aprotinin. Half of the culture was harvested and replaced by fresh medium every other day.
  • the proteins were purified from the harvested expression medium using Ni-NTA affinity chromatography.
  • the proteins were eluted with phosphate buffered saline containing 300 mM imidazole and 0.1% (v/v) protease inhibitor cocktail (Sigma, P-8849).
  • the proteins were further purified using size-exclusion chromatography.
  • a Superdex 200 Increase (GE healthcare) column was used.
  • a Superose 6 Increase (GE healthcare) column was used.
  • Each biotinylation reaction contained 200 pM biotin, 500 pM ATP, 500 pM MgCh, 30 pg/mL BirA, 0.1% (v/v) protease inhibitor cocktail and not more than 100 pM of the protein- AviTag substrate.
  • the reactions were incubated at 30 °C for 2 hours.
  • the biotinylated proteins were then purified by size-exclusion chromatography. 2.
  • Fab-phage clones specific for the SARS-CoV-2 S protein were isolated from phage- displayed antibody libraries (Persson et al. (2013) J Mol Biol 22;425(4):803-l 1) by multiple rounds of binding selections with the RBD immobilized in wells of neutravidin-coated microwell plates, as described (Persson et al. (2013)). Individual colonies were isolated from Escherichia coli infected with phage outputs from rounds 3 and 4, and individual Fab-phage clones were amplified. Antibody variable domains were sequenced by PCR amplification from phage supernatants for extraction of DNA sequences encoding antibody complementarity determining regions.
  • Phage clone variable domain DNA was amplified by PCR and subcloned into pSCSTa-hlgl and pSCSTl-hk vectors.
  • Vectors for the heavy and light chains were transfected into HEK293F cells (Invitrogen, Grand Island, NY) using FectoPro according to the manufacturer’s instructions (Polyplus Transfection, NY). Cell cultures were incubated at 37 °C for 4-5 days post-transfection. The cell cultures were centrifuged, and the supernatants were applied to a protein-A affinity column ( ⁇ 2 mL packed beads per 600 mL culture) (Pierce, ThermoScientific, Rockford, IL).
  • IgG proteins were eluted with 100 mM glycine, pH 2.0 and neutralized with 2 M Tris, pH 7.5.
  • the eluent underwent buffer exchange and concentration in to PBS, pH 7.4 by centrifugation in a 50 kDa centrifugal concentrator.
  • the plates were washed with PBST (3.2 mM Na 2 HPO 4 , 0.5 mM KH 2 PO 4 , 1.3 mM KCl, 135 mM NaCl, 0.05% TWEENTM 20, pH 7.4.) and incubated for 30 minutes with anti-k-HRP (horse radish peroxidase) antibody conjugate (1 :7500 dilution in PBT).
  • PBST 3.2 mM Na 2 HPO 4 , 0.5 mM KH 2 PO 4 , 1.3 mM KCl, 135 mM NaCl, 0.05% TWEENTM 20, pH 7.4.
  • anti-k-HRP human radish peroxidase
  • S protein was immobilized in 384-well plates as above but at a concentration of 5 pg/well, then wells were quenched with 100 pg/mL biotin.
  • Immobilized S protein was blocked with 100 nM IgG for 30 minutes and 100 nM biotinylated ACE2 was added to both IgG- blocked and non-blocked wells in parallel. Following a 30 minutes incubation, the plates were washed withPBST, and HRP/streptavidin conjugate (1 : 10000 dilution in PBST) was added and incubated for 30 min. The plates were washed with PBST, developed with TMB substrate, and quenched with 0.5 M H2SO4 before measuring absorbance at 450 nm.
  • FIG 1A depicts binding of unique Fab-phage clones to immobilized RBD blocked by solution-phase ACE2. The signal was normalized to the signal in the absence of ACE2 (control).
  • FIG IB depicts serial dilutions of IgGs binding to immobilized SARS-CoV-2 S protein. The EC50 values for each curve are shown in parentheses (in qg/mL and qM).
  • FIG 1C depicts binding of Fab-phage to immobilized RBD or negative controls, as indicated.
  • FIG ID depicts binding of ACE2 to immobilized S protein the presence (white bars) or absence (black bars) of 100 qM IgG
  • SARS-CoV-2 strain 2019 n-CoV/USA_WAl/2020 was obtained from the Centers for Disease Control and Prevention (gift of Natalie Thornburg).
  • Virus stocks were produced in Vero CCL81 cells (ATCC) and titrated by focusforming assay on Vero E6 cells.
  • Serial dilutions of mAbs were incubated with IO2 focusforming units (FFU) of SARS-CoV-2 for 1 h at 37°C.
  • FFU focusforming units
  • MAb-virus complexes were added to Vero E6 cell monolayers in 96-well plates and incubated at 37 °C for 1 h.
  • SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies). Data were processed using Prism software (GraphPad Prism 8.0).
  • Linkers of the various antibody formats can be of variable length and composition
  • CDR sequences in the various formats are those of clone 15031, the CDRs in these various formats may be replaced with the CDRs of clones 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049.

Abstract

Described herein are synthetic monoclonal antibodies, built on a human IgG framework, e.g., Trastuzumab, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2 infection and proliferation. The antibodies that exhibit neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells are bound to the receptor binding domain (RBD).

Description

COVID-19-BINDING IMMUNOGLOBULINS AND METHODS FOR THEIR USE
[0001] SEQUENCE LISTING
[0000.1] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on October 6, 2021, is named 120733_PD759WO_SL.txt and is 81,112 bytes in size.
[0002] BACKGROUND
[0003] Pathogenic strains of coronaviruses have caused serious zoonotic infections in humans three times in the last 20 years. SARS-CoV-2, the cause of the ongoing pandemic of Coronavirus Disease 2019 (COVID-19), is a novel coronavirus belonging to Coronaviridae family, beta-coronavirus genus and Sarbecovirus subgenus. It has a linear, positive-sense, single-stranded RNA genome of approximately 30 kilobases that is 86% identical to that of the SARS-CoV. The SARS-CoV-2 genome shows a high level of homology with SARS-like coronavirus isolated from bats (RaTG13 in particular) and a strain isolated from pangolins (99% identity), and the virus may have originated from recombination between CoVs that infect pangolins and bats (Xiao, K., et al. (2020) Isolation of SARSCoV-2-related coronavirus from Malayan pangolins. Nature).
[0004] SARS-CoV-2 utilizes an extensively glycosylated spike (S) protein that protrudes from the viral membrane, to mediate host-cell entry (Yan, R., et al. (2020) Structural basis for the recognition of SARS-CoV-2 by full-length human ACE2. Science 367, 1444-1448.) The S protein contains 1,282 amino acids organized into 2 subunits (SI and S2) and forms a homotrimer on the virus surface. The receptor binding domain (RBD), located in the SI subunit, recognizes the host cell receptor, angiotensin converting enzyme 2 (ACE2), the binding of which facilitates cleavage of the S2 subunit into S2' and S2" by cell-surface proteases, which in turn enables fusion and internalization of the virus (Hoffmann, et al. (2020) SARS-CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically Proven Protease Inhibitor; Cell 181, 271-280.) Being responsible for binding to the host ACE2 receptor, the RBD of the S protein presents neutralizing epitopes for CO VID-19 (Wu, et al., (2020) A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2. Science; Shi, et al. (2020) A human neutralizing antibody targets the receptor binding site of SARS-CoV-2, Nature, and; Pinto, et al. (2020)). Cross- neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody, Nature 583, 290-295) and many studies have shown that the S protein is a major determinant of the immune response in both convalescent patients and in positive asymptomatic individuals.
[0005] As of July 17, 2021, the ongoing COVID-19 viral pandemic has tallied more than 188,000,000 confirmed cases and caused over 4,000,000 deaths worldwide (www.who.int). As of October 2021, a number of vaccines have been approved for use worldwide, including JNJ-78436735 (Johnson & Johnson’s Janssen (J&J/Janssen) COVID-19 Vaccine), as well as mRNA-based vaccines such as mRNA-1273 (Moderna COVID-19 Vaccine) and BNT162b2 (Pfizer-BioNTech COVID-19 Vaccine). In addition, over 6 billion COVID-19 vaccine doses have been administered to date. However, worldwide vaccine availability, vaccine hesitancy, and the possibility of vaccine resistance due to viral mutations and novel variants may become a barrier to eradicating the virus and without an effective treatment the virus continues to cause severe illness and death. Several pharmacological treatments are currently under assessment in randomized clinical trials, including many repurposed existing drugs, such as remdesivir, hydroxychloroquine as well as alpha-interferon, and several novel agents targeting key virus-host interactions. Current therapeutic strategies are mainly supportive and aimed at delaying the spread of the virus and to reduce the impact of the disease. Although licensed vaccines are now being applied (Poland, G.A., Ovsyannikova, I.G., Kennedy, R.B., (2020). SARS-CoV-2 immunity: review and applications to phase 3 vaccine candidates. The Lancet, 396, 1595-1606), their effectiveness across demographics, availability worldwide, and resistance to adoption by the public remains an unknown. Given the severity of the disease, and its rapid spread in the population, more effective therapies and vaccines are needed as soon as possible to deal with this pandemic (Hung et al. (2020)). Triple combination of interferon beta- lb, lopinavir-ritonavir, and ribavirin in the treatment of patients admitted to hospital with COVID-19: an open-label, randomized, phase 2 trial. Lancet 395.). Almost certainly, COVID-19 will remain a serious human health concern for the foreseeable future and there is an urgent need for the development of therapeutics capable of treating patients and those at high risk for infection and/or with a poor prognosis.
[0006] SUMMARY OF INVENTION
[0007] Herein we describe antibodies and binding fragments thereof that bind to the spike protein of SARS-CoV-2 (also referred to interchangeably herein as COVID-19), and inhibit or compete for ACE2 binding. The antibodies described herein exhibit neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WAl in Vero E6 cells, and also bind to the receptor binding domain (RBD) of SARS-CoV-2. The antibodies and binding fragments described herein may be engineered to reduce or eliminate effector function.
[0008] An embodiment of this invention is a full-length SARS-CoV-2-binding antibody, (for example, a monoclonal antibody or a chimeric antibody), or a monovalent binding fragment thereof, (for example, a Fab, a F(ab)2, a F(ab’)2, a Fv, a disulfide linked Fv, a scFv, a disulfide linked scFv), and/or a single chain domain antibody, (for example, a VHH, or a scFab), or a diabody (see e.g., Figure 4). The antibody and binding fragments thereof comprise the heavy chain complementarity determining regions (CDRs) and/or light chain CDRs of an antibody identified as 15031, 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049. Table 2 and Table 3 present the amino acid and nucleotide sequences of the heavy chain and light chain complementarity determining regions (CDR1, CDR2 and CDR3) of the antibodies identified as 15031, 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049. The antibodies described herein may be interchangeably referenced throughout this specification and accompanying figures by a 5- digit nomenclature (for example, 15031, 15032, 15033, etc.) and/or may be identified by the last two to four digits (for example, 31, 32, 33-7, 33-A2, etc.).
[0009] An embodiment of this invention is a method for detecting SARS-CoV-2, a SARS- CoV-2 S polypeptide or fragment thereof in a sample, e.g., a biological sample from a subject, or a sample not from a subject, e.g., wastewater or a body of water receiving wastewater, using an antibody of this invention.
[0010] An embodiment of this invention is a method for inhibiting SARS-CoV-2 infection of a cell having a receptor for SARS-CoV-2, e.g., ACE2, by contacting the cell with an effective amount of an antibody or binding fragment described herein.
[0011] An embodiment of this invention is a method for inhibiting SARS-COV-2 S protein binding to an ACE2 receptor by administering an antibody, or an antibody binding fragment, of this invention to a subject in need thereof.
[0012] An embodiment of this invention is a method for treating as subject infected with SARS-COV-2 with an effective amount of an antibody or binding fragments and pharmaceutical compositions comprising the antibodies and/or binding fragments described herein. [0013] Another embodiment includes a nucleic acid encoding an antibody and/or binding fragments described herein.
[0014] A further embodiment is a vector comprising a nucleic acid of this invention.
[0015] Another embodiment includes a recombinant cell producing an antibody, binding fragments, nucleic acid, or vector of this invention.
[0016] Another embodiment is an immunoassay comprising or using an antibody and/or binding fragments described herein.
[0017] In an embodiment, the immunoassay is an enzyme linked immunosorbent assay (ELISA).
[0018] Another aspect of this invention is a kit comprising an antibody and/or binding fragments described herein and the nucleic acid molecules and/or vectors comprising the nucleic acid molecules that encode the antibodies and/or binding fragments described herein. The kit may include additional reagents, controls, and instructions for how to use the kit.
