WO2022132904A1 - Anticorps monoclonaux humains ciblant le sars-cov-2 - Google Patents

Anticorps monoclonaux humains ciblant le sars-cov-2 Download PDF

Info

Publication number
WO2022132904A1
WO2022132904A1 PCT/US2021/063525 US2021063525W WO2022132904A1 WO 2022132904 A1 WO2022132904 A1 WO 2022132904A1 US 2021063525 W US2021063525 W US 2021063525W WO 2022132904 A1 WO2022132904 A1 WO 2022132904A1
Authority
WO
WIPO (PCT)
Prior art keywords
seq
antibody
nos
domain
amino acid
Prior art date
Application number
PCT/US2021/063525
Other languages
English (en)
Inventor
Joshua Hoong Yu TAN
Peter Dobbs CROMPTON
Hyeseon CHO
Kristina Kay GONZALES-WARTZ
Mary Elizabeth Peterson
Original Assignee
The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by The United States Of America, As Represented By The Secretary, Department Of Health And Human Services filed Critical The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Publication of WO2022132904A1 publication Critical patent/WO2022132904A1/fr

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • SARS-CoV-2 BACKGROUND The novel coronavirus SARS-CoV-2 emerged in Wuhan, China in 2019 and has infected almost 70 million individuals and caused 1.5 million deaths worldwide. SARS-CoV-2 enters the human body through the upper respiratory tract where virus titers peak within the first week. The virus then invades the lower respiratory tract reaching maximum viral load during the second week. SARS-CoV-2-specific antibodies can be detected within the first two weeks of infection. In vitro and in vivo experiments, along with case studies in humans, strongly support a role for SARS-CoV-2-neutralizing antibodies in protection against a SARS-CoV-2 infection.
  • Isolated monoclonal antibody or antigen binding fragments are disclosed that specifically bind to a coronavirus spike protein and neutralize SARS-CoV-2.
  • the antibody or antigen binding fragment includes one of: a) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)1, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)1, a LCDR2, and a LCDR3 of the V H and V L set forth as SEQ ID NOs: 1 and 5, respectively; b) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)1, a HCDR2, and a HCDR3, and a light chain complementarity determining region (LCDR)1, a LCDR2, and a LCDR3 of the V H and VL set forth as SEQ ID NOs: 9 and 13, respectively; c) a heavy chain variable region and a light chain variable region comprising a heavy chain complementarity determining region (HCDR)1, a HCDR2, and a HCDR3, and a light chain
  • the monoclonal antibody can bind either the N-terminal domain (NTD) or the In some embodiments, the monoclonal antibody specifically binds the N-terminal domain (NTD) or the receptor binding domain (RBD) of the coronavirus spike protein.” In further embodiments, disclosed are bispecific antibodies that include these antibodies and/or antigen binding fragments. In more embodiments, methods are disclosed for inhibiting a SARS-CoV-2 infection in a subject. In further embodiments, methods are disclosed for detecting SARS-CoV-2 in a biological sample.
  • FIG.2A-2G Characteristics of monoclonal antibodies from SARS-CoV-2 convalescent donors.
  • A VH, VK and V L gene usage of antibodies from plasmablasts and memory B cells (MBCs). Up to the top four genes in each chart are shown with different colors (genes that were tied for 4 th and lower are not highlighted).
  • B Heavy and light chain gene mutations of antibodies from plasmablasts and MBCs. Data indicate mean ⁇ SD; Mann-Whitney U-test.
  • PB plasmablast. Figs.3A-3G.
  • A Isoaffinity plot of antibodies targeting SARS-CoV-2 RBD.
  • B Neutralization potency versus affinity of anti-RBD antibodies.
  • C Isoaffinity plot of antibodies targeting SARS-CoV-2 NTD.
  • D Neutralization potency versus affinity of anti-NTD antibodies.
  • E Epitope binning of anti-RBD antibodies.
  • ACE2 was only used as an analyte (competitor) and not as a ligand, while all other antibodies were tested as both ligands and analytes. Full lines indicate two-way competition while dotted lines indicate one-way competition.
  • N-glycans at the N343 glycan site are represented by sticks.
  • G Epitope binning of anti-NTD antibodies. All antibodies were tested as both ligands and analytes. Full lines indicate two-way competition while dotted lines indicate one-way competition. The number and percentage of neutralizing antibodies (IC 50 ⁇ 10 ⁇ g/mL) in each bin are shown.
  • Figs.4A-4E Bispecific antibodies against SARS-CoV-2.
  • A Scheme of the dual variable domain (DVD) immunoglobulin. In our bispecific antibody naming system, the first name refers to the antibody used to make the outer binding site and the second refers to the antibody at the inner binding site.
  • GS or EL refers to the type of linker connecting the two antigen-binding sites, see the Examples Section for details.
  • B Binding of individual and bispecific antibodies to various domains from SARS-CoV-1 and SARS-CoV-2. Area under the curve (AUC) values are shown after subtraction with the negative control antigen.
  • C Neutralization potency of bispecific antibodies with authentic and pseudotyped SARS-CoV-2.
  • D Neutralization curves of CV1206_521_GS and CV664_993_GS with authentic and pseudotyped SARS- CoV-2.
  • E 3D reconstructions from negative stain EM images of CV1206_521_GS. Fig.5. Image of nanopens containing B cells.
  • the lower panel shows positive signals from binding of secreted antibodies to SARS-CoV-2 spike-coated beads.
  • Figs.6A-6C Summary of anti-SARS-CoV-2 monoclonal antibodies
  • Fig.7 Neutralization potency of IgG and IgA forms of the same monoclonal antibodies (originally isolated as IgA).
  • Figs.8A-8B Pseudovirus and SARS-CoV-2 neutralization of benchmark and new antibodies.
  • A Pseudovirus neutralization of antibody panel. Error bars show standard deviation.
  • Figs.9A-9B The lower panel shows positive signals from binding of secreted antibodies to SARS-CoV-2 spike-coated beads.
  • Figs.6A-6C Summary of anti-SARS-CoV-2 monoclonal antibodies
  • Fig.7 Neutralization potency of IgG and IgA forms of the same
  • FIG.10 Comparison of affinity of antibodies from plasmablasts and memory B cells.
  • A Comparison of affinity, association rates and dissociation rates of SARS-CoV-2 RBD-specific antibodies.
  • B Comparison of affinity, association rates and dissociation rates of SARS-CoV-2 NTD- specific antibodies.
  • Fig.10. RBD epitope binning heat map. A dark grey square indicates competition while a light grey square indicates non-overlapping binding. The rows show antibodies attached to the SPR chip (ligands) while the columns show antibodies added after antigen binding (analytes). Fig.11. NTD epitope binning heat map. A dark grey square indicates competition while a light grey square indicates non-overlapping binding.
  • Figs.12A-12C Screening of antibody combinations for synergy in neutralizing SARS-CoV-2.
  • A Screen of antibody combinations for synergy in neutralizing authentic SARS-CoV-2. Only non- overlapping pairs based on epitope binning data were tested. The numbers outside the brackets show the observed neutralization percentages while numbers in the brackets show the expected values. Combinations with an observed neutralization percentage of >5% the expected value are highlighted.
  • B Neutralization of authentic SARS-CoV-2 (FRNA assay) by titrations of CV503 and CV664, as well as CV664 and CV993.
  • GS or EL refers to the type of linker connecting the two antigen-binding sites, see the Examples Section.
  • B Size exclusion chromatography of bispecific antibodies, compared to the control standard IgG CV503.
  • C Sensograms showing binding of bispecific antibodies to RBD attached to ligand IgGs (which are conjugated to the SPR chip). An increase in signal after RBD binding indicates attachment of the second antibody.
  • A Neutralization of authentic SARS-CoV-2 with bispecific antibodies in comparison with single constituent antibodies.
  • B Neutralization of pseudotyped SARS-CoV-2 with bispecific antibodies in comparison with single constituent antibodies.
  • Fig.15 The reason for SARS-CoV-2.
  • B Blinded clinical scores assigned to hamsters throughout the course of disease are shown. A, a.m.; P, p.m. Points represent means ⁇ SD.
  • C Weight change is shown for hamsters that were administered with bispecific antibodies at 1 mg per hamster, 12 hours before intranasal virus exposure at 5 log10 PFU.
  • SEQ ID NOs: 2, 3 and 4 are the amino acid sequence of the IMGT (IMMUNOGENETICS® information system) HCDR1, HCDR2, and HCDR3 of CV503.
  • SEQ ID NO: 5 is the is the amino acid sequence of the light chain variable (V L ) domain (lambda) of CV503.
  • SEQ ID NOs: 6, 7 and 8 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR3 of CV503.
  • SEQ ID NO: 9 is the amino acid sequence of the V H domain of CV664.
  • SEQ ID NOs: 10, 11 and 12 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV664.
  • SEQ ID NO: 13 is the is the amino acid sequence of the V L domain (kappa) of CV664.
  • SEQ ID NOs: 14, 15 and 16 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR3 of CV664.
  • SEQ ID NO: 17 is the amino acid sequence of the V H domain of CV993.
  • SEQ ID NOs: 18, 19 and 20 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV993.
  • SEQ ID NO: 21 is the is the amino acid sequence of the V L domain (lambda) of CV993.
  • SEQ ID NOs: 22, 23 and 24 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR2 of CV993.
  • SEQ ID NO: 25 is the amino acid sequence of the V H domain of CV521.
  • SEQ ID NOs: 26, 27, and 28 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV521.
  • SEQ ID NO: 29 is the is the amino acid sequence of the V L (lambda) domain of CV521.
  • SEQ ID NOs: 30, 31 and 32 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR2 of CV521.
  • SEQ ID NO: 33 is the amino acid sequence of the V H domain of CV1182.
  • SEQ ID NOs: 34, 35 and 36 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV1182.
  • SEQ ID NO: 37 is the is the amino acid sequence of the V L domain (kappa) of CV1182.
  • SEQ ID NOs: 38, 39 and 40 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR2 of CV1182.
  • SEQ ID NO: 41 is the amino acid sequence of the V H domain of CV1206.
  • SEQ ID NOs: 42, 43 and 44 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV1206.
  • SEQ ID NO: 45 is the is the amino acid sequence of the V L domain (kappa) of CV1206.
  • SEQ ID NOs: 46, 47 and 48 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR2 of CV1206.
  • SEQ ID NO: 49 is the amino acid sequence of the V H domain of CV532.
  • SEQ ID NOs: 50, 51 and 52 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV532.
  • SEQ ID NO: 53 is the is the amino acid sequence of the V L domain (lambda) of CV532.
  • SEQ ID NOs: 54, 55 and56 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR3 of CV532.
  • SEQ ID NO: 57 is the amino acid sequence of the V H domain of CV635.
  • SEQ ID NOs: 58, 59 and 60 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV635.
  • SEQ ID NO: 61 is the is the amino acid sequence of the V L domain (lambda) of CV635.