[0019] BRIEF DESCRIPTION OF THE FIGURES
[0020] Figure 1 - Characterization of IgGs by ELISA. FIG 1A depicts binding of unique Fab-phage clones to immobilized RBD blocked by solution-phase ACE2. Signal was normalized to the signal in the absence of ACE2 (control). FIG IB depicts serial dilutions of IgGs binding to immobilized SARS-CoV-2 S protein. The ECso values for each curve are shown in parentheses (in qg/mL and qM). FIG 1C depicts binding of Fab-phage to immobilized RBD or negative controls, as indicated. FIG ID depicts binding of ACE2 to immobilized S protein the presence (white bars) or absence (black bars) of 100 qM IgG.
[0021] Figure 2 - Binding kinetics for IgGs and SARS-CoV-2 S protein. Bio-layer interferometry (BLI) was used to evaluate antibody binding kinetics to immobilized S protein over a range of IgG concentrations (67 - 0.8 qM). Evolved sensor signals were fit and values for binding kinetics were extracted, and are shown in the table at the bottom (Table 1), along with ECso values derived from ELISA curves in Figure IB. As shown, the antibodies herein have dissociation constant (KD) for the SARS-COV-2 or a SARS-COV-2 S polypeptide of about or less than 0.8±0.1 qM, about or less than 0.4±0.1 qM or about or less than 0.2±0.1 qM.
[0022] Figure 3 - Neutralization of virus by IgGs. Microneutralization assays were performed in St Louis (Top) or Rome (Bottom). IgG-mediated neutralization of virus was assessed in a focal reduction neutralization assay and expressed as % relative infection, and infection curves were fitted to extract IC50 values, which are shown in parentheses (in rg/mL and r|M).
[0023] Figure 4 depicts various formats of the antibody, and binding fragments of this invention. Black lines = linkers. Spheres represent the cysteines of an intermolecular disulphide bond.
[0024] Figure 5 - Characterization of IgG 15031. (A) Expressed and Protein A-purified human IgGl antibody derived from phage clone 15031 was assessed for binding to SARS CoV-2 S-protein by ELISA versus control capture protein (neutravidin) over a range of IgG concentrations to estimate EC50 value (shown inset) (B) Binding kinetics of the interaction between IgG 15031 and SARS CoV-2 S-protein (left panel) was also assessed by biolayer interferometry to estimate the equilibrium dissociation constant (KD) (shown insert) in comparison to a non-specific control protein (right panel) (C) Analytical SEC of IgG 15031 and the control clinical benchmark IgG (trastuzumab) (D) Antibody-mediated neutralization was assessed in a focus reduction neutralization assay following incubation of serial dilutions of antibody with the Washington strain of SARS CoV-2 (2019 n-CoV/USA_WAl/2020). Samples were run in triplicate, using trastuzumab as a non-neutralizing isotype control and the infection signal normalized relative to the control antibody. Estimated IC50 values for the neutralizing antibody obtained from the fit curves are shown inset.
[0025] Figure 6 - Characterization of IgG 15032. (A) Expressed and Protein A-purified human IgGl antibody derived from phage clone 15032 was assessed for binding to SARS CoV-2 S-protein by ELISA versus control capture protein (neutravidin) over a range of IgG concentrations to estimate EC50 value (shown inset) (B) Binding kinetics of the interaction between IgG 15032 and SARS CoV-2 S-protein (left panel) was also assessed by biolayer interferometry to estimate the equilibrium dissociation constant (KD) (shown inset) in comparison to a non-specific control protein (right panel) (C) Analytical SEC of IgG 15032 and the control clinical benchmark IgG (trastuzumab) (D) Antibody-mediated neutralization was assessed in a focus reduction neutralization assay following incubation of serial dilutions of antibody with the Washington strain of SARS CoV-2 (2019 n-CoV/USA_WAl/2020). Samples were run in triplicate, using trastuzumab as a non-neutralizing isotype control and the infection signal normalized relative to the control antibody at the same concentration. Estimated IC50 values for the neutralizing antibody obtained from the fit curves are shown inset. [0026] Figure 7 - Characterization of IgG 33 (A) Expressed and Protein A-purified human IgGl antibody derived from phage clone 15033 was assessed for binding to SARS CoV-2 S- protein by ELISA versus control capture protein (neutravidin) over a range of IgG concentrations to estimate ECso value (shown inset) (B) Binding kinetics of the interaction between IgG 15033 and SARS CoV-2 S-protein (left panel) was also assessed by biolayer interferometry to estimate the equilibrium dissociation constant (KD) (shown inset) in comparison to a non-specific control protein (right panel) (C) Analytical SEC of IgG 15033 and the control clinical benchmark IgG (trastuzumab) (D) ) Antibody-mediated neutralization was assessed in a focus reduction neutralization assay following incubation of serial dilutions of antibody with the Washington strain of SARS CoV-2 (2019 n- CoV/USA_WAl/2020). Samples were run in triplicate, using trastuzumab as a nonneutralizing isotype control and the infection signal normalized relative to the control antibody at the same concentration. Estimated ICso values for the neutralizing antibody obtained from the fit curves are shown inset.
[0027] Figure 8 - Characterization of IgG 15033-7. (A) Affinity matured IgG 15033-7, obtained following optimization of IgG 15033, was expressed and Protein A-purified then assessed for binding to Fc-tagged SARS CoV-2 RBD protein ELISA versus control capture protein (neutravidin) over a range of IgG concentrations (B) The binding of IgGs 15033 and 15033-7 versus SARS CoV-2 S-protein was assessed by biolayer interferometry and representative sensor traces shown for a single IgG concentration. The equilibrium dissociation constant and associated binding rate constants for each is reported in table formats (C) Analytical SEC of IgG 15033-7 and the control clinical benchmark IgG (trastuzumab) (D) IgG 15033-7 was evaluated in a pseudoviral infection assay that employs VLPs pseudotyped with S-protein bearing RBD mutations corresponding to clinically- observed variants of concern to estimate their potency and breadth of neutralization against the indicated variants. Samples were run in triplicate, and the infection signal normalized to a no antibody control. Estimated ICso values for the neutralizing antibody obtained from the fit curves are shown inset.
[0028] Figure 9 - Characterization of IgG 15033A2. (A) Affinity matured IgG 15033A2, obtained following optimization of IgG 15033-7, was expressed and Protein A-purified then assessed for binding to Fc-tagged SARS CoV-2 RBD protein by ELISA versus control BSA protein over a range of IgG concentrations (B) The binding of Fab 15033A2 versus Fc-tagged SARS CoV-2 RBD protein or control protein was assessed by biolayer interferometry over a range of Fab concentrations to obtain the equilibrium dissociation constant (KD) (shown inset) (C) Analytical SEC of IgG 15033A2 and the control clinical benchmark IgG (trastuzumab) (D) Evaluation of IgG 15033A2 in a pseudoviral infection assay that employs VLPs pseudotyped with S-protein bearing RBD mutations corresponding to clinically- observed variants of concern to estimate their potency and breadth of neutralization against the indicated variants. Samples were run in triplicate, and the infection signal normalized to a no antibody control. Estimated IC50 values for the neutralizing antibody obtained from the fit curves are shown inset.
[0029] Figure 10 - Comparing the potency of optimized IgG 15033A2 to IgG 15033-7 by pseudovirus neutralization. IgG 15033-7 and the optimized variant obtained by maturation of IgG 15033-7 (IgG 33 A2) were assessed by pseudovirus neutralization assay against a variant (MB61) toward which IgG 15033-7 showed partially compromised potency. Serial dilutions of the two antibodies were pre-incubated with equivalent concentrations of VLPs in triplicate in parallel to confirm enhanced neutralization.
[0030] Figure 11 - Characterization of IgG 36. (A) Expressed and Protein A-purified human IgGl derived from phage clone 15036 was assessed for binding to SARS CoV-2 S-protein by ELISA versus negative control protein (BSA) over a range of IgG concentrations to estimate the EC50 value (shown inset) (B) The binding of IgGs 15036 versus Fc-tagged SARS CoV-2 RBD protein was assessed by biolayer interferometry and sensor traces shown for serial dilutions of IgG. The equilibrium dissociation constant and associated binding rate constants for each is reported in table format below (C) Analytical SEC of IgG 15036 and the control clinical benchmark IgG (trastuzumab) (D) IgG 15036 was evaluated in a pseudoviral infection assay that employs VLPs pseudotyped with S-protein bearing RBD mutations corresponding to clinically-observed variants of concern to estimate their potency and breadth of neutralization against the indicated variants. Samples were run in triplicate, and the infection signal normalized to a no antibody control. Estimated IC50 values for the neutralizing antibody obtained from the fit curves are shown inset.
[0031] Figure 12 - Characterization of IgG 15046. (A) Expressed and Protein A-purified human IgGl derived from phage clone 15046 was assessed for binding to SARS CoV-2 S- protein by ELISA versus negative control protein (BSA) over a range of IgG concentrations (B) The binding of IgG 46 versus Fc-tagged SARS CoV-2 RBD protein was assessed by biolayer interferometry and sensor traces shown for serial dilutions of IgG. The equilibrium dissociation constant and associated binding rate constants for each is reported in table format below (C) Analytical SEC of IgG 15046 and the control clinical benchmark IgG (trastuzumab).
[0032] Figure 13 - Characterization of IgG 15046A1. (A) Affinity matured IgG 15046A1, obtained following optimization of IgG 15046, was expressed and Protein A-purified then assessed for binding to SARS CoV-2 S-protein by ELISA versus negative control protein (BSA) over a range of IgG concentrations to estimate ECso value (shown inset) (B) The binding of IgG 15046A1 versus SARS CoV-2 RBD protein or a biotinylated negative control protein was assessed by biolayer interferometry and sensor traces shown for serial dilutions of IgG. The equilibrium dissociation constant and associated binding rate constants for each is reported in table format below (C) Analytical SEC of IgG 15046A1 and the control clinical benchmark IgG (trastuzumab) (D) IgG 15046A1 and the parental IgG 46 was evaluated in a pseudoviral infection assay that employs VLPs pseudotyped with wild-type S-protein corresponding to S-protein from the original Wuhan strain of virus. Samples were run in triplicate, and the infection signal normalized relative to VLP infection in the absence of antibody.
[0033] Figure 14 - Epitope mapping of IgG 15036 and 15046. (A) IgG obtained from selections against SARS CoV2 S-protein were assessed for binding to both SARS and SARS CoV-2 RBD protein and a negative control protein by biolayer interferometry. The estimated equilibrium dissociation constants are reported in table format (B) A phage blocking ELISA was used to map the relative epitopes of antibodies 15033, 15036 and 15046 and previously published antibody (Yuan, Science 2020). Plate immobilized S-protein was incubated with a saturating concentration of the indicated blocking IgG (legend right). Fab-phage supernatant (x-axis) was added to S-protein-coated wells in the presence or absence of the indicated blocking antibody and binding evaluated by development with phage-binding anti -Ml 3 antibodies. Phage binding signals were normalized to the isotype control antibody well and plotted. In each case, Fab-phage clone binding was inhibited by its corresponding blocking IgG, expected as a positive control for blockade.
[0034] Figure 15 - Biophysical properties of IgG 15036 and 15046 (A) Poly-reactivity of IgGs 15036 and 15046 were assessed by ELISA against a panel of proteins known to be ‘sticky’ and the binding to which is predictive of poor pharmacokinetics. Following exposure of 50 nM IgG to the indicated protein (legend right), binding was detected by development with an anti-human Fc antibody. Binding signals were plotted for the two test samples as well as a positive (non-sticky) IgG control (trastuzumab) and a negative (sticky) control IgG (IgG 6606) for comparison. (B) Analytical SEC of the indicated IgGs were conducted to assess monodispersity and elution times relative to the clinical benchmark IgG (trastuzumab).
[0035] Figure 16 - Neutralization of SARS CoV2 variants by pseudovirus infection assay - comparison with clinically authorized antibodies. Neutralization assays were conducted using of a panel of VLPs pseudotyped with SARS-CoV2 S-protein bearing RBD mutations found in clinically isolated variants. Inhibition profiles for were generated for (A) IgG 15033A2, and two clinically authorized antibodies (B) bamlanivimab (Eli-Lilly) and (C) casirivimab (Regeneron) to estimate their potency and breadth of neutralization against the indicated variants. Samples were run in triplicate, normalized to samples run in the absence of antibody and error bars plotted as the standard deviation from the average of triplicate readings.
[0036] Figure 17 - Neutralization of authentic SARS CoV-2 virus by IgG 33-7 versus clinically authorized antibodies. A focus reduction neutralization assay was conducted using serial 10-fold dilutions of IgG or the indicated bi-specific IgG against an authentic SARS CoV-2 strain isolated in the USA (Washington strain (2019 n-CoV/USA_WAl/2020) and an isogenic version of the SARS-CoV-2 strain isolated in S. Africa (B.1.351) bearing multiple S-protein mutations including in the RBD. Samples were run in triplicate and error bars are expressed as the standard deviation of the average infection signal.
[0037] DETAILED DESCRIPTION OF THE INVENTION
[0038] Definitions
[0039] The term "amino acid" includes all of the naturally occurring amino acids as well as modified amino acids.
[0040] The term an "affinity matured" antibody or “maturation of an antibody” refers to an antibody with one or more alterations in one or more hypervariable regions (HVRs), compared to a parent or source antibody which does not possess such alterations, such alterations resulting in an improvement in the affinity of the antibody for antigen or to other desired properties of the molecule. The term "amino acid" includes all of the naturally occurring amino acids as well as modified amino acids.
[0041] The term "antibody" as used herein is intended to include human antibodies, monoclonal antibodies, polyclonal antibodies, antibody binding fragments, single chain and other chimeric antibodies. The antibody may be from recombinant sources and/or produced in transgenic animals. The basic antibody structural unit is known to comprise a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. A “whole antibody” or “full-length” refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen binding portion thereof. Each heavy chain is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL or CL1. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL 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, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Herein, the assignment of amino acids to each domain is as defined in accordance with INTERNATIONAL IMMLNOGENETICS INFORMATION SYSTEM (IMGT) numbering (Lefranc et al., IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains, Development and Comparative Immunology. 2003; 27:55-77). In general, antibody molecules obtained from humans relate to any of the classes IgG, IgM, IgA, IgE and IgD, which differ from one another by the nature of the heavy chain present in the molecule. Certain classes have subclasses as well, such as IgGi, IgG2, and others. Furthermore, in humans, the light chain may be a kappa (k) chain or a lambda (X) chain.
[0042] The term “antibody binding fragment”, or "binding fragment" or “antigen binding fragment” as used herein is intended to include without limitations Fab, Fab', F(ab')2, scFv, scFab, dsFv, ds-scFv, diabodies and single domain antibodies, e.g., VHH. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, scFab, VHH, dsFv, ds-scFv, diabodies, and other fragments can also be synthesized by recombinant techniques, see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883 and Holliger et al. (1993) PNAS. USA 90:6444-6448.
[0043] An antigen binding site or “binding site” is formed by amino acid residues of the N- terminal variable regions of the heavy chain (VH) and N-terminal variable regions of the light chains (VL). The three CDRs of a light chain and the CDRs of a heavy chain are disposed relative to each other in three-dimensional space to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of a bound antigen. In single domain antibodies, also known as nanobodies, the binding site is formed by only one heavy variable domain (VH). “VHH” as used herein refers to a human VH that has been engineered to be independent of the light chain (Nilvebrant et al. Curr Pharm Des. (2016) 22(43):6527-6537; Barthelemy et al. Journal of Biological Chemistry (2007) 283:3639-3654. Nilvebrant et al. J Mol Biol. (2021) 433(21):Epub).
[0044] The term "capture antibody" as used herein refers to an antibody or binding fragment thereof bound to a solid support and used to capture the target antigen in a sample, for example SARS-COV-2 or a SARS-COV-2 S polypeptide, e.g., an SI and or S2 subunit, optionally soluble SARS-COV-2 S polypeptide by forming a complex with the target antigen.
[0045] By "consisting essentially of', it is meant a limitation of the scope of composition or method described to the specified materials or steps that do not materially affect the basic and novel characteristic(s) of the subject invention. For example, in some cases antibodies or binding fragments "consisting essentially of' a disclosed sequence has the amino acid sequence of the disclosed sequence plus or minus about 5 amino acid residues at the boundaries of the sequence based upon the sequence from which it was derived, e.g. about 5 residues, 4 residues, 3 residues, 2 residues or about 1 residue less than the recited bounding amino acid residue, or about 1 residue, 2 residues, 3 residues, 4 residues, or 5 residues more than the recited bounding amino acid residue.
[0046] By "consisting of', it is meant the exclusion from the composition, method, or kit of any element, step, or ingredient not specified in the claim. For example, antibodies or binding fragments "consisting of' a disclosed sequence consists only of the disclosed amino acid sequence. [0047] A "conservative amino acid substitution" as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-chain length for one another. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size; alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, and valine are all typically considered to be small.
[0048] The “COVID-19 S polypeptide” as used herein is an extensively glycosylated spike (S) protein of SARs-CoV-2 that protrudes from the viral membrane, to mediate host-cell entry. The S polypeptide contains 1,282 amino acids organized into 2 subunits (SI and S2) and the receptor binding domain (RBD), is located in the SI subunit.
[0049] The term "denatured" as used herein refers to a polypeptide that has lost tertiary and/or secondary structure (e.g. fully unfolded protein), for example when exposed to denaturing conditions in SDS sample loading buffer.
[0050] The term "detectable tag" or “detectable label” as used herein refers to moieties such as peptide sequences, a radio-opaque or a radioisotope label, such as 3H, 14C; 32P; 35S; 123I, 125I, 133I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion that can be appended or introduced into recombinant protein.
[0051] The term "detection antibody" as used herein refers to an antibody or binding fragment thereof that binds a capture antibody or a target antigen, for example SARS-CoV-2 or a SARS-CoV-2 S polypeptide, or optionally a target antigen already in a complex with a capture antibody. For example, the detection antibody binds the capture antibody: SARS- CoV-2 or a SARS-CoV-2 S polypeptide complex at an epitope on the target antigen that is different than the one recognized by the capture antibody. [0052] “Diabody” or “Diabodies” as used herein are dimeric antibody fragments. In each polypeptide of the diabody, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fv fragments, the linker between the VL and VH is too short for intramolecular pairing and as such each antigen-binding site is formed by pairing of the VH and VL of one polypeptide with the VH and VL of the other polypeptide. Diabodies thus have two antigen-binding sites, and can be monospecific or bispecific, (see e.g., Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994) Structure 2: 1121-1123; Kontermann and Dubel eds., Antibody Engineering (2001) Springer-Verlag. New York. 790 pp. (ISBN 3-540-41354-5) incorporated herein by reference.
[0053] The term “effective amount” as used herein refers generally to an amount of the antibodies and binding fragments described herein, or a pharmaceutical composition comprising the antibodies or binding fragments, sufficient at dosages and periods of time necessary to achieve the desired result, e.g., to ameliorate at least one physical adverse effect of SARS-COV-2 infection or proliferation. This might be an amount sufficient to interfere with the binding of SARS-COV-2 to its target, and/or delay onset of, one or more symptoms of the disease. The amount required to be administered will depend on the binding affinity of the antibody for its specific antigen, and will also depend on the rate at which an administered antibody is depleted from the free volume other subject to which it is administered. Common ranges for therapeutically effective dosing of an antibody or antibody fragment of the invention may be, by way of nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight and from about 2 mg/kg to about 30 mg/kg. Common dosing frequencies may range, for example, from twice daily to once a week.
[0054] The term "epitope" as used herein refers to the site on the antigen that is recognized by the antibodies or binding fragments disclosed herein. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An antibody is said to specifically bind an antigen when the dissociation constant is <1 pM; e.g., <100 r|M, preferably <10 r|M and more preferably <1 qM.
[0055] The term the “Fc domain” refers to the constant region of immunoglobulin molecules and is also called the fragment crystallizable region. The Fc domain is composed of two identical protein fragments, derived from the second and third constant domains of the antibody's two heavy chains and the Fc domains of IgGs bear a highly conserved N- glycosylation site. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity.
[0056] The term "heavy chain complementarity determining region" as used herein refers to regions of hypervariability within the heavy chain variable region of an antibody molecule. The heavy chain variable region has three complementarity determining regions termed heavy chain complementarity determining region 1 (CDR1), heavy chain complementarity determining region 2 (CDR2) and heavy chain complementarity determining region 3 (CDR3) from the amino terminus to carboxy terminus. All CDRs and framework regions (FRs) disclosed herein, amino acid sequences of CDRs and FRs disclosed herein, and CDR- encoding or FR-encoding nucleic acid sequences disclosed herein, are intended to be defined in accordance with IMGT numbering (Lefranc et al. IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains. Development and Comparative Immunology. 2003; 27:55-77).
[0057] The term "heavy chain variable region" or “heavy chain variable domain” or “VH” as used herein refers to the variable domain of the heavy chain comprising the heavy chain complementarity determining region 1, heavy chain complementarity determining region 2 and heavy chain complementarity determining region 3.
[0058] The term "host cell" refers to a cell into which a recombinant DNA expression cassette or vector can be introduced to produce a recombinant cell. Host cells include but are not limited to, prokaryotic cells such as Escherichia coli and Bacillus subtilis, fungal cells such as yeast and Aspergillus, insect cells such as S2 drosophila cells and Sf9, or animal cells, including human cells, e.g., fibroblast cells, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, or HEK293 cells.
[0059] The term “inhibit” or “inhibition” or “inhibiting” includes its generally accepted meaning which includes prohibiting, preventing, restraining, alleviating, ameliorating, and slowing, stopping or reversing progression or severity of an infection. As such, the present method includes both therapeutic and/or prophylactic administration, as appropriate. Inhibition of virus pathogenesis or virus infection of the cell (for example, inhibition of viral cell entry, viral replication, and/or virus proliferation) is indicated when the antibody inhibits virus pathogenesis, infection and/or proliferation, in vitro or in vivo, by at least 20%, more preferably by at least 30%, even more preferably by at least 40%, at least 50%, at least 60, at least 70%, at least 80% or at least 90%, as compared to a non-specific, control antibody.
[0060] The term "isolated antibody or binding fragment thereof' or "isolated and purified antibody or binding fragment thereof' refers to an antibody or binding fragment thereof that is substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized and/or other antibodies, for example directed to a different epitope.
[0061] The term "KD" refers to the dissociation constant of a complex for example of a particular antibody-antigen interaction.
[0062] The term "light chain complementarity determining region" as used herein refers to regions of hypervariability within the light chain variable region of an antibody molecule. Light chain variable regions have three complementarity determining regions termed light chain complementarity determining region 1 (CDR1), light chain complementarity determining region 2 (CDR2) and light chain complementarity determining region 3 (CDR3) from the amino terminus to the carboxy terminus.
[0063] The term "light chain variable region" or “light chain variable domain” or “VL” as used herein refers to the variable domain of the light chain comprising the light chain complementarity determining region 1, light chain complementarity determining region 2 and light chain complementarity determining region 3.
[0064] The term "monoclonal antibody" (MAb), as used herein, refers to a population of antibody molecules that contain only one molecular species of antibody molecule consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of the monoclonal antibody are identical in all the molecules of the population. MAbs contain an antigen binding site capable of immunoreacting with a particular epitope of the antigen characterized by a unique binding affinity for it.
[0065] The term "native" or "natively folded" as used herein refers to a protein in its native conformation (e.g. 3D conformation) or in a conformation sufficient to confer functionality, including for example partially unfolded protein capable of binding a receptor or ligand.
[0066] The term "nucleic acid sequence" as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term "nucleic acid sequences" includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The term "isolated nucleic acid" as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5' and 3' ends of the nucleic acid) from which the nucleic acid is derived.
[0067] As used herein the term “paratope” includes the antigen binding site in the variable region of an antibody that binds to an epitope.
[0068] The term "polypeptide" as used herein refers to a polymer consisting of a large number of amino acid residues bonded together in a chain. The polypeptide can form a part or the whole of a protein. The polypeptide may be arranged in a long, continuous and unbranched peptide chain. The polypeptide may also be arranged in a biologically functional way. The polypeptide may be folded into a specific three-dimensional structure that confers it a defined activity. The term "polypeptide" as used herein is used interchangeably with the term "protein".
[0069] The term "polypeptide fragment" as used herein refers to a polypeptide that has an amino terminal and/or carboxy-terminal deletion, but where the remaining amino acid sequence is identical to the corresponding positions in the naturally-occurring sequence deduced, for example, from a full-length cDNA sequence.
[0070] The term "isolated polypeptide" as used herein refers to substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized.
[0071] The term "reference agent" as used herein refers to an agent that can be used in an assay and that can be for example a standard amount of SARS-CoV-2 or a SARS-CoV-2S polypeptide used as a reference for example for detecting SARS-COV-2 or a SARS-CoV-2 polypeptide in a sample or for screening or for diagnosing a subject having or suspected of having a SARS-CoV-2 infection.