  • SEQ ID NOs: 62, 63, and 64 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR3 of CV635.
  • SEQ ID NO: 65 is the amino acid sequence of the V H domain of CV085.
  • SEQ ID NOs: 66, 67 and 68 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV085.
  • SEQ ID NO: 69 is the is the amino acid sequence of the V L domain Kappa) of CV085.
  • SEQ ID NOs: 70, 71, and 72 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR3 of CV085.
  • SEQ ID NO: 73 is the amino acid sequence of the V H domain of CV576.
  • SEQ ID NOs: 74, 75 and 76 are the amino acid sequence of the IMGT HCDR1, HCDR2, and HCDR3 of CV576.
  • SEQ ID NO: 77 is the is the amino acid sequence of the V L domain (kappa)of CV576.
  • SEQ ID NOs: 78, 79 and 80 are the amino acid sequence of the IMGT LCDR1, LCDR2, and LCDR3 of CV576.
  • SEQ ID NO: 81 is an exemplary nucleic acid sequence encoding the V H of CV503.
  • SEQ ID NO: 82 is an exemplary nucleic acid sequence encoding the V L of CV503.
  • SEQ ID NO: 83 is an exemplary nucleic acid sequence encoding the V H of CV664.
  • SEQ ID NO: 84 is an exemplary nucleic acid sequence encoding the V L of CV664.
  • SEQ ID NO: 85 is an exemplary nucleic acid sequence encoding the V H of CV993.
  • SEQ ID NO: 86 is an exemplary nucleic acid sequence encoding the V L of CV993.
  • SEQ ID NO: 87 is an exemplary nucleic acid sequence encoding the V H of CV521.
  • SEQ ID NO: 88 is an exemplary nucleic acid sequence encoding the V L of CV521.
  • SEQ ID NO: 89 is an exemplary nucleic acid sequence encoding the V H of CV1182.
  • SEQ ID NO: 90 is an exemplary nucleic acid sequence encoding the V L of CV1182.
  • SEQ ID NO: 91 is an exemplary nucleic acid sequence encoding the V H of CV1206.
  • SEQ ID NO: 92 is an exemplary nucleic acid sequence encoding the V L of CV1206.
  • SEQ ID NO: 93 is an exemplary nucleic acid sequence encoding the V H of CV532.
  • SEQ ID NO: 94 is an exemplary nucleic acid sequence encoding the V L of CV532.
  • SEQ ID NO: 95 is an exemplary nucleic acid sequence encoding the V H of CV635.
  • SEQ ID NO: 96 is an exemplary nucleic acid sequence encoding the V L of CV635.
  • SEQ ID NO: 97 is an exemplary nucleic acid sequence encoding the V H of CV085.
  • SEQ ID NO: 98 is an exemplary nucleic acid sequence encoding the V L of CV085.
  • SEQ ID NO: 99 is an exemplary nucleic acid sequence encoding the V H of CV576.
  • SEQ ID NO: 100 is an exemplary nucleic acid sequence encoding the V L of CV576.
  • SEQ ID NO: 101 is the amino acid sequence of the V H of CV503_521_GS, with the linker sequence underlined.
  • SEQ ID NO: 102 is the amino acid sequence of the V L of CV503_521_GS, with the linker sequence underlined.
  • SEQ ID NO: 103 is the amino acid sequence of the V H of CV503_993_EL, with the linker sequence underlined.
  • SEQ ID NO: 104 is the amino acid sequence of the V L of CV503_993_EL, with the linker sequence underlined.
  • SEQ ID NO: 105 is the amino acid sequence of the V H of CV1206_521_GS, with the linker sequence underlined.
  • SEQ NO: 106 is the amino acid sequence of the V L of CV1206_521_GS, with the linker sequence underlined.
  • SEQ ID NO: 107 is the amino acid sequence of the V H of CV664_993_GS, with the linker sequence underlined.
  • SEQ ID NO: 108 is the amino acid sequence of the V L of CV664_993_GS, with the linker sequence underlined.
  • SEQ ID NO: 109 is the amino acid sequence of the V H of CV503_664_GS, with the linker sequence underlined.
  • SEQ ID NO: 110 is the amino acid sequence of the V L of CV503_664_GS, with the linker sequence underlined.
  • SEQ ID NO: 111 is the amino acid sequence of the V H of CV1206_521_EL, with the linker sequence underlined.
  • SEQ ID NO: 112 is the amino acid sequence of the V L of CV1206_521_EL, with the linker sequence underlined.
  • SEQ ID NO: 113 is the amino acid sequence of the V H of CV521_1182_GS, with the linker sequence underlined.
  • SEQ ID NO: 114 is the amino acid sequence of the V L of CV521_1182_GS, with the linker sequence underlined.
  • SEQ ID NO: 115 is the amino acid sequence of the V H of CV521_503_GS, with the linker sequence underlined.
  • SEQ ID NO: 116 is the amino acid sequence of the V L of CV521_503_GS, with the linker sequence underlined.
  • SEQ ID NO: 117 is the amino acid sequence of the V H of CV993_521_GS, with the linker sequence underlined.
  • SEQ ID NO: 118 is the amino acid sequence of the V L of CV993_521_GS, with the linker sequence underlined.
  • SEQ ID NO: 119 is the amino acid sequence of the V H of CV503_664_EL, with the linker sequence underlined.
  • SEQ ID NO: 120 is the amino acid sequence of the V L of CV503_664_EL, with the linker sequence underlined.
  • SEQ ID NO: 121 is a nucleic acid sequence encoding the V H domain of CV503_521_GS.
  • SEQ ID NO: 122 is a nucleic acid sequence encoding the V L domain of CV503_521_GS.
  • SEQ ID NO: 123 is a nucleic acid sequence encoding the V H domain of CV503_993_EL.
  • SEQ ID NO: 124 is a nucleic acid sequence encoding the V L domain of CV503_993_EL.
  • SEQ ID NO: 125 is a nucleic acid sequence encoding the V H domain of CV521_503_GS.
  • SEQ ID NO: 126 is a nucleic acid sequence encoding the V L domain of CV521_503_GS.
  • SEQ ID NO: 127 is a nucleic acid sequence encoding the V H domain of CV993_521_GS.
  • SEQ ID NO: 128 is a nucleic acid sequence encoding the V L domain of CV993_521_GS.
  • SEQ ID NO: 129 is a nucleic acid sequence encoding the V H domain of CV503_664_EL.
  • SEQ ID NO: 130 is a nucleic acid sequence encoding the V L domain of CV503_664_EL.
  • SEQ ID NO: 131 is a nucleic acid sequence encoding the V H domain of CV1206_521_EL.
  • SEQ ID NO: 132 is a nucleic acid sequence encoding the V L domain of CV1206_521_EL.
  • SEQ ID NO: 133 is a nucleic acid sequence encoding the V H domain of CV521_1182_GS.
  • SEQ ID NO: 134 is a nucleic acid sequence encoding the V L domain of CV521 1182 GS.
  • SEQ ID NO: 135 is a nucleic acid sequence encoding the V H domain of CV1206_521_GS.
  • SEQ ID NO: 136 is a nucleic acid sequence encoding the V L domain of CV1206_521_GS.
  • SEQ ID NO: 137 is a nucleic acid sequence encoding the V H domain of CV664_993_GS.
  • SEQ ID NO: 138 is a nucleic acid sequence encoding the V L domain of CV664_993_GS.
  • SEQ ID NO: 139 is a nucleic acid sequence encoding the V H domain of CV503_664_GS.
  • SEQ ID NO: 140 is a nucleic acid sequence encoding the V L domain of CV503_664_GS.
  • SEQ ID NO: 141 is the GS linker.
  • SEQ ID NO: 142 is the EL linker (when outer V is heavy chain) Note that there are 3 different EL linkers, one for heavy chain, one where the outer is kappa and one where the outer is lambda. Seq 126 is the heavy chain linker.
  • SEQ ID NO: 143 is the EL linker (when outer V is kappa)
  • SEQ ID NO: 144 is the EL linker (when outer V is lambda) DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS
  • Monoclonal antibodies, and antigen binding fragments thereof, that specifically bind the spike protein of SARS-CoV-2 are disclosed herein.
  • the monoclonal antibody binds the receptor binding domain (RBD) of the spike protein.
  • the monoclonal antibody specifically binds the NTD domain of the spike protein.
  • Bispecific antibodies are also disclosed. These monoclonal antibodies and bispecific antibodies can be used to inhibit a coronavirus infection, specifically a SARS-Cov-2 infection. The monoclonal antibodies and bispecific antibodies also can be used to detect a coronavirus infection, specifically a SARS-CoV-2 infection.
  • an antigen includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.”
  • the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control.
  • Administration The introduction of an agent, such as a disclosed antibody, into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravascular, the agent (such as antibody) is administered by introducing the composition into a blood vessel of the subject.
  • Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.
  • Amino acid substitution The replacement of one amino acid in a polypeptide with a different amino acid.
  • Antibody and Antigen Binding Fragment An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as a coronavirus spike protein, such as a spike protein from SARS-CoV-2.
  • antibody is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen-binding activity.
  • Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen.
  • antigen binding fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab') 2 ; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.
  • Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Dübel (Eds.), Antibody Engineering, Vols.1-2, 2 nd ed., Springer-Verlag, 2010). Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies). An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different.
  • a naturally-occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.
  • a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda ( ⁇ ) and kappa ( ⁇ ).
  • V H refers to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab.
  • V L refers to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.
  • the V H and V L contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5 th ed., NIH Publication No.91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991).
  • CDRs complementarity-determining regions
  • amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th ed., NIH Publication No.91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991; “Kabat” numbering scheme), Al-Lazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio., 273(4):927-948, 1997; “Chothia” numbering scheme), and Lefranc et al.
  • IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains Dev. Comp. Immunol., 27(1):55-77, 2003; “IMGT” numbering scheme).
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located.
  • a V H CDR3 is the CDR3 from the V H of the antibody in which it is found
  • a V L CDR1 is the CDR1 from the V L of the antibody in which it is found.
  • Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3.
  • Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.
  • a disclosed antibody includes a heterologous constant domain.
  • the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutation) to increase half-life.
  • a “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts.
  • each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen.
  • the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.
  • the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.
  • monoclonal antibodies are isolated from a subject.
  • Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual, 2 nd ed.
  • a “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment.
  • the non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical.
  • a “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species.
  • a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody.
  • a “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species.
  • a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome.
  • Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et al. Phage display: A Laboratory Manuel. 1 st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).
  • Antibody or antigen binding fragment that neutralizes SARS-CoV-2 An antibody or antigen binding fragment that specifically binds to a SARS-CoV-2 antigen (such as the spike protein) in such a way as to inhibit a biological function associated with SARS-CoV-2 that inhibits infection.
  • the antibody can neutralize the activity of SARS-CoV-2.