[0072] The term "sample from a subject” or “biological sample" as used herein refers to any biological fluid, cell or tissue sample from a subject, which can be assayed for SARS-CoV-2 or a SARS-CoV-2 S polypeptide. For example, the sample can comprise lung or nasal secretions, lung lavage, urine, serum, plasma or cerebrospinal fluid. The sample can for example be a "post-treatment" sample wherein the sample is obtained after one or more treatments, or a "base-line sample" which is for example used as a base line for assessing disease progression.
[0073] The term "sandwich ELISA" as used herein refers to an ELISA comprising a solid support and a capture antibody or binding fragment thereof (specific for the antigen) immobilized onto the solid support. In such an ELISA an amount of target antigen in a sample is bound by the capture antibody (e.g. SARS-CoV-2 or a SARS-CoV-2 S polypeptide comprised in a sample). The bound antigen is detected by a second antibody or binding fragment thereof, i.e. a detection antibody or binding fragment thereof, which recognizes an epitope that is different from the one recognized by the capture antibody. The capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex) is detected by the detection antibody which can be covalently linked to an enzyme or can itself be detected by addition of a secondary antibody which is linked to an enzyme. For example, the capture antibody and/or the detection antibody can comprise CDR regions disclosed herein.
[0074] “SARs-CoV-2” or “COVID-19” as used herein is the novel coronavirus belonging to Coronaviridae family, beta-coronavirus genus and Sarbecovirus subgenus and the cause of the ongoing pandemic of Coronavirus Disease 2019 (COVID-19).
[0075] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide (Fv fragment has the antigen-binding site made of the VH and VL regions, but they lack the constant regions of Fab (CHI and CL) regions) further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired structure for antigen binding. For a review of scFv and other antibody fragments, see James D. Marks, Antibody Engineering, Chapter 2, Oxford University Press (1995) (Carl K. Borrebaeck, Ed.). [0076] The “COVID-19 S polypeptide” as used herein is an extensively glycosylated spike (S) protein of SARs-CoV-2 that protrudes from the viral membrane, to mediate host-cell entry. The S polypeptide contains 1,282 amino acids organized into 2 subunits (SI and S2) and the receptor binding domain (RBD), is located in the SI subunit.
[0077] The terms "subject", "individual", "host", and "patient", are used interchangeably herein and refer to any mammalian subject, e.g., a human being, a monkey, an ape, or a dog or cat, for whom diagnosis, treatment, or therapy is desired.
[0078] The terms "treatment", "treating" and the like are used herein to generally means obtaining a desired pharmacologic and/or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. "Treatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., slowing or arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. Such treatment is desirably performed prior to complete loss of function in the affected tissues. The subject therapy may be administered before the symptomatic stage of the disease, during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease.
[0079] The term "variant" as used herein includes one or more amino acid and/or nucleotide modifications in a sequence (polypeptide or nucleic acid respectively) for example, one or more modifications of a light chain or a heavy chain complementarity determining region (CDR) disclosed herein that perform substantially the same function as the light chain and heavy chain CDRs disclosed herein in substantially the same way. For instance, variants of the CDRs disclosed herein have the same function of being able to specifically bind to an epitope on SARS-CoV-2 or a SARS-CoV-2 S polypeptide or in the case of nucleotide modifications, encode CDRs that have same function of being able to specifically bind to an epitope on SARS-CoV-2 or a SARS-CoV-2 S polypeptide. For example, in the case of nucleotide modifications, codon optimized and degenerate sequences are included. Variants of CDRs disclosed herein include, without limitation, amino acid substitutions as well as additions and deletions to the CDR sequences disclosed herein. For example, the substitution, addition or deletion can be 1, 2, 3, 4, 5, 6, 7, or 8 amino acids and/or the corresponding number of nucleotides.
[0080] As used in this invention, the term "vector" refers to a nucleic acid delivery vehicle or plasmid that can be engineered to contain a nucleic acid molecule, e.g., a nucleic acid sequence encoding the binding molecules described herein. The vector that can express protein when inserted with a polynucleotide is called an expression vector. Vectors can be inserted into the host cell by transformation, transduction, or transfection, so that the carried genetic substances can be expressed in the host cell. Vectors are well known to the technical personnel in the field, including but not limited to: plasmid; phagemid; cosmid; artificial chromosome such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or Pl derived artificial chromosome (PAC); phage such as /.phage or Ml 3 phage and animal viruses etc. Animal viruses may include but not limited to, reverse transcriptase virus (including lentivirus), adenovirus, adeno-associated virus, herpes virus (e. g. herpes simplex virus), chicken pox virus, baculovirus, papilloma virus, and papova virus (such as SV40). A vector can contain multiple components that control expression of the binding molecules described herein, including but not limited to, promoters, e.g., viral or eukaryotic promoters, e.g., a CMV promoter, signal peptides, e.g., TRYP2 signal peptide, transcription initiation factor, enhancer, selection element, and reporter gene. In addition, the vector may also contain replication initiation site(s).
[0081] The term "level" as used herein refers to an amount (e.g. relative amount or concentration) of SARS-CoV-2 or a SARS-CoV-2 S polypeptide that is detectable or measurable in a sample. For example, the SARS-CoV-2 or a SARS-CoV-2 S polypeptide level can be a concentration such as pM or a relative amount such as 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.4, 2.6, 2.8, 3.0, 3.2, 3.4, 3.6, 3.8, 4.0, 4.2, 4.4, 4.6, 4.8, 5.0 and/or 10 times a control level, where for example, the control level is the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a healthy or asymptomatic subject.
[0082] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and/or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and/or steps. For example, a composition comprising antibodies or binding fragments is a composition that may comprise other elements in addition to the antibodies or binding fragments, e.g. functional moieties such as polypeptides, small molecules, or nucleic acids bound, e.g. covalently bound, to antibodies or binding fragments; agents that promote the stability of the antibodies or binding fragments composition, agents that promote the solubility of the antibodies or binding fragments composition, adjuvants, etc. as will be readily understood in the art, with the exception of elements that are encompassed by any negative provisos. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.
[0083] In understanding the scope of the present disclosure, the term "consisting" and its derivatives, as used herein, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
[0084] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about." Further, it is to be understood that "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.
[0085] The terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of plus or minus 0.1 to 10%, 1-10%, or preferably 1-5%, of the number to which reference is being made if this deviation would not negate the meaning of the word it modifies.
[0086] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
[0087] II. Antibody and/or Binding Fragment Thereof
[0088] The present disclosure relates to an antibody and/or binding fragment thereof as well as methods of making and use for example for diagnosing and/or prognosing SARS-CoV-2 infection and/or proliferation.
[0089] Described herein are antibodies and antibody binding fragments that specifically bind SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g., S protein subunits SI and S2. The antibodies described herein exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WAl in Vero E6 cells, and also bound to the receptor binding domain (RBD).
[0090] Accordingly, an embodiment of this invention is an antibody and/or binding fragment thereof that specifically binds SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g., S protein subunits SI or S2.
[0091] In some embodiments, the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention may be at least 50%, at least 55%, at least 60%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the heavy chain (hlgl) CDRs of an antibody of Table 2. In some embodiments, the antibodies and antigen-binding fragments thereof contain a heavy chain variable region having a CDR amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% 98%, or at least 99% identical to the amino acid sequence of the heavy chain (hlgl) CDRs of an antibody of Table 2. In some embodiments, the amino acid sequence of the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention consist essentially of the amino acid sequence of the heavy chain (hlgl) CDRs of an antibody of Table 2.
[0092] In some embodiments, the light chain CDRs of the antibodies and antigen-binding fragments of this invention may be at least 50%, at least 55%, at least 60%, at least 75%. at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identical to the amino acid sequence of the light chain (hk) CDRs of an antibody of Table 2. In some embodiments, the antibodies and antigen-binding fragments thereof contain a light chain variable region having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2. In some embodiments, the amino acid sequence of the heavy chain CDRs of the antibodies and antigen-binding fragments of this invention consist essentially of the amino acid sequence of light chain (hk) CDRs of an antibody of Table 2.
[0093] In some embodiments, the antibodies and antigen-binding fragments thereof comprise (a) a VH domain comprising three heavy chain variable region CDRs, the CDRs having an amino acid sequence at least 90%/o, at least 91%, at least 92%, at least 93%, at least 94%, 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of three heavy chain (hlgl) CDRs of an antibody of Table 2, and (b) a VL domain comprising three light chain CDRs having an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identical to the amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2.
[0094] In some embodiments, the antibodies and antigen-binding fragments thereof comprise (a) a VH domain comprising three heavy chain variable region CDRs, the CDRs having an amino acid sequence of three heavy chain (hlgl) CDRs of an antibody of Table 2, and/or (b) a VL domain comprising three light chain CDRs having an amino acid sequence of the light chain (hk) CDRs of an antibody in Table 2.
[0095] In an embodiment of the invention the antibody comprises an amino acid sequence at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identical to the amino acid sequence of a molecule of Table 4.
[0096] In an embodiment of the invention the antibody comprises an amino acid sequence of the molecules of Table 4. In an embodiment of the invention the antibody consists essentially of an amino acid sequence of the molecules of Table 4. Although the amino acid and nucleotide sequences of the molecules in Table 4 comprise the heavy chain and light chain CDRs of antibody 15031, embodiments of this invention include the amino acid and nucleotide of the molecules in Table 4 wherein the CDRs of 15031 are replaced with the CDRs of antibodies 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049 set forth in Tables 2 and 3.
[0097] A further aspect is an antibody and/or binding fragment of this invention, wherein the antibody and/or binding fragment thereof specifically binds to SARS-CoV-2 or a SARS- CoV-2 S polypeptide in an unfixed or mildly fixed sample.
[0098] In an embodiment, the antibody and/or binding fragment thereof comprises a VL domain comprising a CDR3 and a VH domain comprising a CDR1, a CDR2 and a CDR3, wherein the amino acid sequences of the CDRs comprise one or more of the CDR sequences set forth in Table 2.
[0099] In an embodiment, the antibody and/or binding fragment thereof comprises a light chain variable region comprising an hk CDR3 of Table 2 and the heavy chain variable region comprises a hlgl CDR1, a CDR2 and a CDR3 of an antibody set forth in Table 2.
[0100] In an embodiment, the antibody, binding fragment thereof, or CDRs disclosed herein may have one or more amino acid substitutions, including but not limited to conservative amino acid substitutions.
[0101] The antibody comprising the light chain and heavy chain CDRs set forth in Table 2 can be any class of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; and any isotype, including: IgGl, IgG2, IgG3 and IgG4.
[0102] Chimeric antibodies can be prepared using routine recombinant techniques. For example, an Fab of an IgG of Table 4 may be reformatted into another full length IgG by subcloning the variable domains of the immunoglobulin’s light and heavy chains into mammalian expression vectors and producing the IgG protein using human embryonic kidney cells (HEK293T). As described elsewhere any cell type suitable for expressing an antibody can be used.
[0103] In one embodiment, the antibody may be a full-length immunoglobulin molecule, e.g., a chimeric antibody, a human antibody, a humanized antibody, a polyclonal antibody, or a minibody, and the antibody binding fragment may be a Fab, a Fab', a F(ab)2, a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a disulfide linked scFv, a single chain domain antibody scFab or VHH, or a diabody, that does not comprise an Fc domain or fragment thereof .
[0104] The antigen binding fragments, e.g., Fab, Fab' and F(ab')2, scFv, scFab, dsFv, ds-scFv, VHH, or diabodies, and other fragments can be synthesized or expressed by recombinant techniques. Antibodies can also be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments.
[0105] In an embodiment, the antibody is a synthetic monoclonal antibody or a chimeric antibody comprising one or more CDRs selected from the CDRs of Table 2. In an embodiment, the antibody is a chimeric antibody comprising a VL domain comprising the hk CDR1, CDR2 and CDR3 from an antibody of Table 2 and/or a VH domain comprising the hlgl CDR1, CDR2 and CDR3 from an antibody of Table 2.
[0106] In an embodiment, the SARS-CoV-2 binding molecule is a VHH fragment, a scFv, an Fab or a diabody that comprises the hlgl CDR1, CDR2 and CDR3 and/or the hk CDR1, CDR2 or CDR3 from an antibody of Table 2.
[0107] The antibodies and antibody fragments described herein may be isolated antibodies and antibody fragments.
[0108] In an embodiment of the full-length antibodies of this invention the Fc domain of the antibody is engineered such that it does not target the cell that binds the antibody for ADCC or CDC-dependent death. In an embodiment of the invention the Fc domain is dimerized via a knob-in-hole configuration. Methods are well known in the art for mitigating antibody effector function, including for example amino acid substitutions in the Fc regions, e.g., the N297G (NG) and D265A, N297G (DANG) variants or L234A, L235A, P329G (LALA-PG) substitutions, see e.g., Lo et al. “Effector Attenuating Substitutions that Maintain Antibody Stability and Reduce Toxicity in Mice. The Journal of Biological Chemistry Vol. 292, No. 9, pp. 3900 -3908, March 3, 2017, incorporated herein by reference.
[0109] In an embodiment of this invention the antibody and/or binding fragment thereof is linked, e.g., conjugated or recombinantly fused, to a therapeutic moiety, e.g., a cytotoxin, a chemotherapeutic drug, an immunosuppressant or a radioactive metal ion.
[0110] In a further embodiment, the antibody and/or binding fragment is labelled and/or conjugated or fused to a tag, for example to produce a detection tool or a diagnostic agent. For example, the detectable tag can be a purification tag such as a His-tag, a HA-tag, a GST- tag, biotin or a FLAG-tag.
[0111] The label is preferably capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque or a radioisotope, such as 3H, 14C; 32P; 35S; 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion.
[0112] Another aspect of the disclosure relates to an antibody complex comprising the antibody and/or binding fragment described herein and SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
[0113] Yet another aspect of this invention is a nucleic acid encoding an antibody and/or binding fragment described herein. In an embodiment, the nucleic acid encodes an antibody and/or binding fragment comprising a light chain variable region comprising a CDR1, CDR2 and CDR3 and a heavy chain variable region comprising a CDR1 CDR2 and CDR3, wherein one or more of the CDRs are the light chain and heavy chain CDRs set forth in Table 2.
[0114] The degeneracy of the genetic code allows for different nucleic acids to encode the same amino acid sequence. Accordingly, also included are nucleotide sequences that encode an antibody disclosed herein that specifically binds SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
[0115] Also included in another embodiment are codon degenerate or optimized sequences. In another embodiment, the nucleic acid sequences are at least 70%, at least 75%, at least 80%, at least 90% and at least 95% identical to nucleic acid sequences of Table 3 or Table 4 encoding the molecules set forth in Table 2 or Table 4.
[0116] In an embodiment of this invention, the nucleic acid is an isolated nucleic acid.
[0117] Another aspect of this invention is a vector comprising the nucleic acid herein disclosed. In an embodiment, the vector is an isolated vector.
[0118] The vector can be any vector suitable for producing an antibody and/or binding fragment thereof, including for example vectors described herein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses).
[0119] A further aspect of this invention is a recombinant cell producing the antibody and/or binding fragment thereof herein disclosed or the vector herein disclosed.
[0120] The recombinant cell can be generated using any cell suitable for producing a polypeptide, for example suitable for producing an antibody and/or binding fragment thereof. [0121] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the invention may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.
[0122] More particularly, bacterial host cells suitable for producing recombinant antibody producing cells include E. coli, B. subtilis, Salmonella typhimurium, and various species within the genus Pseudomonas, Streptomyces, and Staphylococcus, as well as many other bacterial species well known to one of ordinary skill in the art. Suitable bacterial expression vectors preferably comprise a promoter which functions in the host cell, one or more selectable phenotypic markers, and a bacterial origin of replication. Representative promoters include the R-lactamase (penicillinase) and lactose promoter system, the trp promoter and the tac promoter. Representative selectable markers include various antibiotic resistance markers such as the kanamycin or ampicillin resistance genes. Suitable expression vectors include but are not limited to bacteriophages such as lambda derivatives or plasmids such as pBR322, the pUC plasmids pUC18, pUC19, pUC118, pUC119, and pNH8A, pNH16a, pNH18a, and Bluescript M13 (Stratagene, La Jolla, Calif.).
[0123] Suitable yeast and fungi host cells include, but are not limited to, Saccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus. Examples of vectors for expression in yeast S. cerevisiae include pYepSecl, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for the transformation of yeast and fungi are well known to those of ordinary skill in the art.
[0124] Suitable mammalian cells include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g, ATCC No. CCL 2), 293 (ATCC No. 1573), NS-1 cells and any derivatives of these lines.
[0125] In an embodiment, the mammalian cells used to produce a recombinant antibody are selected from CHO, HEK293 cells or FREESTYLE™ 293-F cells (Life technologies). FREESTYLE 293-F cell line is derived from the 293 cell line and can be used with the FREESTYLE™ MAX 293 Expression System, FREESTYLE™ 293 Expression System or other expression systems.
[0126] Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences.
[0127] In an embodiment, the vector is designed for production of a light chain or a heavy chain, e.g., an IgGl heavy chain.
[0128] Suitable insect cells include cells and cell lines from Bombyx or Spodotera species. Baculovirus vectors available for expression of proteins in cultured insect cells (SF 9 cells) include the pAc series and the pVL series.
[0129] The recombinant expression vectors may also contain genes which encode a fusion moiety (i.e. a "fusion protein") which provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including for example tags and labels described herein. Further, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
[0130] "Operatively linked" is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate regulatory sequences is dependent on the host cell chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
[0131] In an embodiment, expression of the antibody or binding fragment thereof is under the control of an inducible promoter. Examples of inducible non-fusion expression vectors include pTrc (28) and pET l id. [0132] The recombinant expression vectors may also contain a marker gene which facilitates the selection of host cells transformed or transfected with a recombinant molecule of the invention. Examples of selectable marker genes are genes encoding a protein such as G418 and hygromycin which confer resistance to certain drugs, P-galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. Transcription of the selectable marker gene is monitored by changes in the concentration of the selectable marker protein such as - galactosidase, chloramphenicol acetyltransferase, or firefly luciferase. If the selectable marker gene encodes a protein conferring antibiotic resistance such as neomycin resistance transformant cells can be selected with G418. Cells that have incorporated the selectable marker gene will survive, while the other cells die. This makes it possible to visualize and assay for expression of recombinant expression vectors of the invention and in particular to determine the effect of a mutation on expression and phenotype. It will be appreciated that selectable markers can be introduced on a separate vector from the nucleic acid of interest. Other selectable markers include fluorescent proteins such as GFP which may be cotransduced with the nucleic acid of interest.
[0133] Yet another aspect is a composition, e.g., a pharmaceutical comprising the antibody and/or binding fragment thereof, the nucleic acid herein disclosed or the recombinant cell herein disclosed, optionally in combination with a suitable diluent or carrier or excipient. The pharmaceutical composition may consist or consist essentially of the antibodies or binding fragment or the nucleic acid molecules expression cassettes and vectors encoding the antibodies or binding fragment described herein, and a pharmaceutically acceptable diluent, carrier or excipient. Suitable carriers, diluents and excipients, and their formulations are described in Remington: The Science and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company, Easton, Pa. 1995. Typically, an appropriate amount of a pharmaceutically-acceptable salt is used in the formulation to render the formulation isotonic. Examples of the pharmaceutically-acceptable carrier include, but are not limited to, saline, Ringer's solution and dextrose solution. The pH of the solution is preferably from about 5 to about 8, and more preferably from about 7 to about 7.5. Further carriers include sustained release preparations such as semipermeable matrices of solid hydrophobic polymers containing the agonist, which matrices are in the form of shaped articles, e.g., films, liposomes or microparticles. Delivery systems for administering therapeutic agents, e.g., antibodies and fragments thereof, are known in the art, see e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), Langer, 1990, Science 249: 1527-1533; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:20; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321 :574; Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228: 190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 7 1 : 105); U.S. Pat. Nos. 5,679,377; 5,916,597; 5,912,015; 5,989,463; 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253). Examples of polymers used in sustained release formulations include, but are not limited to, polyphydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene- co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, polypvinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides, poly(lactide-co-glycolides) (PLGA), and polyorthoesters. In one embodiment, the polymer used in a sustained release formulation is inert, free of leachable impurities, stable on storage, sterile, and biodegradable. It will be apparent to those persons skilled in the art that certain carriers may be more preferable depending upon, for instance, the route of administration and concentration of the antibodies or binding fragment being administered.
[0134] The composition can be a lyophilized powder or aqueous or non-aqueous solution or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes. Other components that may be present in such compositions include, but are not limited to, water, surfactants (pharmaceutically acceptable non-ionic surfactants such as, for example, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 80 (polyoxyethylene (20) sorbitan monooleate), zwitterionic, or ionic surfactants), alcohols, polyols, glycerin and vegetable oils.
[0135] Suitable diluents for nucleic acids include but are not limited to water, saline solutions and ethanol. Suitable diluents for polypeptides, including antibodies or fragments thereof and/or cells include but are not limited to saline solutions, pH buffered solutions and glycerol solutions or other solutions suitable for freezing polypeptides and/or cells.
[0136] The composition can further comprise stabilizing agents, for example reducing agents, hydrophobic additives, and protease inhibitors which are added to physiological buffers.
[0137] III. Methods [0138] Additional aspects of the invention are methods for using, producing or isolating an antibody and/or binding fragment thereof described herein with specific binding affinity to SARS-CoV-2 or a SARS-CoV-2 S polypeptide.
[0139] It has been reported the symptoms of COVID-19 infections include, e.g., fever, cough, shortness of breath, chills, repeated shaking with chills, muscle pain, headache, sore throat, loss of taste or smell, and can lead to physical complications, e.g., pneumonia and trouble breathing, organ failure in several organs, heart problems, severe lung condition, e.g., acute respiratory distress syndrome, blood clots, acute kidney injury and additional viral and bacterial infections. An embodiment of the invention relates to a method for treating, preventing, mitigating, or delaying the progression of a SARS-CoV-2 infection, or ameliorating a symptom or complication of a CO VID-19 infection, comprising administering an effective amount of an antibody or binding fragment of this invention to a subject having or suspected of having a CO VID-19 infection.
[0140] A method for treating, preventing, mitigating, or delaying the progression of a SARS- CoV-2 infection includes reducing viral load in a subject in need thereof. The antibody or binding fragment of the present invention may be administered systemically or locally, e.g., by injection (e.g. subcutaneous, intravenous, intraperitoneal, intrathecal, intraocular, etc.), inhalation, implantation, topically, or orally. Depending on the route of administration, the antibody or binding fragment may be coated in a material to protect the molecules from the action of acids and other natural conditions which may inactivate the molecules. The antibody or binding fragment described herein may be dissolved or suspended in a pharmaceutically acceptable, preferably aqueous carrier. In addition, the composition comprising the binding molecule can contain excipients, such as buffers, binding agents, blasting agents, diluents, flavors, lubricants, etc. An extensive listing of excipients that can be used in such a composition, can be, for example, taken from A. Kibbe, Handbook of Pharmaceutical Excipients (Kibbe, 2000). The antibody or binding fragment of this invention can also be administered in conjunction with other therapeutic regimens or agents for treatment of COVID-19 infections and the associated symptoms or complications, e.g., convalescent serum therapy, small molecules, oleandrin, neutralizing monoclonal antibodies, recombinant antibodies, cytokines, or antivirals, e.g., LY-CoV555 (Eli Lilly), e.g., recombinant anti-CD47/PD-Ll bispecific antibody (IBI322)( Innovent Biologies, Inc. ), remdesivir, Actemra, Oluminant (baricitinib), merimepodib, dexamethaone (glucocorticoid), Aviptadil (RLF-100) , hydroxy cl oropuine, Avigan (favipiravir, Avifavir), Bucillamine , niclosamide, aviptadil, PIKfyve kinase inhibitor, tyrosine kinase inhibitor efineptakin alfa, interferon beta, or SLV213 (Selva Therapeutics).
[0141] Effective dosages and schedules for administering the antibodies and fragments thereof of this invention and nucleic acids that encode them described herein may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of such antibodies and fragments thereof that must be administered will vary depending on, for example, the subject that will receive the binding molecule, the route of administration, the particular type of antibody or fragment thereof used and other drugs being administered. Guidance in selecting appropriate doses for antibodies and fragments thereof of this invention is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone, eds., Noges Publications, Park Ridge, N.J., (1985) ch. 22 and pp. 303-357; Smith, Antibodies in Human Diagnosis and Therapy, Haber, eds., Raven Press, New York (1977) pp. 365-389. The dosage ranges for the administration of the compositions are those large enough to produce the desired effect. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of the inflammation in the patient and can be determined by one of skill in the art. The dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days. While individual needs vary, determination of optimal ranges of effective amounts of the antibody, fragments thereof, nucleic acid and vector is within the skill of the art.
[0142] An embodiment of the invention relates to a method for preventing or delaying the cleavage of the S2 subunit, or the fusion of SARS-CoV-2 with the cell and internalization of SARS-CoV-2 by a cell, e.g., a cell having an ACE2 receptor, by contacting the cell with an effective amount of an antibody or antibody binding fragment of this invention.