  • an antibody or antigen binding fragment that neutralizes SARS-CoV-2 may interfere with the virus by binding it directly and limiting entry into cells.
  • an antibody may interfere with one or more post-attachment interactions of the pathogen with a receptor, for example, by interfering with viral entry using the receptor.
  • an antibody that is specific for a coronavirus spike protein neutralizes the infectious titer of SARS-CoV-2.
  • an antibody or antigen binding fragment that specifically binds to SARS- CoV-2 and neutralizes SARS-CoV-2 inhibits infection of cells, for example, by at least 50% compared to a control antibody or antigen binding fragment.
  • a “broadly neutralizing antibody” is an antibody that binds to and inhibits the function of related antigens, such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity antigenic surface of antigen.
  • the antibody can bind to and inhibit the function of an antigen from more than one class and/or subclass of the pathogen.
  • a pathogen such as a virus
  • the antibody can bind to and inhibit the function of an antigen, such as the spike protein from coronaviruses including SARS-CoV-2.
  • Biological sample A sample obtained from a subject.
  • Biological samples include all clinical samples useful for detection of disease or infection in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum), cerebrospinal fluid; as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin.
  • a biological sample is obtained from a subject having or suspected of having a SARS-CoV-2 infection.
  • Bispecific antibody A recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes.
  • Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains.
  • the antigen binding domains can be linked using a linker.
  • the antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof.
  • a bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain.
  • Conditions sufficient to form an immune complex Conditions which allow an antibody or antigen binding fragment to bind to its cognate epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols or those conditions encountered in vivo.
  • the conditions employed in the methods are “physiological conditions” which include reference to conditions (e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, more typically pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C.
  • physiological conditions e.g., temperature, osmolarity, pH
  • Osmolarity is within the range that is supportive of cell viability and proliferation.
  • the formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging (MRI), computed tomography (CT) scans, radiography, and affinity chromatography.
  • Conjugate A complex of two molecules linked together, for example, linked together by a covalent bond.
  • an antibody is linked to an effector molecule; for example, an antibody that specifically binds to SARS-CoV-2 covalently linked to an effector molecule, such as a detectable label.
  • the linkage can be by chemical or recombinant means.
  • the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule.
  • a peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.” Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein.
  • a SARS-CoV-2-specific antibody can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for spike protein binding, and/or SARS-CoV-2 neutralization activity.
  • the term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some embodiments less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.
  • Non-conservative substitutions are those that reduce an activity or function of the antibody, such as the ability to specifically bind to a coronavirus spike protein.
  • Placement in direct physical association includes both in solid and liquid form, which can take place either in vivo or in vitro.
  • Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as an antibody.
  • Contacting can also include contacting a cell for example by placing an antibody in direct physical association with a cell.
  • Control A reference standard. In some embodiments, the control is a negative control, such as sample obtained from a healthy patient not infected a coronavirus.
  • control is a positive control, such as a tissue sample obtained from a patient diagnosed with a coronavirus infection.
  • the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values).
  • a difference between a test sample and a control can be an increase or conversely a decrease.
  • the difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference.
  • a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.
  • Coronavirus A family of positive-sense, single-stranded RNA viruses that are known to cause severe respiratory illness. Viruses currently known to infect human from the coronavirus family are from the alphacoronavirus and betacoronavirus genera.
  • betacoronaviruses include SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Human coronavirus HKU1 (HKU1-CoV), Human coronavirus OC43 (OC43-CoV), Murine Hepatitis Virus (MHV-CoV), Bat SARS-like coronavirus WIV1 (WIV1-CoV), and Human coronavirus HKU9 (HKU9- CoV).
  • SARS-CoV-2 Middle East respiratory syndrome coronavirus
  • SARS-CoV Severe Acute Respiratory Syndrome coronavirus
  • HKU1-CoV Human coronavirus HKU1
  • OC43-CoV Human coronavirus OC43
  • MHV-CoV Murine Hepatitis Virus
  • WIV1-CoV Bat SARS-like coronavirus WIV1
  • HKU9- CoV Human coronavirus HKU9
  • Non-limiting examples of alphacoronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and Transmissible gastroenteritis coronavirus (TGEV).
  • a non-limiting example of a deltacoronaviruses is the Swine Delta Coronavirus (SDCV).
  • SDCV Swine Delta Coronavirus
  • the viral genome is capped, polyadenylated, and covered with nucleocapsid proteins.
  • the coronavirus virion includes a viral envelope containing type I fusion glycoproteins referred to as the spike (S) protein.
  • S spike
  • Most coronaviruses have a common genome organization with the replicase gene.
  • Degenerate variant refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.
  • Detectable marker A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as an antibody, to facilitate detection of the second molecule.
  • the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination).
  • detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI).
  • Dual variable domain immunoglobulin A bi-specific antibody that includes two heavy chain variable domains and two light chain variable domains. Unlike IgG, however, both heavy and light chains of a DVD-immunoglobulin molecule contain an additional variable domain (VD) connected via a linker sequence at the N-termini of the VH and VL of an existing monoclonal antibody (mAb).
  • VD additional variable domain
  • DVD-immunoglobulin molecule contains four antigen recognition sites, see Jakob et al., Mabs 5: 358-363, 2013, incorporated herein by reference, see Fig.1 for schematic and space-filling diagrams.
  • a DVD-IGTM molecule functions to bind two different antigens on each DFab simultaneously.
  • Effective amount A quantity of a specific substance sufficient to achieve a desired effect in a subject to whom the substance is administered. For instance, this can be the amount necessary to inhibit a coronavirus infection, such as a SARS-CoV-2 infection, or to measurably alter outward symptoms of such an infection.
  • a desired response is to inhibit or reduce or prevent SARS-CoV-2 infection.
  • the SARS-CoV-2 infection does not need to be completely eliminated or reduced or prevented for the method to be effective.
  • administration of an effective amount of the immunogen can induce an immune response that decreases the SARS-CoV-2 infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the SARS-CoV-2) by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infection), as compared to a suitable control.
  • administering can reduce or inhibit a SAR-CoV-2 infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the coronavirus or by an increase in the survival time of infected subjects, or reduction in symptoms associated with the infection) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infection), as compared to a suitable control.
  • a SAR-CoV-2 infection for example, as measured by infection of cells, or by number or percentage of subjects infected by the coronavirus or by an increase in the survival time of infected subjects, or reduction in symptoms associated with the infection
  • a desired amount for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (el
  • the effective amount of an antibody or antigen binding fragment that specifically binds the coronavirus spike protein that is administered to a subject to inhibit infection will vary depending upon a number of factors associated with that subject, for example the overall health and/or weight of the subject.
  • An effective amount can be determined by varying the dosage and measuring the resulting response, such as, for example, a reduction in pathogen titer.
  • Effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays.
  • An effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining an effective response.
  • an effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks.
  • the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • a unit dosage form of the agent can be packaged in an amount, or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.
  • Effector molecule A molecule intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted, or a detectable marker. Effector molecules can include, for example, polypeptides and small molecules. Some effector molecules may have or produce more than one desired effect.
  • Epitope An antigenic determinant.
  • an epitope is the region of an antigen to which B and/or T cells respond.
  • An antibody can bind to a particular antigenic epitope, such as an epitope on a coronavirus spike protein.
  • Expression Transcription or translation of a nucleic acid sequence.
  • an encoding nucleic acid sequence (such as a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some examples is processed to become mRNA.
  • An encoding nucleic acid sequence may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment.
  • a heterologous gene is expressed when it is transcribed into an RNA.
  • a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization or degradation of specific protein molecules after they are produced.
  • Expression Control Sequences Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked.
  • Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcriptional terminators, a start codon (ATG) in front of a protein-encoding gene, splice signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • control sequences is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.
  • Expression control sequences can include a promoter.
  • Expression vector A vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.
  • a polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host.
  • the expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.
  • Fc region The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains.
  • an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain.
  • the Fc region is typically understood to include immunoglobulin domains C ⁇ 2 and C ⁇ 3 and optionally the lower part of the hinge between C ⁇ 1 and C ⁇ 2.
  • the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to Kabat.
  • the Fc region includes immunoglobulin domains C ⁇ 2 and C ⁇ 3 and optionally the lower part of the hinge between C ⁇ 1 and C ⁇ 2.
  • Heterologous Originating from a different genetic source.
  • a nucleic acid molecule that is heterologous to a cell originated from a genetic source other than the cell in which it is expressed.
  • a heterologous nucleic acid molecule encoding a protein, such as an scFv is expressed in a cell, such as a mammalian cell.
  • Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known in the art, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.
  • Host cell Cells in which a vector can be propagated and its DNA expressed. The cell may be prokaryotic or eukaryotic.
  • IgA A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype comprises IgA 1 and IgA 2 . IgA antibodies can exist as monomers, polymers (referred to as pIgA) of predominantly dimeric form, and secretory IgA. The constant chain of wild-type IgA contains an 18-amino-acid extension at its C-terminus called the tail piece (tp).
  • IgG A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class comprises IgG 1 , IgG 2 , IgG 3 , and IgG 4 .
  • Immune complex The binding of antibody or antigen binding fragment (such as a scFv) to a soluble antigen forms an immune complex.
  • an immune complex can be detected through conventional methods, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.
  • Inhibiting or treating a disease Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a SARS-CoV-2 infection.
  • Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop.
  • the term “ameliorating,” with reference to a disease or pathological condition refers to any observable beneficial effect of the treatment.
  • Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection.
  • the beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease.
  • a “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.
  • reduces is a relative term, such that an agent reduces a disease or condition if the disease or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent.
  • prevents does not necessarily mean that an agent completely eliminates the disease or condition, so long as at least one characteristic of the disease or condition is eliminated.
  • a composition that reduces or prevents an infection can, but does not necessarily completely, eliminate such an infection, so long as the infection is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% the infection in the absence of the agent, or in comparison to a reference agent.
  • isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods.
  • the term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids.
  • An isolated nucleic acid, peptide or protein, for example an antibody can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.
  • Kabat position A position of a residue in an amino acid sequence that follows the numbering convention delineated by Kabat et al. (Sequences of Proteins of Immunological Interest, 5 th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No.91-3242, 1991).
  • Linker A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link a detectable marker to an antibody.
  • Non-limiting examples of peptide linkers include glycine-serine linkers.
  • the terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide, such as an scFv.
  • the linkage can be either by chemical or recombinant means.
  • Nucleic acid refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.
  • Nucleic acid molecule or sequence: A deoxyribonucleotide or ribonucleotide polymer or combination thereof including without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA or RNA.
  • the nucleic acid can be double stranded (ds) or single stranded (ss). Where single stranded, the nucleic acid can be the sense strand or the antisense strand.
  • Nucleic acids can include natural nucleotides (such as A, T/U, C, and G), and can include analogs of natural nucleotides, such as labeled nucleotides.
  • cDNA refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.
  • Encoding refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom.