[0143] Yet another aspect relates to a method for screening or for diagnosing a subject having or suspected of having a SARS-CoV-2 infection, the method comprising measuring the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample from a subject by contacting the sample with an antibody or binding fragment of this invention, wherein detecting a level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide bound by the antibody or binding fragment is indicative that the subject is infected with SARS-CoV-2. The method may further comprise the step of comparing the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample bound by the antibody or binding fragment with a negative or positive control, wherein an increased level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample bound by the antibody or binding fragment as compared to a negative control, e.g., a value derived from a group of subjects without SARS-CoV-2 infection, or convalescing from a SARS-CoV-2 infection, is indicative that the subject is infected with SARS-CoV-2 or wherein a level of SARS-CoV-2 similar to or exceeding the level of SARS-CoV-2 of the positive control, e.g., a value derived from a group of subjects known to have a SARS-CoV-2 infection, is indicative that the subject is infected with SARS- CoV-2.
[0144] An additional aspect of the invention is a method for prognosing CO VID-19 progression or lack thereof (e.g. recovery or worsening of disease). Accordingly an aspect is a method of prognosing a likelihood of recovery after CO VID-19 infection, the method comprising: a) measuring a level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample from a subject by contacting the sample with the antibodies or antibody fragments described herein; and b) comparing the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide bound by the antibodies or fragments thereof in the sample with a control, for example a control value derived from a group of subjects who recovered from or in whom the COVID-19 disease symptoms regressed, wherein a decreased level of SARS-CoV-2 in the sample bound by the antibodies or binding fragments compared to the control is indicative that the subject has an increased likelihood of recovery after COVID-19 infection. The level of SARS-CoV-2 may be measured by, e.g., measuring the amount of SARS-CoV-2 or a SARS-CoV-2 S polypeptide bound to the antibodies or antibody fragments of this invention by e.g., immunoprecipitation, an immunoassay, e.g. ELISA, immunoblot detection. The antibody-based detection can also be combined with a mass spectrophometric assay, for example as in the case of a particle-based flow cytometric assay.
[0145] In an embodiment, the control is a control value derived from a group of subjects recovered from COVID-19 infection and an increased level of SARS-CoV-2 or a SARS- CoV-2 S polypeptide in the sample compared to the control is indicative that the subject has a decreased likelihood of recovery after CO VID-19 infection and/or an increased likelihood of COVID-19 infection and associated adverse effects progressing.
[0146] A further embodiment of the invention is a method for prognosing the likelihood of progression of a COVID-19 infection and associated adverse effects, the method comprising: a) measuring a level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample from a subject by contacting the sample with the antibodies or antibody fragments described herein; and b) comparing the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample bound to the antibodies or antibody fragments described herein with a control, wherein an increased level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample compared to the control, for example wherein the control is a control value derived from a group of subjects who did not recover or progressed is indicative that the subject has an decreased likelihood of recovering from a SARS-CoV-2 infection.
[0147] In an embodiment, the control is a control value derived from a group of subjects who recovered and an increased level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in the sample compared to the control is indicative that the subject has a decreased likelihood of recovery after SARS-CoV-2 and/or an increased likelihood of SARS-CoV-2 infection and associated adverse effects progressing.
[0148] The sample can for example be taken after the subject has received a treatment and compared for example to a pre-treatment sample. Alternatively, the patient can be monitored after a repeating interval to assess for example if treatment or other intervention is necessary. In an embodiment, the test is repeated and plotted to assess the subject's progression.
[0149] The sample may be a tissue sample, e.g. lung tissue, or a biological fluid such as blood, or a fraction thereof such as plasma or serum, or a lung secretion or a pulmonary lavage. In an embodiment the biological fluid is saliva or a nasal secretion.
[0150] In another embodiment, the sample is selected from a fresh sample such as a fresh (e.g. not frozen) biological fluid sample or tissue sample, a onetime frozen biological fluid sample or tissue sample (e.g. frozen a single time at the time of obtaining the sample)) or a repeat frozen sample (e.g. frozen and thawed and frozen biological fluid sample or repeat frozen tissue sample). In an embodiment, the sample comprises native or natively folded SARS-CoV-2 polypeptide. In an embodiment, the sample is a fixed sample such as a mildly fixed sample wherein the fixation induces limited denaturation and/or unfolding.
[0151] In another embodiment, the isolated and purified antibody and/or binding fragment thereof is affinity matured. Affinity maturation can be performed as described for the initial selection, with antigen adsorbed to plastic plates, using a for example a phage library comprising variants of the CDR sequences.
[0152] A person skilled in the art will appreciate that several methods can be used to isolate and produce antibodies and/or binding fragments thereof with specific binding affinity to SARS-CoV-2 or a SARS-CoV-2 polypeptide, e.g. SARS-CoV-2 S polypeptide. A method that can be used is a phage display method. For example, SARS-CoV-2 or a SARS-CoV-2 S polypeptide is produced in order to isolate and characterize the antibody and/or binding fragment thereof. Phage from a human Fab phage-displayed library are selected following several rounds of panning. Phage with specific binding affinity to the SARS-CoV-2 or a SARS-CoV-2 S polypeptide, as determined by ELISA, are sequenced and cloned into vectors designed for production of light chain or heavy chain. The heavy chain can be for example an IgG, or an IgG isotype such as an IgGl or an IgG4. Antigen binding fragments and IgG polypeptides are then affinity purified by using, for example, Protein A affinity columns.
[0153] In another embodiment, a nucleic acid encoding an antibody or antibody binding fragment described herein is expressed in a host cell to make the antibody and/or binding fragment. In an embodiment, the method comprises: a) expressing in a host cell a nucleic acid encoding an antibody and/or binding fragment; b) culturing the host cell to produce the antibody and/or binding fragment; and c) isolating and/or purifying the antibody and/or binding fragment from the host cell.
[0154] In some embodiments, a nucleic acid encoding a single chain antibody is expressed. In other embodiments, multiple nucleic acids are expressed, for example a nucleic acid encoding an antibody light chain and a nucleic acid encoding an antibody heavy chain.
[0155] Suitable host cells and vectors are described above. Vectors and nucleic acids encoding an antibody or antibody biding fragment described herein may be introduced into mammalian cells via conventional techniques such as calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection and other liposome-based transfection agents, electroporation or microinjection.
[0156] Nucleic acid encoding an antibody or antibody biding fragment described herein may be directly introduced into mammalian cells using delivery vehicles such as retroviral vectors, adenoviral vectors and DNA virus vectors.
[0157] V. Assays
[0158] The SARS-CoV-2 or SARS-CoV-2 S polypeptide specific antibodies disclosed herein can be used in a variety of assays for binding, detecting and measuring SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample. For example, the antibodies can be used in an ELISA, as well as immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA) (Muhammad S. Alam, Nov 2018) Current Protocols in Immunology Vol 123, Issue 1., mass spectroscopy -based techniques, particle-based flow cytometric assay, fluorescence-activated cell sorting (FACS) and ELISA.
[0159] Immunodetection methods as described herein generally involve the detection or measuring of antibody :SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g. an SI of S2 subunit, complexes using antibodies and/or binding fragments thereof disclosed herein. The detection of such complexes is well known in the art and may be achieved through different methods, for example by using a detectable label or marker, such as a radioactive, fluorescent or enzymatic tag. Detection of these complexes may also involve the use of a ligand such as a secondary antibody and/or binding fragment thereof specific for SARS-CoV-2 or a SARS- CoV-2 S polypeptide or for the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex.
[0160] They can also be used to make detection assays, for example a sandwich ELISA, as one antibody can be used as a capture reagent to isolate the SARS-CoV-2 or a SARS-CoV-2 S polypeptide while another antibody binding a distinct epitope can be used as a detection reagent.
[0161] Accordingly, another aspect is an immunoassay comprising one or more antibodies and/or binding fragments thereof described herein.
[0162] In an embodiment, the immunoassay is an enzyme linked immunosorbent assay (ELISA). Antibodies and/or binding fragments thereof may be used in the context of detection assays such as ELISAs, for example sandwich ELISAs. The antibodies and binding fragments of this invention may be used as capture and detection antibodies for the detection of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in solution. For example, the full-length antibodies or binding fragments of this invention that recognize the S protein, e.g., the SI subunit, are immobilized and incubated with SARS-CoV-2 or a SARS-CoV-2 S polypeptide or subunit. The samples are then incubated with biotinylated IgGs recognizing epitopes on the virus, polypeptide, or subunit.
[0163] In an embodiment, the ELISA is a sandwich ELISA comprising a capture antibody and a detection antibody that bind to different epitopes. For example, the capture antibody is an antibody or binding fragment which specifically binds a first epitope of SARS-CoV-2 or a SARS-CoV-2 S polypeptide, e.g., a SI subunit or the RBD, and/or the detection antibody is an antibody or binding fragment which specifically binds a second epitope of the SARS- CoV-2 or a SARS-CoV-2 S polypeptide, e.g., the SI subunit or the RBD, wherein the first and second epitopes are different. In an embodiment, one or both of the capture or detection antibodies is an antibody and/or binding fragment thereof herein.
[0164] In one embodiment, the immunoassay is for the detection and/or measuring of SARS- CoV-2 or a SARS-CoV-2 S polypeptide, e.g., the SI or S2 subunit, in a sample, wherein the method of making the immunoassay comprises: a) coating a solid support with the capture antibody; b) contacting the capture antibody with the sample under conditions to form a capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex; c) removing unbound sample; d) contacting the capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex with the detection antibody; e) removing unbound detection antibody; and f) detecting and/or measuring the capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex, wherein one or both of the capture or detection antibodies is an antibody and/or binding fragment thereof herein.
[0165] In an embodiment, the ELISA is a competitive ELISA. In an embodiment, the ELISA is a direct ELISA. In an embodiment, the ELISA is an indirect ELISA.
[0166] As used herein, "solid supports" include any material to which SARS-CoV-2 or a SARS-CoV-2 S polypeptide and antibodies and/or binding fragments thereof herein disclosed are capable of binding to. For example, the solid support can include plastic, glass, polystyrene, nylon, polypropylene, nylon, polyethylene, dextran, amylases, natural and modified celluloses and polyacrylamides. For example, the solid support is a microtiter plate, magnetic beads, latex beads or array surfaces.
[0167] In embodiments of the immunoassays described herein, the sample may be contacted with an antibody and/or binding fragment thereof under appropriate conditions, for example, at a given temperature and for a sufficient period of time, to allow effective binding of SARS-CoV-2 or a SARS-CoV-2 S polypeptide to the antibody or binding fragment, thus forming an antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex, such as a capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex. For example, the contacting step is carried out at room temperature for about 30 minutes, about 60 minutes, about 2 hours or about 4 hours. For example, the contacting step is carried out at about 4°C. overnight.
[0168] The antibody and/or binding fragment as described herein may be, e.g., complexed with SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a suitable buffer. For example, the buffer has a pH of about 5.0 to about 10.0. For example, the buffer has a pH of 4.5, 6.5 or 7.4. For example, the buffer is a HBS-EP buffer, a KRH buffer or Tris-buffered saline. For example, the buffer comprises BSA and/or TWEEN™20.
[0169] Any unbound material in a sample may be removed by washing so that only the formed antibody :SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex remains on the solid support. For example, the unbound sample is washed with phosphate-buffered saline, optionally comprising bovine serum albumin (BSA).
[0170] In an embodiment, the detection antibody is labelled and/or conjugated to a tag.
[0171] For example, the detection antibody directly labelled and/or conjugated. For example, the detection antibody is indirectly labelled and/or conjugated. Indirect labels include for example fluorescent or chemiluminescent tags, metals, dyes or radionuclides attached to the antibody. Indirect labels include for example horseradish peroxidase, alkaline phosphatase (AP), beta-galactosidase and urease. For example, HRP can be used with a chromogenic substrate, for example tetramethybenzidine, which produces a soluble product in the presence of hydrogen peroxide that is detectable at 450 nm.
[0172] An embodiment of the invention is an assay for detecting and/or measuring level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide in a sample, the assay comprising: a) contacting a sample with the antibody and/or binding fragment described herein under conditions to form an antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex; and b) detecting and/or measuring the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex. A further embodiment is an assay for detecting and/or measuring SARS-CoV-2 or a SARS-CoV-2 S polypeptide the method comprising: a) contacting a sample, the sample being a biological fluid, with the antibody and/or binding fragment of the antibody as described herein under conditions to form an antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex; and b) detecting and/or measuring the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex. The assay for detecting and/or measuring the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide is performed under non-denaturing or mildly denaturing conditions.
[0173] In an embodiment, the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex is detected directly for example by using an antibody labeled with a detectable tag or fusion moiety. In an embodiment, the complex is detected indirectly using a secondary antibody specific for the antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex. [0174] In an embodiment, the assay for detecting or measuring the level of SARS-CoV-2 or a SARS-CoV-2 S polypeptide is an immunoprecipitation, immunoblot, immunohistochemistry or immunocytochemistry, proximity ligation assay (PLA), mass spectroscopy -based techniques and fluorescence-activated cell sorting (FACS), and ELISA.
[0175] Detecting can be performed using methods that are qualitative or measured using quantitative methods, for example by comparing to a standard or standard curve.
[0176] The sample used in the methods described herein may be a biological sample from a subject, e.g., a human or other primate, or the sample may be from another source. The biological sample from a subject may be a tissue or a fluid, e.g., blood and its component parts, serum or plasma, saliva, a lung secretion or a lung lavage, or urine etc. Other samples may be, e.g., wastewater or a body of water where wastewater drains, e.g., a river, canal, pond, bay etc.
[0177] IV. Kits
[0178] A further aspect of this invention relates to a kit comprising i) an antibody and/or binding fragment described herein, ii) a nucleic acid encoding the an antibody and/or binding fragment described herein, iii) a composition comprising the an antibody and/or binding fragment described herein or the nucleic acid encoding the antibody or fragment, or iv) a recombinant cell described herein, comprised in a vial such as a sterile vial or other housing and optionally a reference agent, and/or instructions for use thereof.
[0179] In an embodiment, the kit comprises components and/or is for use in performing an assay described herein.
[0180] For example, the kit is an ELISA kit and can comprise a first antibody, e.g. a capture antibody, for example attached to a solid support, and a second antibody, e.g. a detection antibody, that binds to SARS-CoV-2 or a SARS-CoV-2 S polypeptide and/or binds the capture antibody: SARS-CoV-2 or a SARS-CoV-2 S polypeptide complex, and that is conjugated to a detectable label. The first and/or second antibody may be an antibody or antibody fragment of this invention. Any combination of antibodies or antibody fragments described herein can be used.
[0181] In an embodiment, the kit is a diagnostic kit and the instructions are directed to a method described herein. [0182] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well- known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofection). Enzymatic reactions and purification techniques are performed according to manufacturer's specifications or as commonly accomplished in the art or as described herein. The foregoing techniques and procedures are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification. See e.g., Sambrook et al. Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures utilized in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.
[0183] The following non-limiting examples are illustrative of the present disclosure:
Examples
[0184] To identify antibodies that neutralize SARS-CoV-2 as a basis for therapeutic development, we conducted phage-display selections against the RBD of the S protein, isolated numerous clones that bound to the SARS-CoV-2 RBD or the full-length S protein ectodomain, and screened to identify those that blocked ACE2 binding. In these studies, a library containing 1010 Fab-phage clones was used for selections (Persson et al. (2013) CDR- H3 diversity is not required for antigen recognition by synthetic antibodies. J. Mol. Biol. 425, 803-811.). Among the high affinity Fab-phage clones, those that blocked ACE2 were selected for further characterization and expressed as full-length human IgG proteins with a framework engineered to possess high thermostability and low immunogenicity for therapeutic applications (Persson et al. (2013)). Several IgGs were found to exhibit sub- nanomolar affinities for the SARS-CoV-2 S protein and from this group antibody number 15033 emerged as a best neutralizer with an ability to neutralize SARS- CoV-2 with high potency.
1. Expression and purification of antigens
[0185] The previously reported piggyBac transposase-based expression plasmid PB-T-PAF (Li et al., (2013) Simple piggyBac transposon-based mammalian cell expression system for inducible protein production. Proc. Natl. Acad. Sci. U.S.A. 110, 5004-5009) was modified to generate two new vectors, one containing a CMV promotor (PB-CMV) and the other a TRE promotor (PB-TRE). To facilitate nuclear export of mRNA, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) was also added to each of these plasmids. Each of the protein ORFs was cloned into both expression vectors. The PB-CMV version was used for large- scale transient expression. The PB-TRE version was used for establishing inducible stable cell lines using the piggyBac transposase-based method (Li, Z., et al. (2013) Simple piggyBac transposon-based mammalian cell expression system for inducible protein production. Proc. Natl. Acad. Sci. U.S. A. 110, 5004-5009.).
[0186] cDNAs encoding the SARS-CoV and SARS-CoV-2 spike proteins were human codon optimized and synthesized by Genscript. cDNA encoding human ACE2 was obtained from MGC clone 47598. The soluble SARS-CoV-2 spike ectodomain trimer included residues 1- 1211, followed by a foldon trimerization motif (Tao et al. (1997) Structure of bacteriophage T4 fibritin: A segmented coiled coil and the role of the C-terminal domain. Structure 5, 789- 798), a 6xHis tag (SEQ ID NO: 1) and an AviTag biotinylation motif (Fairhead, M., and Howarth, M. (2015) Site-specific biotinylation of purified proteins using BirA. Methods Mol. Biol. 1266, 171-184.). Residues 682-685 (RRAR (SEQ ID NO: 100)) were mutated to SSAS (SEQ ID NO: 101) to remove the furin cleavage site on the SARS-CoV-2 spike protein.
Residues 986-987 (KV) were mutated to two proline residues to stabilize the pre-fusion form of the spike (Pallesen et al. (2017) Immunogenicity and structures of a rationally designed prefusion MERS-CoV spike antigen. Proc. Natl. Acad. Sci. U. S.A. 114, E7348-E7357.). The SARS-CoV-2 receptor binding domain (RBD, residues 328-528), the soluble human ACE2 construct (residues 19-615), and the SARS-CoV RBD (residues 315-514) were eachfollowed by a 6xHis tag (SEQ ID NO: 1) and an AviTag.
[0187] Freestyle 293-F cells were grown in Freestyle 293 expression medium (Thermo Fisher) in suspension culture. Upon transfection, 300 mL cells were seeded to 1-L shaker flasks at a cell density of 106 cells/mL. 15 mL Opti-MEM medium (Thermo Fisher) containing 300 pg of PB-CMV plasmid DNA was mixed with 15 mL Opti-MEM medium containing 400 pL 293fectin reagent (Thermo Fisher). The mixture was incubated for 5 minutes before being added to the shaker flask. Two days post transfection, each 300 mL culture was expanded to 3 shaker flasks containing a total of 900 mL medium, and the expression was continued for another 4 days.
[0188] Freestyle 293-F cells or a GnTl-knockout Freestyle 293-F cell line was used for generating the stable cell lines. The cells were transfected on 6-well plates. Each well contained 106 cells growing in 2 mL Freestyle 293 expression medium. For producing doxycycline-inducible cell lines, 2 pg of the PB- TRE expression plasmid, 0.5 pg of the PB- rtTA-neomycin helper plasmid (Li et al. (2013)) and 0.5 pg of the piggyBactransposase plasmid pCyL43 (Wang et al. (2008) Chromosomal transposition of PiggyBac in mouse embryonic stem cells. Proc. Natl. Acad. Sci. U. S. A. 105, 9290-9295.) were co-transfected to each well using the Lipofectamine 2000 reagent (Thermo Fisher). These cells were then selected using 2 pg/mL puromycin and 200 pg/mL G418 for two weeks. The ACE2- expressing cell line was constructed by co-transfecting the Freestyle 293-F cells with the PB- CMV plasmid encoding the full length human ACE2 and the pCyL43 plasmid. This cell line was selected using 2 pg/mL puromycin.
[0189] For stable expression, the cell lines were grown as suspension cultures in 1-L shaker flasks. Each flask contained 300 mL Freestyle 293 expression medium supplemented with 1 pg/mL doxycycline and 1 pg/mL aprotinin. Half of the culture was harvested and replaced by fresh medium every other day.
[0190] The proteins were purified from the harvested expression medium using Ni-NTA affinity chromatography. The proteins were eluted with phosphate buffered saline containing 300 mM imidazole and 0.1% (v/v) protease inhibitor cocktail (Sigma, P-8849). The proteins were further purified using size-exclusion chromatography. For the RBDs and ACE2, a Superdex 200 Increase (GE healthcare) column was used. For the spike ectodomain, a Superose 6 Increase (GE healthcare) column was used.
[0191] Each biotinylation reaction contained 200 pM biotin, 500 pM ATP, 500 pM MgCh, 30 pg/mL BirA, 0.1% (v/v) protease inhibitor cocktail and not more than 100 pM of the protein- AviTag substrate. The reactions were incubated at 30 °C for 2 hours. The biotinylated proteins were then purified by size-exclusion chromatography. 2. Fab-phage selections
[0192] Fab-phage clones specific for the SARS-CoV-2 S protein were isolated from phage- displayed antibody libraries (Persson et al. (2013) J Mol Biol 22;425(4):803-l 1) by multiple rounds of binding selections with the RBD immobilized in wells of neutravidin-coated microwell plates, as described (Persson et al. (2013)). Individual colonies were isolated from Escherichia coli infected with phage outputs from rounds 3 and 4, and individual Fab-phage clones were amplified. Antibody variable domains were sequenced by PCR amplification from phage supernatants for extraction of DNA sequences encoding antibody complementarity determining regions.
3. Expression and purification of IgG proteins
[0193] Phage clone variable domain DNA was amplified by PCR and subcloned into pSCSTa-hlgl and pSCSTl-hk vectors. Vectors for the heavy and light chains were transfected into HEK293F cells (Invitrogen, Grand Island, NY) using FectoPro according to the manufacturer’s instructions (Polyplus Transfection, NY). Cell cultures were incubated at 37 °C for 4-5 days post-transfection. The cell cultures were centrifuged, and the supernatants were applied to a protein-A affinity column (~2 mL packed beads per 600 mL culture) (Pierce, ThermoScientific, Rockford, IL). IgG proteins were eluted with 100 mM glycine, pH 2.0 and neutralized with 2 M Tris, pH 7.5. The eluent underwent buffer exchange and concentration in to PBS, pH 7.4 by centrifugation in a 50 kDa centrifugal concentrator.
4. Binding and competition ELISAs
[0194] For capture of biotinylated antigens, 384-well microplate wells were coated overnight at 4 °C with 2 pg/mL neutravidin in PBS pH 7.4. After coating, wells were blocked with 0.2% BSA in phosphate buffered saline (PBS) for one hour and washed 4 times with 0.05% TWEEN™ in PBS (PT buffer). Solutions of S protein (10 nM) or RBD (50 nM) or biotinylated control (50 nM) in PBS were used to immobilize target by incubating for 15 minutes at room temperature and washing with PT buffer. IgGs were diluted into PBT, applied to the wells, and incubated at room temperature for 30 min. The plates were washed with PBST (3.2 mM Na2HPO4, 0.5 mM KH2PO4, 1.3 mM KCl, 135 mM NaCl, 0.05% TWEEN™ 20, pH 7.4.) and incubated for 30 minutes with anti-k-HRP (horse radish peroxidase) antibody conjugate (1 :7500 dilution in PBT). The wells were washed 4-6 times with PBST and developed as described above. The absorbance at 450 nm was determined. To estimate ECso values, data were fit to standard four-parameter logistic equations using Graphpad Prism (GraphPad Software, La Jolla, CA).
[0195] For competition ELISAs, S protein was immobilized in 384-well plates as above but at a concentration of 5 pg/well, then wells were quenched with 100 pg/mL biotin.
Immobilized S protein was blocked with 100 nM IgG for 30 minutes and 100 nM biotinylated ACE2 was added to both IgG- blocked and non-blocked wells in parallel. Following a 30 minutes incubation, the plates were washed withPBST, and HRP/streptavidin conjugate (1 : 10000 dilution in PBST) was added and incubated for 30 min. The plates were washed with PBST, developed with TMB substrate, and quenched with 0.5 M H2SO4 before measuring absorbance at 450 nm.
[0196] The results of these ELISAs are presented in Figure 1. FIG 1A depicts binding of unique Fab-phage clones to immobilized RBD blocked by solution-phase ACE2. The signal was normalized to the signal in the absence of ACE2 (control). FIG IB depicts serial dilutions of IgGs binding to immobilized SARS-CoV-2 S protein. The EC50 values for each curve are shown in parentheses (in qg/mL and qM). FIG 1C depicts binding of Fab-phage to immobilized RBD or negative controls, as indicated. FIG ID depicts binding of ACE2 to immobilized S protein the presence (white bars) or absence (black bars) of 100 qM IgG
5. Binding kinetics
[0197] To determine the affinity and binding kinetics of IgGs for the S protein, BLI experiments were performed on an Octet HTX instrument (ForteBio) at 1000 rpm and 25 °C. Biotinylated S protein was first captured on SA biosensors (ForteBio) from a 2 pg/mL solution in PBT, in parallel with an identical concentration of an unrelated biotinylated control protein, followed by a 180 s quench step with 100 pg/mL biotin. After equilibrating with PBT, loaded biosensors were dipped for 600 s into wells containing serial 3 -fold dilutions of IgG and subsequently were transferred back into assay buffer for 600 s dissociation. Binding response data were reference subtracted and were fitted with 1 : 1 binding model using ForteBio’ s Data Analysis software 9.0.