  • a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system.
  • coding strand the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings
  • non-coding strand used as the template for transcription
  • a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.
  • a first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter such as the CMV promoter
  • operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.
  • Pharmaceutically acceptable carriers The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.
  • parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • injectable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • solid compositions e.g., powder, pill, tablet, or capsule forms
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection.
  • the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial.
  • Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).
  • Polypeptide A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D- optical isomer can be used, the L-isomers being preferred.
  • the terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins.
  • a polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced.
  • a polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal end. In some embodiments, the polypeptide is a disclosed antibody or a fragment thereof.
  • a purified peptide preparation is one in which the peptide or protein (such as an antibody) is more enriched than the peptide or protein is in its natural environment within a cell.
  • a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.
  • a recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques.
  • a recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence.
  • a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell.
  • the nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.
  • SARS-CoV-2 Also known as Wuhan coronavirus or 2019 novel coronavirus, SARS-CoV-2 is a positive-sense, single stranded RNA virus of the genus betacoronavirus that has emerged as a highly fatal cause of severe acute respiratory infection.
  • the viral genome is capped, polyadenylated, and covered with nucleocapsid proteins.
  • the SARS-CoV-2 virion includes a viral envelope with large spike glycoproteins.
  • the SARS-CoV-2 genome like most coronaviruses, has a common genome organization with the replicase gene included in the 5'-two thirds of the genome, and structural genes included in the 3'-third of the genome.
  • the SARS-CoV-2 genome encodes the canonical set of structural protein genes in the order 5' - spike (S) - envelope (E) - membrane (M) and nucleocapsid (N) - 3'.
  • Symptoms of SARS-CoV-2 infection include fever and respiratory illness, such as dry cough and shortness of breath. Cases of severe infection can progress to severe pneumonia, multi-organ failure, and death. The time from exposure to onset of symptoms is approximately 2 to 14 days.
  • Standard methods for detecting viral infection may be used to detect SARS-CoV-2 infection, including but not limited to, assessment of patient symptoms and background and genetic tests such as reverse transcription-polymerase chain reaction (rRT-PCR). The test can be done on patient samples such as respiratory or blood samples.
  • SARS Spike (S) protein A class I fusion glycoprotein initially synthesized as a precursor protein of approximately 1256 amino acids in size for SARS-CoV, and 1273 for SARS-CoV-2. Individual precursor S polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease between approximately position 679/680 for SARS-CoV, and 685/686 for SARS-CoV-2, to generate separate S1 and S2 polypeptide chains, which remain associated as S1/S2 protomers within the homotrimer and is therefore a trimer of heterodimers.
  • the S1 subunit is distal to the virus membrane and contains the receptor-binding domain (RBD) that is believed to mediate virus attachment to its host receptor.
  • the S2 subunit is believed to contain the fusion protein machinery, such as the fusion peptide, two heptad-repeat sequences (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and the cytosolic tail domain.
  • a diagram of the SARS-CoV structure, showing the N-terminal domain (NTD), and the RBD is shown in Fig. 3F.
  • sequence identity The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the percentage identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences.
  • Homologs and variants of a V L or a V H of an antibody that specifically binds a target antigen are typically characterized by possession of at least about 75% sequence identity, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest. Any suitable method may be used to align sequences for comparison. Non-limiting examples of programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math.2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol.48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.
  • Biol.215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.
  • Blastn is used to compare nucleic acid sequences
  • blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site. Generally, once two sequences are aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences.
  • the percent sequence identity between the two sequences is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.
  • bind When referring to an antibody or antigen binding fragment, refers to a binding reaction which determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biologics.
  • an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, for example a coronavirus spike protein and does not bind in a significant amount to other proteins present in the sample or subject.
  • a target protein peptide or polysaccharide
  • the epitope may be present on SARS-CoV-2 spike protein, such that the antibody binds to the spike protein on both types of virus, but does not bind to other proteins.
  • Specific binding can be determined by standard methods. See Harlow & Lane, Antibodies, A Laboratory Manual, 2 nd ed., Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity.
  • K D refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction.
  • K D refers to the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.
  • An antibody that specifically binds to an epitope on a coronavirus spike protein an antibody that binds substantially to the coronavirus spike protein, such as the NTD or RBD of a spike protein from SARS- CoV-2, including viruses, substrate to which the spike protein is attached, or the protein in a biological specimen. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody and a non-target. Typically, specific binding results in a much stronger association between the antibody and a spike protein than between the antibody other different coronavirus proteins (such as MERS or SARS-CoV), or from non-coronavirus proteins.
  • MERS or SARS-CoV
  • Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope.
  • Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein.
  • immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.
  • Subject Living multi-cellular vertebrate organisms, a category that includes human and non- human mammals, such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, and the like.
  • a subject is a human.
  • the subject is a human.
  • a subject is selected that is in need of inhibiting a SARS-CoV-2 infection.
  • the subject is either uninfected and at risk of the SARS-CoV-2 infection or is infected and in need of treatment.
  • Transformed A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques.
  • transformed and the like encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.
  • Vector An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence.
  • Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replication- incompetent, or a virus or bacterium or other microorganism that may be replication-competent.
  • a vector is sometimes referred to as a construct.
  • Recombinant DNA vectors are vectors having recombinant DNA.
  • a vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication.
  • a vector can also include one or more selectable marker genes and other genetic elements.
  • Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses.
  • a viral vector comprises a nucleic acid molecule encoding a disclosed antibody or antigen binding fragment that specifically binds to a coronavirus spike protein and neutralizes the coronavirus.
  • the viral vector can be an adeno-associated virus (AAV) vector.
  • AAV adeno-associated virus
  • the disclosed antibodies can inhibit a coronavirus infection in vivo, and can be administered prior to, or after, an infection with a coronavirus, such as SARS-CoV-2.
  • Bispecific antibodies including the variable domains of these antibodies are also provided.
  • compositions comprising the antibodies and antigen binding fragments and a pharmaceutically acceptable carrier.
  • Nucleic acids encoding the antibodies, antigen binding fragments, variable domains, and expression vectors (such as adeno-associated virus (AAV) viral vectors) comprising these nucleic acids are also provided.
  • the antibodies, antigen binding fragments, nucleic acid molecules, host cells, and compositions can be used for research, diagnostic, treatment and prophylactic purposes.
  • the disclosed antibodies and antigen binding fragments can be used to diagnose a subject with a coronavirus infection or can be administered to inhibit a coronavirus infection in a subject.
  • A. Monoclonal Antibodies that Specifically Bind a Coronavirus Spike protein and Antigen Binding Fragments Thereof refers to isolated monoclonal antibodies that include heavy and/or light chain variable domains (or antigen binding fragments thereof) comprising a CDR1, CDR2, and/or CDR3 with reference to the IMGT numbering scheme (unless the context indicates otherwise).
  • Various CDR numbering schemes (such as the Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions.
  • a monoclonal antibody that comprises the heavy and light chain CDRs of any one of the antibodies described herein. In some embodiment, a monoclonal antibody is provided that comprises the heavy and light chain variable regions of any one of the antibodies described herein. Table 1. IMGT CDRs of Antibodies and SEQ ID NOs.
  • CV993 binds to both SARS-CoV-1 and SARS-CoV-2. All other antibodies bind only SARS-CoV-2. a. Monoclonal antibody CV503 In some embodiments, the antibody or antigen binding fragment is based on or derived from the CV503 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV503 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 1, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3, and 4, respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7, and 8, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3, and 4, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7, and 8, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 1, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 5, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 1, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • the antibody or antigen binding fragment is based on or derived from the CV664 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV664 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11, and 12, respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15, and 16, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11, and 12, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15, and 16, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 9, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • Monoclonal antibody CV993 the antibody or antigen binding fragment is based on or derived from the CV993 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV993 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2 and/or SARS-CoV-1.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 17, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 21, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2 and/or SARS-CoV-1.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 18, 19, and 20 respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 22, 23, and 24, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2 and/or SARS-CoV-1.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 18, 19, and 20, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 22, 23, and 24, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 17, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 21, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2 and/or SARS-CoV-1.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 17, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 21, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2 and/or SARS-CoV-1.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • Monoclonal antibody CV521 In some embodiments, the antibody or antigen binding fragment is based on or derived from the CV521 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV521 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 25, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27, and 28 respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31, and 32, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27, and 28, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31, and 32, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 25, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 25, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 25, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • the antibody or antigen binding fragment is based on or derived from the CV1182 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV1182 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 38, 39, and 40, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35, and 36, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 38, 39, and 40, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 33, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 33, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 37, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • the antibody or antigen binding fragment is based on or derived from the CV1206 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV1206 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 41, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 45, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 41 and 45, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 42, 43, and 44 respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 46, 47, and 48, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 42, 43, and 44, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 46, 47, and 48, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 41, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 41, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 41, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 45, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 41 and 45, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • the antibody or antigen binding fragment is based on or derived from the CV532 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV532 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 49, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 53, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 49 and 53, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 50, 51, and 52 respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 54, 55 and 56, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 50, 51, and 52, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 54, 55, and 56, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 49, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 49, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 53, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 53, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 49, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 53, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 49 and 53, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • Monoclonal Antibody CV635 In some embodiments, the antibody or antigen binding fragment is based on or derived from the CV635 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV635 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 57, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 61, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 57 and 61, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 58, 59, and 60 respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 62, 63, and 64, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 58, 59, and 60, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 62, 63, and 64, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 57, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 57, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 61, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 61, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 57, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 61, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 57 and 61, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication. i. Monoclonal antibody CV085
  • the antibody or antigen binding fragment is based on or derived from the CV805 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV085 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 65, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 69, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 65 and 69, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 66, 67, and 68 respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 70, 71, and 72, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 66, 67, and 68, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 70, 71, and 72, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 65, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 65, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 69, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 69, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 65, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 69, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 65 and 69, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication.
  • Monoclonal Antibody CV576 In some embodiments, the antibody or antigen binding fragment is based on or derived from the CV576 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the HCDR1, the HCDR2, and the HCDR3, and the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the CV576 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 73, and specifically binds to a coronavirus spike, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 77, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 73 and 77, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 74, 75, and 76 respectively, and/or a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 78, 79, and 80, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 74, 75, and 76, respectively, a V L comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 78, 79, and 80, respectively, wherein the V H comprises an amino acid sequence at least 90% identical to SEQ ID NO: 73, such as 95%, 96%, 97%, 98% o 99% identical to SEQ ID NO: 73, and wherein the V L comprises an amino acid sequence at least 90% identical to SEQ ID NO: 77, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 77, and the antibody or antigens binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the antibody or antigen binding fragment comprises a V H comprising the amino acid sequence set forth as SEQ ID NO: 73, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V L comprising the amino acid sequence set forth as SEQ ID NO: 77, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the antibody or antigen binding fragment comprises a V H and a V L comprising the amino acid sequences set forth as SEQ ID NOs: 73 and 77, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.