[0198] The binding kinetics for IgGs and SARS-CoV-2 S protein are presented in Figure 2 and Table 1.
Table 1
Figure imgf000046_0001
,
6. Virus neutralization assays
[0199] For assays conducted in St Louis, SARS-CoV-2 strain 2019 n-CoV/USA_WAl/2020 was obtained from the Centers for Disease Control and Prevention (gift of Natalie Thornburg). Virus stocks were produced in Vero CCL81 cells (ATCC) and titrated by focusforming assay on Vero E6 cells. Serial dilutions of mAbs were incubated with IO2 focusforming units (FFU) of SARS-CoV-2 for 1 h at 37°C. MAb-virus complexes were added to Vero E6 cell monolayers in 96-well plates and incubated at 37 °C for 1 h. Subsequently, cells were overlaid with 1% (w/v) methylcellulose in MEM supplemented with 2% FBS. Plates were harvested 30 hours later by removing overlays and fixed with 4% PFA in PBS for 20 minutes at room temperature. Plates were washed and sequentially incubated with 1 pg/mL anti-S antibody CR3022 (Yan et al. (2020) A highly conserved cryptic epitope in the receptor binding domains of SARS-CoV-2 and SARS-CoV. Science 368, 630-633) and HRP- conjugated goat anti-human IgG in PBS supplemented with 0.1% saponin and 0.1% BSA. SARS-CoV-2-infected cell foci were visualized using TrueBlue peroxidase substrate (KPL) and quantitated on an ImmunoSpot microanalyzer (Cellular Technologies). Data were processed using Prism software (GraphPad Prism 8.0).
[0200] For assays conducted in Rome, SARS-CoV-2 strain 2019-nCoV/Italy-INMI was used. Antibodies were diluted to a concentration of 10 pg/ml in serum -free medium, and titrated in duplicate in two-fold serial dilutions. Equal volumes (50 pl) of approximately 100 TCID50/well virus and antibody dilutions, were mixed and incubated at 37 °C for 30 min. Subsequently, 96-well tissue culture plates with sub-confluent Vero E6 cell monolayers were infected with 100 pl/well of virus- serum mixtures in duplicate and incubated at 37 °C and 5% CO2 for two days. Then, the supernatant of each plate was carefully discarded and 120 pl of a Crystal Violet solution containing 2% Formaldehyde was added to each well. After 30 minutes fixation, the fixing solution was removed and cell viability was measured by photometer at 595 nm (Synergy HTXBiotek).
[0201] The results of these neutralization assays are depicted in Figure 3. [0202] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the inventions. Various substitutions, alterations and modifications may be made to the invention without departing from the spirit and scope of the invention. Other aspects, advantages, and modifications are within the scope of the invention.
[0203] The contents of all references, issued patents, and published patent applications cited through this application are hereby incorporated by reference. The appropriate component, process and methods of those patents, applications and other documents may be selected for the invention and embodiments thereof.
[0204] To clarify the use of and to hereby provide notice to the public, the phrases "at least one of <A>, <B>, . . . and <N>" or "at least one of <A>, <B>, ... or <N>" or "at least one of <A>, <B>, . . . <N>, or combinations thereof' or "<A>, <B>, . . . and/or <N>" are defined by the Applicant in the broadest sense, superseding any other implied definitions hereinbefore or hereinafter unless expressly asserted by the Applicant to the contrary, to mean one or more elements selected from the group comprising A, B, ... and N. In other words, the phrases mean any combination of one or more of the elements A, B, ... or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional elements not listed. Unless otherwise indicated or the context suggests otherwise, as used herein, "a" or "an" means "at least one" or one or more.
CDR Overview
Table 2: Lead antibody clones - CDR amino acid sequences
Figure imgf000048_0001
Figure imgf000048_0003
Table 3 : Lead antibody clones - CDR nucleotide sequences
Figure imgf000048_0002
Figure imgf000048_0004
Figure imgf000049_0001
Table 4:
Notes -
CDRs in the nucleotide and amino acid sequences are shown as underlined
Linkers in the nucleotide and amino acid sequences are shown as underlined, italicized and BOLD
Linkers of the various antibody formats can be of variable length and composition
Although all the CDR sequences in the various formats are those of clone 15031, the CDRs in these various formats may be replaced with the CDRs of clones 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, and 15049.
1. LEAD VHH 15031 clone example
Figure imgf000050_0001
2. LEAD scFv 15031 clone example
Figure imgf000050_0002
Figure imgf000051_0002
3. LEAD Fab 15031 clone example
Figure imgf000051_0001
4. LEAD Diabody 15031 clone example
Figure imgf000052_0001
5. LEAD hlgGl 15031 clone example
Figure imgf000053_0001
6. LEAD hlgGl 15032 clone example
Figure imgf000054_0001
7. LEAD hlgGl 15033 clone example
Figure imgf000055_0001
8. LEAD hlgGl 15033-7 clone example
Figure imgf000056_0001
9. LEAD hlgGl 15033-A2 clone example
Figure imgf000057_0001
10. LEAD hlgGl 15036 clone example
Figure imgf000058_0001
11. LEAD hlgGl 15046 clone example
Figure imgf000059_0001
12. LEAD hlgGl 15046-Al clone example
Figure imgf000060_0001

Claims

WE CLAIM
1. An isolated antibody that specifically binds SARS-CoV-2 S protein, comprising three light chain complementary determining regions CDRs that are at least 75% identical to the light chain CDRs of an antibody in Table 2 (hk CDR1, CDR2 and CDR3), and/or three heavy chain CDRs that are at least 75% identical to the heavy chain CDRs of an antibody in Table 2 (hlgl CDR1, CDR2, and CDR3).
2. The isolated antibody of claim 1, wherein the isolated antibody comprises hk CDR1, CDR2 and CDR3 of an antibody in Table 2 and hlgl CDR1, CDR2, and CDR3 of an antibody in Table 2.
3. The isolated antibody of claim 1, wherein the antibody comprises the amino acid sequence of an antibody in Table 4.
4. The isolated antibody of claim 1, wherein the antibody consists essentially of the amino acid sequence of a molecule of Table 4.
5. The isolated antibody of claim 1, wherein the antibody comprises the amino acid sequence of an antibody in Table 4, wherein the CDRs of the antibody are replaced with the CDRs of antibody 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, or 15049.
6. The isolated antibody of claim 1 consisting essentially of the amino acid sequence of a molecule in Table 4, with the proviso that the CDRs of the molecules are replaced with the CDRs of antibody 15032, 15033, 15033-7, 15033-A2, 15034, 15036, 15046, 15046-Al, or 15049.
7. The isolated antibody of claim 1, wherein the antibody is an IgG or a binding fragment of an IgG.
8. The isolated antibody of claim 1, wherein the antibody is an IgGl or a binding fragment of an IgGl .
9. The isolated antibody of claim 1, wherein the isolated antibody is a monoclonal antibody, a humanized antibody a polyclonal antibody, or a chimeric antibody.
10. The isolated antibody of claim 1 wherein the antibody is an antibody fragment selected from the group consisting of Fab, an F(ab)2, a F(ab’)2, an Fv, a disulfide linked Fv, a scFv, a disulfide linked scFv, a single chain domain antibody, scFab, a VHH, or a diabody
59 The isolated antibody of claim 7, wherein the antibody comprises antigen binding fragments consisting essentially of,
(a) a VHH
(b) an scFv;
(c) an Fab;
(d) a diabody; and/or
(e) an IgG comprising the heavy chain and light chain CDRs of an antibody of Table 2. An antibody conjugate comprising the antibody of any one of claims 1-11 linked to one or more therapeutic moiety or labeling moiety. The antibody conjugate of claim 12 wherein the labeling moiety is radio-opaque or a radioisotope, such as 3H, 14C; 32P; 35S; 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion. A pharmaceutical composition comprising the antibody of any one of claims 1-11 and a pharmaceutically acceptable carrier or excipient. A pharmaceutical composition comprising the antibody conjugate of claim 12 and a pharmaceutically acceptable carrier or excipient. A nucleic acid encoding an antibody of any one of claims 1-11. A vector comprising the nucleic acid of claim 16. A recombinant cell comprising the nucleic acid of claim 16 or a vector comprising said nucleic acid. A composition comprising the nucleic acid of claim 16 or a vector comprising said nucleic acid. A composition comprising the recombinant cell of claim 18. A method for producing an antibody with specific binding affinity to an epitope of SARS-CoV-2 S protein, the steps comprising:
60 a. expressing in a host cell the nucleic acid according to claim 16; b. culturing the host cell to produce the antibody; and c. isolating and purifying the antibody from the host cell. A method of inhibiting infection of a cell and/or proliferation of SARS-CoV-2 comprising contacting SARS-CoV-2 with an effective amount of an antibody of any one of claims 1-11. A method of inhibiting infection of a cell and/or proliferation of SARS-CoV-2 comprising contacting SARS-CoV-2 with an effective amount of an antibody conjugate of claim 12. The method of claim 23, wherein the SARS-CoV-2 is contacted with the antibody in vitro or in vivo. A method of treating a subject infected with SARS-CoV-2 comprising administering an effective amount of the antibody of any one of claims 1-11 to the subject. The method of claim 26, wherein the antibody is administered by infusion or inhalation. The method of claim 26, wherein the antibody is administered orally, rectally, intravenously, intraperitoneally, intracardially, intraarterially, subcutaneously, intrathecally, intranasally, intraocularly, or topically. The method of claim 26, wherein the antibody is injected directly into the subject’s liver, heart or lungs. A method for determining the amount of SARS-CoV-2 in a sample comprising contacting the sample with an antibody of any one of claims 1-11 and assessing the level of antibody bound to the SARS-CoV-2 or the amount of SARS-CoV-2 bound to the antibody, wherein the amount of bound antibody or SARS-CoV-2 indicates the amount of SARS-CoV-2 in the sample. The method of claim 29, wherein the amount of bound antibody or amount of bound SARS-CoV-2 is compared to a control. The method of claim 29, wherein the amount of bound antibody or amount bound SARS-CoV-2 is assessed by ELISA or a sandwich ELISA.
61 A method for isolating SARS-CoV-2 from a sample comprising contacting a sample suspected of comprising SARS-CoV-2 with an antibody of any one of claims 1-11 and isolating complexes of antibody and SARS-CoV-2 from the sample. The method of claim 32, wherein the antibody is labelled with a detectable label. The method of claim 33, wherein the detectable label is radio-opaque or a radioisotope, such as 3H, 14C; 32P; 35S; 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, betagalactosidase or horseradish peroxidase (HRP); an imaging agent; or a metal ion. The method of claim 32, wherein the sample is a biological sample from a subject, wherein the biological sample is selected from the group consisting of saliva, lung secretions, and blood. The method of claim 32, wherein the sample is saliva, a lung secretion, or blood from a subject suspected of being infected or known to be infected with SARS- CoV-2. The method of claim 32, wherein the sample is a wastewater or a swab of a solid surface. A kit comprising an antibody of any one of claims 1-11, a reference agent, and optionally instructions for use thereof. The kit of claim 38, wherein the antibody comprises the amino acid sequence of an antibody of Table 4. A kit comprising the nucleic acid according to claim 16 or a vector comprising said nucleic acid, a reference agent and optionally instructions for use thereof.
62
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* Cited by examiner, † Cited by third party
Title
SHI, R. ET AL.: "A human neutralizing antibody targets the receptor-binding site of SARS-CoV- 2", NATURE, vol. 584, 26 May 2020 (2020-05-26), pages 120 - 124, XP037211681, DOI: 10.1038/s41586-020-2381-y *
WU YAN; WANG FEIRAN; SHEN CHENGUANG; PENG WEIYU; LI DELIN; ZHAO CHENG; LI ZHAOHUI; LI SHIHUA; BI YUHAI; YANG YANG; GONG YUHUAN; XI: "A noncompetingpair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2", SCIENCE, vol. 368, no. 6496, 12 June 2020 (2020-06-12), pages 1274 - 1278, XP055799109, DOI: 10.1126/ science .abc2241 *
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