  • the coronavirus can be SARS-CoV-2.
  • the disclosed antibodies inhibit viral entry and/or replication. 1. Additional antibodies
  • antibodies that bind to an epitope of interest can be identified based on their ability to cross-compete (for example, to competitively inhibit the binding of, in a statistically significant manner) with the antibodies provided herein in binding assays.
  • antibodies that bind to an epitope of interest can be identified based on their ability to cross-compete (for example, to competitively inhibit the binding of, in a statistically significant manner) with the one or more of the antibodies provided herein in binding assays.
  • Human antibodies that bind to the same epitope on the spike of the coronavirus protein, such as the NTD or RBD of the spike protein, to which the disclosed antibodies bind can be produced using any suitable method.
  • Such antibodies may be prepared, for example, by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge.
  • Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes.
  • the endogenous immunoglobulin loci have generally been inactivated.
  • Antibodies and antigen binding fragments that specifically bind to the same epitope can also be isolated by screening combinatorial libraries for antibodies with the desired binding characteristics. For example, by generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al., J. Mol.
  • repertoires of V H and V L genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994).
  • Phage typically display antibody fragments, either as single-chain Fv (scFv) fragments or as Fab fragments.
  • naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self antigens without any immunization as described by Griffiths et al., EMBO J, 12: 725-734 (1993).
  • naive libraries can also be made synthetically by cloning unrearranged V-gene segments from stem cells, and using PCR primers containing random sequence to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992).
  • Patent publications describing human antibody phage libraries include, for example: U.S. Pat.
  • An antibody or antigen binding fragment of the antibodies disclosed herein can be a human antibody or fragment thereof. Chimeric antibodies are also provided.
  • the antibody or antigen binding fragment can include any suitable framework region, such as (but not limited to) a human framework region from another source, or an optimized framework region.
  • a heterologous framework region such as, but not limited to a mouse or monkey framework region, can be included in the heavy or light chain of the antibodies.
  • the antibody can be of any isotype.
  • the antibody can be, for example, an IgA, IgM or an IgG antibody, such as IgG 1 , IgG 2 , IgG 3 , or IgG 4 .
  • the class of an antibody that specifically binds to a coronavirus spike protein can be switched with another.
  • a nucleic acid molecule encoding V L or V H is isolated such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively.
  • a nucleic acid molecule encoding V L or V H is then operatively linked to a nucleic acid sequence encoding a C L or C H from a different class of immunoglobulin molecule.
  • This can be achieved, for example, using a vector or nucleic acid molecule that comprises a C L or C H chain.
  • an antibody that specifically binds the spike protein, that was originally IgG may be class switched to an IgA. Class switching can be used to convert one IgG subclass to another, such as from IgG 1 to IgG 2 , IgG 3 , or IgG 4 .
  • the disclosed antibodies are oligomers of antibodies, such as dimers, trimers, tetramers, pentamers, hexamers, septamers, octomers and so on.
  • the antibody or antigen binding fragment can be derivatized or linked to another molecule (such as another peptide or protein).
  • the antibody or antigen binding fragment is derivatized such that the binding to the spike protein is not affected adversely by the derivatization or labeling.
  • the antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, a bi-specific antibody or a diabody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • another antibody for example, a bi-specific antibody or a diabody
  • a detectable marker for example, an effector molecule, or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).
  • the antibody or antigen binding fragment specifically binds the coronavirus spike protein with an affinity (e.g., measured by K D ) of no more than 1.0 x 10 -8 M, no more than 5.0 x 10 -8 M, no more than 1.0 x 10 -9 M, no more than 5.0 x 10 -9 M, no more than 1.0 x 10 -10 M, no more than 5.0 x 10- 10 M, or no more than 1.0 x 10 -11 M.
  • K D can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen.
  • RIA radiolabeled antigen binding assay
  • solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I)- labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293(4):865-881, 1999).
  • MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 ⁇ g/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.).
  • a non-adsorbent plate (NUNCTM Catalog #269620) 100 ⁇ M or 26 pM [ 125 I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res.57(20):4593-4599, 1997).
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour).
  • KD can be measured using surface plasmon resonance assays using a BIACORE®- 2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at ⁇ 10 response units (RU).
  • CM5 carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride
  • NHS N- hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 ⁇ g/ml ( ⁇ 0.2 ⁇ M) before injection at a flow rate of 5 l/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20TM) surfactant (PBST) at 25° C at a flow rate of approximately 25 l/min.
  • TWEEN-20TM polysorbate 20
  • association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (K D ) is calculated as the ratio k off /k on . See, e.g., Chen et al., J. Mol. Biol.293:865-881 (1999).
  • a multi-specific antibody, or a bi-specific antibody, such as a dual variable domain antibody (DVD-IGTM) is provided that comprises an antibody or antigen binding fragment that specifically binds a coronavirus spike protein, as provided herein.
  • DVD-IGTM dual variable domain antibody
  • the bispecific tetravalent immunoglobulin known as the dual variable domain immunoglobulin or DVD-immunoglobulin molecule is disclosed in Wu et al., MAbs.2009;1:339–47, doi: 10.4161/mabs.1.4.8755, incorporated herein by reference. See also Nat Biotechnol.2007 Nov;25(11):1290- 7.
  • a DVD- immunoglobulin molecule includes two heavy chains and two light chains. Unlike IgG, however, both heavy and light chains of a DVD-immunoglobulin molecule contain an additional variable domain (VD) connected via a linker sequence at the N-termini of the VH and V L of an existing monoclonal antibody (mAb). Thus, when the heavy and the light chains combine, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites, see Jakob et al., Mabs 5: 358-363, 2013, incorporated herein by reference, see Fig. 1 for schematic and space-filling diagrams.
  • VD variable domain
  • mAb monoclonal antibody
  • a DVD-immunoglobulin molecule functions to bind two different antigens on each DFab simultaneously.
  • the outermost or N-terminal variable domain is termed VD1 and the innermost variable domain is termed VD2; the VD2 is proximal to the C-terminal CH1 or CL.
  • VD1 The outermost or N-terminal variable domain
  • VD2 the innermost variable domain
  • CL the C-terminal CH1 or CL.
  • DVD- immunoglobulin molecules can be manufactured and purified to homogeneity in large quantities, have pharmacological properties similar to those of a conventional IgG 1 , and show in vivo efficacy. Any of the disclosed monoclonal antibodies can be included in a DVD-immunoglobulin format.
  • the VD1 includes the V H and V L of one of CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576.
  • the VD2 includes the V H and V L of one of CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576.
  • the VD1 and the VD2 include the V H and V L of one of CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576, wherein the VD1 and the VD2 are different.
  • Table 2 The following are exemplary bispecific antibodies in dual variable domain immunoglobulin format: Table 2.
  • VD1 outer variable domain
  • VD2 inner domain
  • VD2 linker
  • IgG constant domain is linked to the inner domain.
  • Exemplary amino acid sequences for these bispecific antibodies are provided as SEQ ID NOs: 101-120, and exemplary nucleic acid sequences ending the heavy and light chain variable domains of the DVD- immunoglobulins are provided as SEQ ID NOs: 121-140.
  • the amino acid sequences of exemplary linkers are provided in SEQ ID NOs: 141-144.
  • the bispecific antibody is a DVD-immunoglobulin includes up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions in one of the heavy and light chain variable domains set forth as SEQ ID NOs: 101-120.
  • the bispecific antibody includes a heavy chain domain and/or light chain domain at least 95%, such s 96%, 97%, 98% or 99% identical to one of SEQ ID NOs: 121-140. a.
  • the VD1 of the DVD-immunoglobulin includes the V H and the V L of CV503 as the VD1
  • the VD2 of the DVD-immunoglobulin includes V H and the V L of CD521.
  • Embodiments of the V H and V L domains of these antibodies are disclosed above; any of these embodiments can be used in the DVD-immunoglobulin bispecific antibody.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a first V H domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second V H domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NOs: 6, 7, and 8, a linker, and a second V L domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a first V H domain comprising SEQ ID NO: 1, a linker, a second V H domain comprising SEQ ID NO: 25, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NO: 5 a linker, a second VL domain comprising SEQ ID NO: 29, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a CV503 V H domain, a GS linker, a CV521 V H domain, and an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: CV503 V L domain, a GS linker, CV521 V L domain, and an IgG or IgA light chain constant domain.
  • CV521_1182_GS In some embodiments the VD1 of the DVD-immunoglobulin includes the V H and the V L of CV521 as the VD1, and the VD2 of the DVD-immunoglobulin includes V H and the V L of CD1182.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NOs: 26, 27 and 28, a linker, a second V H domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, such as an IgG constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NOs: 30, 31 and 32, a linker, a second V L domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a first V H domain comprising SEQ ID NO: 25, a linker, a second V H domain comprising SEQ ID NO: 33, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NO: 29 a linker, a second V L domain comprising SEQ ID NO: 37, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a CV521 V H domain, a GS linker, a CV1182 V H domain, an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: CV521 V L domain, a GS linker, CV1182 V L domain, and an IgG or IgA light chain constant domain.
  • CV503_993_EL In some embodiments the VD1 of the DVD-immunoglobulin includes the V H and the V L of CV503 as the VD1, and the VD2 of the DVD-immunoglobulin includes V H and the V L of CD993. Embodiments of the V H and V L domains of these antibodies are disclosed above; any of these embodiments can be used in the DVD-immunoglobulin bispecific antibody.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second V H domain comprising SEQ ID NOs:18, 19, and 20, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second V L domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be an EL linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NO: 1, a linker, a second V H domain comprising SEQ ID NO: 17, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a V L domain comprising SEQ ID NO: 5 a linker, a second VL domain comprising SEQ ID NO: 21, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be an EL linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a CV503 V H domain, an EL linker, a CV993 V H domain, and an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a CV503 V L domain, an EL linker, a CV993 V L domain, and an IgG or IgA light chain constant domain.
  • VD1 of the DVD-immunoglobulin includes the V H and the V L of CV1206 as the VD1
  • the VD2 of the DVD-immunoglobulin includes V H and the V L of CD521.
  • Embodiments of the V H and V L domains of these antibodies are disclosed above; any of these embodiments can be used in the DVD-immunoglobulin bispecific antibody.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second V H domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, such as an IgG constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second V L domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the linker can be an EL linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NO: 41, a linker, a second V H domain comprising SEQ ID NO: 25, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a V L domain comprising SEQ ID NO: 45 a linker, and second V L domain comprising SEQ ID NO: 29, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the linker can be an EL linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a CV1206 V H domain, a GS linker, a CV521 V H domain, an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a CV1206 V L domain, GS linker, a CV521 V L domain, and an IgG or IgA light chain constant domain.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a CV1206 V H domain, an EL linker, a CV521 V H domain, and an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a CV1206 V L domain, an EL linker, a CV521 V L domain, and an IgG or IgA light chain constant domain.
  • CV521_503_GS the VD1 of the DVD-immunoglobulin includes the V H and the V L of CV521 as the VD1, and the VD2 of the DVD-immunoglobulin includes V H and the V L of CD503.
  • Embodiments of the V H and V L domains of these antibodies are disclosed above; any of these embodiments can be used in the DVD-immunoglobulin bispecific antibody.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second V H domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a V L domain comprising SEQ ID NOs: 30, 31, and 32 a linker, and a second V L domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a first V H domain comprising SEQ ID NO: 25, a linker, a second V H domain comprising SEQ ID NO: 1, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NO: 29 a linker, and second V L domain comprising SEQ ID NO: 5, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a CV521 V H domain, a GS linker, a CV503 V H domain, and an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a CV521 V L domain, a GS linker, a CV503 V L domain, and an IgG or IgA light chain constant domain.
  • CV664_993_GS In some embodiments the VD1 of the DVD-immunoglobulin includes the V H and the V L of CV664 as the VD1, and the VD2 of the DVD-immunoglobulin includes V H and the V L of CD993.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second V H domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NOs: 14, 15, and 16, a linker, and a second V L domain comprising SEQ ID NOs: 22, 23 and 24, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a first V H domain comprising SEQ ID NO: 9, a linker, a second V H domain comprising SEQ ID NO: 17, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NO: 13, a linker, and second V L domain comprising SEQ ID NO: 21, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a CV664 V H domain, a GS linker, a CV993 V H domain, and an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a CV664 V L domain, a GS linker, a CV993 V L domain, and an IgG or IgA light chain constant domain.
  • CV993_521_GS the VD1 of the DVD-immunoglobulin includes the V H and the V L of CV993 as the VD1
  • the VD2 of the DVD-immunoglobulin includes V H and the V L of CD521.
  • Embodiments of the V H and V L domains of these antibodies are disclosed above; any of these embodiments can be used in the DVD-immunoglobulin bispecific antibody.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second V H domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second V L domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a first V H domain comprising SEQ ID NO: 17, a linker, a second V H domain comprising SEQ ID NO: 25, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a V L domain comprising SEQ ID NO: 21 a linker, and second V L domain comprising SEQ ID NO: 29, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a CV993 V H domain, a GS linker, a CV521 V H domain, and an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a CV993 V L domain, a GS linker, a CV521 V L domain, and an IgG or IgA light chain constant domain.
  • VD1 of the DVD-immunoglobulin includes the V H and the V L of CV503 as the VD1
  • the VD2 of the DVD-immunoglobulin includes V H and the V L of CD664.
  • Embodiments of the V H and V L domains of these antibodies are disclosed above; any of these embodiments can be used in the DVD-immunoglobulin bispecific antibody.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second V H domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second V L domain comprising SEQ ID NOs: 14, 15, and 16, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the linker can be an EL linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order, a first V H domain comprising SEQ ID NO: 1, a linker, a second V H domain comprising SEQ ID NO: 9, and a heavy chain constant domain, such as an IgG or IgA constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order, a V L domain comprising SEQ ID NO: 5 a linker, and second VL domain comprising SEQ ID NO: 13, and a light chain constant domain, such as an IgG or IgA constant domain.
  • the linker can be a GS linker.
  • the linker can be an EL linker.
  • the heavy chain of the bispecific antibody comprises, in N-to C terminal order: a CV503 V H domain, a GS linker, a CV664 V H domain, and an IgG or IgA heavy chain constant domain.
  • the light chain of the bispecific antibody comprises, in N to C terminal order: a CV503 V L domain, a GS linker, a CV664 V L domain, and an IgG or IgA light chain constant domain.
  • any suitable method can be used to design and produce a bispecific antibody, such as crosslinking two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types.
  • multispecific antibodies such as bispecific antibodies
  • suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N- hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate).
  • the multi-specific antibody may have any suitable format that allows for binding to the coronavirus spike protein by the antibody or antigen binding fragment as provided herein.
  • Bispecific single chain antibodies can be encoded by a single nucleic acid molecule.
  • bispecific single chain antibodies as well as methods of constructing such antibodies are provided in U.S. Pat. Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538. Additional examples of bispecific single chain antibodies can be found in PCT application No. WO 99/54440; Mack et al., J. Immunol., 158(8):3965-3970, 1997; Mack et al., Proc. Natl. Acad. Sci.
  • a scFv molecule can be fused to one of the V L -CL (L) or VH-CH1 chains, e.g., to produce a bibody one scFv is fused to the C-term of a Fab chain.
  • Antigen binding fragments are encompassed by the present disclosure, such as Fab, F(ab') 2 , and Fv which include a heavy chain and V L and specifically bind a coronavirus spike protein. These antibody fragments retain the ability to selectively bind with the antigen and are “antigen-binding” fragments.
  • Non- limiting examples of such fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; (3) (Fab') 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab') 2 is a dimer of two Fab' fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the V L and V L expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the V H and the V L linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.
  • V H -domain-linker domain-V L -domain V L -domain-linker domain-V H -domain
  • a dimer of a single chain antibody scFV 2 ), defined as a dimer of a scFV.
  • Antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as an E. coli cell) of DNA encoding the fragment.
  • Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2.
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.
  • Other methods of cleaving antibodies such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • amino acid sequence variants of the antibodies and bispecific antibodies provided herein are provided. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody or bispecific antibody.
  • Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody V H domain and/or V L domain, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding. In some embodiments, variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and the framework regions.
  • Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.
  • the variants typically retain amino acid residues necessary for correct folding and stabilizing between the V H and the V L regions, and will retain the charge characteristics of the residues in order to preserve the low pI and low toxicity of the molecules.
  • Amino acid substitutions can be made in the V H and the V L regions to increase yield.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 1.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 5.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 9.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 13.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 17.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 21.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 29.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 29.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 33.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 37.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 41.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 45.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 49.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 53.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 57.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 61.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 65.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 69.
  • the heavy chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 73.
  • the light chain of the antibody comprises up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) compared to the amino acid sequence set forth as one of SEQ ID NO: 77.
  • the antibody or antigen binding fragment can include up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions (such as conservative amino acid substitutions) in the framework regions of the heavy chain of the antibody/bispecific antibody, or the light chain of the antibody/bispecific antibody, or the heavy and light chains of the antibody/bispecific antibody, compared to known framework regions, or compared to the framework regions of the antibody, and maintain the specific binding activity for the epitope of the spike protein.
  • the antibody can be CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576.
  • the bispecific antibody can be CV503_521_GS, CV521_1182_GS, CV503_993_EL, CV1206_521_GS, CV521_503_GS, CV664_993_GS, CV993_521_GS, CV503_664_GS, CV503_664_EL, or CV1206_521_EL.
  • the bispecific antibody includes up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions in one of SEQ ID NOs: 101-120.
  • substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen.
  • conservative alterations e.g., conservative substitutions as provided herein
  • each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.
  • only the framework residues are modified so the CDRs are unchanged.
  • the V L and V H segments can be randomly mutated, such as within HCDR3 region or the LCDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response.
  • in vitro affinity maturation can be accomplished by amplifying V H and V L regions using PCR primers complementary to the HCDR3 or LCDR3, respectively.
  • the primers have been “spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode V H and V L segments into which random mutations have been introduced into the V H and/or V L CDR3 regions.
  • V H amino acid sequence is one of SEQ ID NOs:1, 9, 17, 25, 33, 41, 49, 57, 65, or 73.
  • V L amino acid sequence is one of SEQ ID NOs: 5, 13, 21, 29, 37, 45, 53, 61, 69, or 77, respectively.
  • an antibody such as CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576
  • antigen binding fragment or bispecific antibody (such as CV503_521_GS, CV521_1182_GS, CV503_993_EL, CV1206_521_GS, CV521_503_GS, CV664_993_GS, CV993_521_GS, CV503_664_GS, CV503_664_EL, or CV1206_521_EL) is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated.
  • Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed.
  • the antibody such as CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576
  • bispecific antibody such as CV503_521_GS, CV521_1182_GS, CV503_993_EL, CV1206_521_GS, CV521_503_GS, CV664_993_GS, CV993_521_GS, CV503_664_GS, CV503_664_EL, or CV1206_521_EL
  • comprises an Fc region the carbohydrate attached thereto may be altered.
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol.15(1):26-32, 1997.
  • the oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure.
  • modifications of the oligosaccharide in an antibody may be made in order to create antibody variants with certain improved properties.
  • variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region.
  • the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%.
  • the amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g.
  • Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about ⁇ 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd).
  • Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005/053742; WO 2002/031140; Okazaki et al., J. Mol.
  • Antibody variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.); U.S. Pat. No.6,602,684 (Umana et al.); and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided.
  • the constant region of the antibody or bispecific antibody comprises one or more amino acid substitutions to optimize in vivo half-life of the antibody.
  • the serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn).
  • the antibody comprises an amino acid substitution that increases binding to the FcRn.
  • Non-limiting examples of such substitutions include substitutions at IgG constant regions T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176(1):346-356, 2006); M428L and N434S (the “LS” mutation, see, e.g., Zalevsky, et al., Nature Biotechnol., 28(2):157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12):1759-1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int.
  • the disclosed antibodies and antigen binding fragments can be linked to or comprise an Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide can include the M428L and N434S substitutions.
  • an antibody or bispecific antibody provided herein may be further modified to contain additional nonproteinaceous moieties.
  • the moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers.
  • Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1,3-dioxolane, poly-1,3,6- trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof.
  • PEG polyethylene glycol
  • copolymers of ethylene glycol/propylene glycol carboxymethylcellulose
  • dextran polyvinyl alcohol
  • Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc. B.
  • the antibodies, antigen binding fragments, and bispecific antibodies that specifically bind to a coronavirus spike protein, as disclosed herein, can be conjugated to an agent, such as an effector molecule or detectable marker. Both covalent and noncovalent attachment means may be used.
  • Various effector molecules and detectable markers can be used, including (but not limited to) toxins and radioactive agents such as 125 I, 32 P, 14 C, 3 H and 35 S and other labels, target moieties, enzymes and ligands, etc.
  • the choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect.
  • the procedure for attaching a detectable marker to an antibody, antigen binding fragment, or bispecific antibody can be conjugated to an agent, such as an effector molecule or detectable marker.
  • Polypeptides typically contain a variety of functional groups, such as carboxyl (-COOH), free amine (-NH 2 ) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on a polypeptide to result in the binding of the effector molecule or detectable marker.
  • the antibody, antigen binding fragment, or bispecific antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization may involve attachment of any suitable linker molecule.
  • the linker is capable of forming covalent bonds to both the antibody or antigen binding fragment and to the effector molecule or detectable marker.
  • Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers may be joined to the constituent amino acids through their side chains (such as through a disulfide linkage to cysteine) or the alpha carbon, or through the amino, and/or carboxyl groups of the terminal amino acids.
  • a suitable method for attaching a given agent to an antibody or antigen binding fragment or bispecific antibody can be determined.
  • the antibody, antigen binding fragment or bispecific antibody can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT, computed axial tomography (CAT), MRI, magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination).
  • detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI).
  • useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5- dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like.
  • Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP).
  • An antibody, antigen binding fragment, or bispecific antibody can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • enzymes that are useful for detection, such as horseradish peroxidase, ⁇ - galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like.
  • an antibody or antigen binding fragment is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned.
  • the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable.
  • An antibody, antigen binding fragment, or bispecific antibody may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.
  • the antibody, antigen binding fragment or bispecific antibody can be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also of use as labels.
  • Antibodies can also be conjugated with lanthanides (such as europium and dysprosium), and manganese.
  • An antibody, antigen binding fragment, or bispecific antibody may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).
  • the antibody, antigen binding fragment or bispecific antibody can also be conjugated with a radiolabeled amino acid, for example, for diagnostic purposes.
  • the radiolabel may be used to detect a coronavirus by radiography, emission spectra, or other diagnostic techniques.
  • labels for polypeptides include, but are not limited to, the following radioisotopes: 3 H, 14 C, 35 S, 90 Y, 99m Tc, 111 In, 125 I, 131 I.
  • the radiolabels may be detected, for example, using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination.
  • Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.
  • the average number of detectable marker moieties per antibody, antigen binding fragment, or bispecific antibody in a conjugate can range, for example, from 1 to 20 moieties per antibody or antigen binding fragment.
  • the average number of effector molecules or detectable marker moieties per antibody or antigen binding fragment in a conjugate range from about 1 to about 2, from about 1 to about 3, about 1 to about 8; from about 2 to about 6; from about 3 to about 5; or from about 3 to about 4.
  • the loading (for example, effector molecule per antibody ratio) of a conjugate may be controlled in different ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number or position of linker-effector molecule attachments.
  • Nucleic acid molecules for example, cDNA or RNA molecules
  • Nucleic acids encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences and V H and V L sequences), sequences available in the art (such as framework or constant region sequences), and the genetic code.
  • nucleic acid molecules can encode the V H , the V L , or both the V H and V L (for example in a bicistronic expression vector) of a disclosed antibody or antigen binding fragment.
  • the nucleic acid molecules encode an scFv.
  • the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell) to produce a disclosed antibody or antigen binding fragment.
  • Nucleic acid molecules encoding an scFv are provided.
  • the genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids which differ in their sequence but which encode the same antibody sequence, or encode a conjugate or fusion protein including the V L and/or V H nucleic acid sequence.
  • an isolated nucleic acid molecule encodes the V H of the CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576 antibody.
  • Exemplary nucleic acid sequence are provided herein.
  • the nucleic acid molecule encodes the V L of the CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576 monoclonal antibody.
  • the nucleic acid molecule can encode a bi-specific antibody, such as in DVD-immunoglobulin format.
  • the nucleic acid molecule encodes the V H of the CV503_521_GS, CV521_1182_GS, CV503_993_EL, CV1206_521_GS, CV521_503_GS, CV664_993_GS, CV993_521_GS, CV503_664_GS, CV503_664_EL, or CV1206_521_EL bispecific antibody.
  • the nucleic acid molecule encodes the V L of the CV503_521_GS, CV521_1182_GS, CV503_993_EL, CV1206_521_GS, CV521_503_GS, CV664_993_GS, CV993_521_GS, CV503_664_GS, CV503_664_EL, or CV1206_521_EL bispecific antibody.
  • the nucleic acid molecule can also encode a conjugate. Exemplary nucleic acid sequences are provided as SEQ ID NOs: 81-100 and 121-140.
  • Nucleic acid molecules encoding the antibodies, antigen binding fragments, bispecific antibodies, and conjugates that specifically bind to a coronavirus spike protein can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. Exemplary nucleic acids can be prepared by cloning techniques.
  • Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • TAS transcription-based amplification system
  • 3SR self-sustained sequence replication system
  • the antibodies, antigen binding fragments, and conjugates can be expressed as individual proteins including the V H and/or V L (linked to an effector molecule or detectable marker as needed), or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011).
  • An immunoadhesin can also be expressed.
  • nucleic acids encoding a V H and V L , and immunoadhesin are provided.
  • the nucleic acid sequences can optionally encode a leader sequence.
  • V H - and V L -encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly 4 -Ser) 3 , such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H domains joined by the flexible linker (see, e.g., Bird et al., Science, 242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci.
  • a flexible linker e.g., encoding the amino acid sequence (Gly 4 -Ser) 3 , such that the V H and V L sequences can be expressed as a contiguous single-chain protein, with the V L and V H domains joined by the flexible linker
  • cleavage site can be included in a linker, such as a furin cleavage site.
  • the single chain antibody may be monovalent, if only a single V H and V L are used, bivalent, if two V H and V L are used, or polyvalent, if more than two V H and V L are used.
  • Bispecific or polyvalent antibodies may be generated that bind specifically to a coronavirus spike protein and another antigen.
  • the encoded V H and V L optionally can include a furin cleavage site between the V H and V L domains.
  • Linkers can also be encoded, such as when the nucleic acid molecule encodes a bi-specific antibody in DVD-IGTM format.
  • One or more DNA sequences encoding the antibodies, antigen binding fragments, bispecific antibodies, or conjugates can be expressed in vitro by DNA transfer into a suitable host cell.
  • the cell may be prokaryotic or eukaryotic.
  • Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host may be used. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.
  • nucleic acids encoding the antibodies, antigen binding fragments, and bispecific antibodies (such as DVD-immunoglobulin antibodies) described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette.
  • the promoter can be any promoter of interest, including a cytomegalovirus promoter.
  • an enhancer such as a cytomegalovirus enhancer, is included in the construct.
  • the cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein.
  • the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein- encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons.
  • the vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).
  • expression cassettes which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator.
  • a strong promoter to direct transcription e.g., a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator.
  • this can include a promoter such as the T7, trp, lac, or lamda promoters, a ribosome binding site, and preferably a transcription termination signal.
  • control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences).
  • the cassettes can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofection for mammalian cells.
  • Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.
  • Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.
  • the antibodies, antigen binding fragments, bispecific antibodies, and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009).
  • the antibodies, antigen binding fragment, and conjugates need not be 100% pure.
  • the polypeptides should be substantially free of endotoxin.
  • the methods include administering to the subject an effective amount (that is, an amount effective to inhibit the infection in the subject) of a disclosed antibody, antigen binding fragment, or bispecific antibody, or a nucleic acid encoding such an antibody, antigen binding fragment, or bispecific antibody, to a subject at risk of a coronavirus infection or having the coronavirus infection.
  • the methods can be used pre-exposure or post-exposure.
  • the antibody or antigen binding fragment can be used in the form of a bi-specific antibody, such as a DVD-IGTM.
  • the infection does not need to be completely eliminated or inhibited for the method to be effective.
  • the method can decrease the infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable coronavirus infection) as compared to the coronavirus infection in the absence of the treatment.
  • the subject can also be treated with an effective amount of an additional agent, such as an anti-viral agent.
  • administering inhibits the establishment of an infection and/or subsequent disease progression in a subject, which can encompass any statistically significant reduction in activity (for example, growth or invasion) or symptoms of the coronavirus infection in the subject.
  • Methods are disclosed herein for the inhibition of a coronavirus replication in a subject. The methods include administering to the subject an effective amount (that is, an amount effective to inhibit replication in the subject) of a disclosed antibody, antigen binding fragment, bispecific antibody, or a nucleic acid encoding such an antibody, antigen binding fragment, or bispecific antibody, to a subject at risk of a coronavirus infection or having a coronavirus infection.
  • the methods can be used pre-exposure or post- exposure. Methods are disclosed for treating a coronavirus infection in a subject. Methods are also disclosed for preventing a coronavirus infection in a subject. These methods include administering one or more of the disclosed antibodies, antigen binding fragments, bispecific antibodies, or nucleic acid molecule encoding such molecules, or a composition including such molecules, as disclosed herein. Antibodies, antigen binding fragments thereof, and bispecific antibodies can be administered by intravenous infusion.
  • Doses of the antibody, antigen binding fragment, or bispecific antibody vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg.
  • the dose of the antibody, antigen binding fragment or bispecific antibody can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg.
  • the antibody, antigen binding fragment, or bispecific antibody is administered according to a dosing schedule determined by a medical practitioner.
  • the antibody, antigen binding fragment or bispecific antibody is administered weekly, every two weeks, every three weeks or every four weeks.
  • the method of inhibiting the infection in a subject further comprises administration of one or more additional agents to the subject. Additional agents of interest include, but are not limited to, anti-viral agents such as hydroxychloroquine, arbidol, remdesivir, favipiravir, baricitinib, lopinavir/ritonavir, Zinc ions, and interferon beta-1b, or their combinations.
  • the method comprises administration of a first antibody that specifically binds to a coronavirus spike protein as disclosed herein and a second antibody that also specifically binds to a coronavirus protein, such as a different epitope of the coronavirus protein
  • the first antibody is one of CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576.
  • the first antibody is one of CV503, CV521, CV1206, CV664, or CV993 and the second antibody is another of CV521, CV1182, CV993, and CV664, wherein the first antibody and second antibody are different.
  • one antibody binds the NTD, and another antibody binds the RBD.
  • the antibodies bind different epitopes on the RBD.
  • An effective amount of one, two, three or four, or five of CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, and CV576 can be administered to a subject.
  • a bispecific antibody such as CV503_521_GS, CV521_1182_GS, CV503_993_EL, CV1206_521_GS, CV521_503_GS, CV664_993_GS, CV993_521_GS, CV503_664_GS, CV503_664_EL, or CV1206_521_EL can be administered to the subject.
  • Combinations of these bispecific antibodies can be administered to the subject.
  • a subject is administered DNA or RNA encoding a disclosed antibody, antigen binding fragment, or bispecific antibody, to provide in vivo antibody production, for example using the cellular machinery of the subject.
  • Any suitable method of nucleic acid administration may be used; non- limiting examples are provided in U.S. Patent No.5,643,578, U.S. Patent No.5,593,972 and U.S. Patent No. 5,817,637.
  • U.S. Patent No.5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism.
  • nucleic acids are direct administration with plasmid DNA, such as with a mammalian expression plasmid.
  • the nucleotide sequence encoding the disclosed antibody, antigen binding fragments thereof, or bispecific antibody can be placed under the control of a promoter to increase expression.
  • the methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of an antibody, or antigen binding fragments thereof.
  • a disclosed antibody or antigen binding fragment is expressed in a subject using the pVRC8400 vector (described in Barouch et al., J. Virol., 79(14), 8828-8834, 2005, which is incorporated by reference herein).
  • a subject (such as a human subject at risk of a coronavirus infection or having a coronavirus infection) can be administered an effective amount of an AAV viral vector that comprises one or more nucleic acid molecules encoding a disclosed antibody, antigen binding fragment, or bispecific antibody.
  • the AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed antibody, antigen binding fragment, or bispecific antibody, and administration of the effective amount of the AAV viral vector to the subject leads to expression of an effective amount of the antibody, antigen binding fragment, or bispecific antibody in the subject.
  • Non-limiting examples of AAV viral vectors that can be used to express a disclosed antibody, antigen binding fragment, or bispecific antibody in a subject include those provided in Johnson et al., Nat. Med., 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541):87-91, 2015, each of which is incorporated by reference herein in its entirety.
  • a nucleic acid encoding a disclosed antibody, antigen binding fragment, or bispecific antibody is introduced directly into tissue.
  • the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS ⁇ Gene Gun.
  • the nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter. Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 ⁇ g/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No.5,589,466). Single or multiple administrations of a composition including a disclosed antibody, antigen binding fragment, or bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, can be administered depending on the dosage and frequency as required and tolerated by the patient.
  • the dosage can be administered once, but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit a coronavirus infection without producing unacceptable toxicity to the patient. Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The effective dose can be determined from cell culture assays and animal studies.
  • the coronavirus spike protein-specific antibody, antigen binding fragment, or bispecific antibody or nucleic acid molecule encoding such molecules, or a composition including such molecules can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intra-arterially, intraperitoneally, intramuscularly, intradermally, or intrathecally.
  • the antibody, antigen binding fragment, bispecific antibody or nucleic acid molecule encoding such molecules, or a composition including such molecules is administered by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal or intrathecal injection once a day.
  • the antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules can also be administered by direct injection at or near the site of disease.
  • a further method of administration is by osmotic pump (e.g., an Alzet pump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows for controlled, continuous and/or slow-release delivery of the antibody, antigen binding fragment, conjugate, or nucleic acid molecule encoding such molecules, or a composition including such molecules, over a pre-determined period.
  • the osmotic pump or mini-pump can be implanted subcutaneously, or near a target site. 2.
  • compositions are provided that include one or more of the coronavirus spike protein-specific antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, that are disclosed herein in a pharmaceutically acceptable carrier.
  • the composition comprises the CV503, CV664, CV993, CV521, CV1182, CV1206, CV532, CV635, CV085, or CV576 antibody disclosed herein, or an antigen binding fragment thereof.
  • the composition can include the bispecific antibody, such as CV503_521_GS, CV521_1182_GS, CV503_993_EL, CV1206_521_GS, CV521_503_GS, CV664_993_GS, CV993_521_GS, CV503_664_GS, CV503_664_EL, or CV1206_521_EL
  • the composition comprises two, three, four or more antibodies, antigen binding fragments, or bispecific antibodies, that specifically bind a coronavirus spike protein.
  • the compositions are useful, for example, for example, for the inhibition or detection of a coronavirus infection, such as a SARS-CoV-2 infection.
  • compositions can be prepared in unit dosage forms, such as in a kit, for administration to a subject.
  • the amount and timing of administration are at the discretion of the administering physician to achieve the desired purposes.
  • the antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules can be formulated for systemic or local administration.
  • the, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules is formulated for parenteral administration, such as intravenous administration.
  • the antibody, antigen binding fragment, bispecific antibody, or conjugate thereof, in the composition is at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) pure.
  • the composition contains less than 10% (such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.
  • compositions for administration can include a solution of the antibody, antigen binding fragment, bispecific antibody, conjugate, or nucleic acid molecule encoding such molecules, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier.
  • a pharmaceutically acceptable carrier such as an aqueous carrier.
  • aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
  • These compositions may be sterilized by any suitable technique.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • a typical composition for intravenous administration comprises about 0.01 to about 30 mg/kg of antibody, antigen binding fragment, bispecific antibody, or conjugate per subject per day (or the corresponding dose of a conjugate including the antibody or antigen binding fragment).
  • Any suitable method may be used for preparing administrable compositions; non-limiting examples are provided in such publications as Remington: The Science and Practice of Pharmacy, 22 nd ed., London, UK: Pharmaceutical Press, 2013.
  • the composition can be a liquid formulation including one or more antibodies, antigen binding fragments, or bispecific antibodies, in a concentration range from about 0.1 mg/ml to about 20 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml.
  • Antibodies, an antigen binding fragment thereof, a bispecific antibody, or a nucleic acid encoding such molecules can be provided in lyophilized form and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration.
  • a solution including the antibody, antigen binding fragment, bispecific antibody, or a nucleic acid encoding such molecules can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight.
  • Antibodies, antigen binding fragments, conjugates, or a nucleic acid encoding such molecules can be administered by slow infusion, rather than in an intravenous push or bolus.
  • a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level.
  • an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30-minute period if the previous dose was well tolerated.
  • Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems.
  • Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles.
  • Microcapsules contain the active protein agent, such as a cytotoxin or a drug, as a central core. In microspheres, the active protein agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 ⁇ m are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively.
  • Capillaries have a diameter of approximately 5 ⁇ m so that only nanoparticles are administered intravenously. Microparticles are typically around 100 ⁇ m in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, Colloidal Drug Delivery Systems, J. Kreuter (Ed.), New York, NY: Marcel Dekker, Inc., pp.219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications, A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp.315-339, 1992. Polymers can be used for ion-controlled release of the compositions disclosed herein.
  • Any suitable polymer may be used, such as a degradable or nondegradable polymeric matrix designed for use in controlled drug delivery.
  • hydroxyapatite has been used as a microcarrier for controlled release of proteins.
  • liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug. 2.
  • Methods of detection and diagnosis Methods are also provided for the detection of the presence of a coronavirus spike protein in vitro or in vivo.
  • the presence of a coronavirus spike protein is detected in a biological sample from a subject and can be used to identify a subject with an infection.
  • the sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens.
  • Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine.
  • the method of detection can include contacting a cell or sample, with an antibody, antigen binding fragment, or bispecific antibody, that specifically binds to a coronavirus spike protein, or conjugate thereof (e.g., a conjugate including a detectable marker) under conditions sufficient to form an immune complex, and detecting the immune complex (e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment.
  • the antibody, antigen binding fragment or bispecific antibody is directly labeled with a detectable marker.
  • the antibody (or antigen binding fragment or bispecific antibody) that binds the coronavirus spike protein is unlabeled and a secondary antibody or other molecule that can bind the primary antibody is utilized for detection.
  • the secondary antibody is chosen that is able to specifically bind the specific species and class of the first antibody.
  • the first antibody is a human IgG
  • the secondary antibody may be an anti-human-IgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
  • Suitable labels for the antibody, antigen binding fragment, bispecific antibody or secondary antibody are known and described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
  • the disclosed antibodies, antigen binding fragments thereof, or bispecific antibodies are used to test vaccines.
  • a vaccine composition including a coronavirus spike protein or fragment thereof assumes a conformation including the epitope of a disclosed antibody.
  • a method for testing a vaccine comprising contacting a sample containing the vaccine, such as a coronavirus spike protein immunogen, with a disclosed antibody, antigen binding fragment, or bispecific antibody, under conditions sufficient for formation of an immune complex, and detecting the immune complex, to detect the vaccine including the epitope of interest in the sample.
  • the detection of the immune complex in the sample indicates that vaccine component, such as the immunogen assumes a conformation capable of binding the antibody or antigen binding fragment.
  • the method can also include the use of an assay that distinguishes between SARS-CoV-2 as some isolated mAbs only bind to SARS-CoV-2 RBD but not the SARS-CoV RBD, and some isolated mAbs bind to both SARS-CoV RBD and SARS-CoV-2 RBD.
  • a comparison is made between the binding of a sample to an antibody that binds SARS-CoV RBD and SARS-CoV-2RBD, and the binding of a sample to an antibody that binds only the SARS-CoV-2 RBD but not the SARS-CoV RBD. If both antibodies bind the sample, then the sample is from a subject infected with SARS-CoV-2. If only the antibody that binds both SARS-CoV RBD and SARS-CoV-2 RBD, then the sample is from a subject infected with SARS-CoV.
  • SARS-CoV-2 is a novel pathogen that triggers a primary immune response during which plasmablasts are the main source of neutralizing antibodies.
  • SARS-CoV-2-specific monoclonal antibodies derived from plasmablasts were studied, as well as memory B cells of patients who had recovered from COVID-19. It was determined that plasmablasts produce high-affinity, potent neutralizing antibodies despite limited VH gene mutations. Of the five most potent antibodies derived from either cell type, four targeted distinct sites of the spike protein, three within the RBD and one on the NTD. A panel of bispecific antibodies was designed that combined non-overlapping specificities. A bispecific antibody targeting both RBD and NTD on multiple spike molecules was designed that was substantially more potent than its individual antibody components.
  • Example 1 Plasmablast and Memory B cell Screen Yields Potent Antibodies and Bispecific Antibodies that Specifically Bind SARS-CoV-2
  • convalescent plasma and plasmablast samples were examined from 126 individuals in New York City who had recovered from PCR-documented SARS-CoV-2 infection. These samples were collected in April 2020 and thus reflect the primary B cell response during the first outbreak in the study area.
  • the plasma was tested for binding to the spike protein of non-SARS-CoV-2 coronaviruses, as well as to the receptor-binding domain (RBD) and N-terminal domain (NTD) of SARS-CoV-2 (Fig.1A). All subjects had detectable levels of antibodies against at least one non-SARS-CoV-2 spike protein, consistent with previous exposure to seasonal coronaviruses. As expected, most subjects had detectable antibodies to the SARS-CoV-2 spike protein (119/126), RBD (106/126) and NTD (122/126), and antibody levels against these targets correlated with each other (Fig.1B).
  • the plasma was tested for neutralization of authentic SARS-CoV-2 and a wide range of neutralizing titers was found, from ⁇ 40 to 765 (Fig.1A).
  • Neutralization potency generally correlated with antibody levels to spike, RBD and NTD, although several plasma samples were non-neutralizing despite high antibody levels to each of these targets (Fig.1C), suggesting that fine epitope specificity is critical for effective neutralization of SARS-CoV-2.
  • Fig.1C To investigate the circulating antibody repertoire in these individuals at higher resolution, a single- cell ex vivo plasmablast secretion assay was developed using the Berkeley Lights Beacon optofluidics device to screen for SARS-CoV-2 spike- and RBD-specific monoclonal antibodies.
  • Circulating plasmablasts CD19 + CD27 ++ CD38 ++ ) from these donors were bulk FACS-sorted, distributed individually into nanoliter-volume pens in a microfluidics chip, and then screened directly for secretion of antibodies that bound to beads coated with SARS-CoV-2 spike or RBD (Fig.5). A total of ⁇ 44,000 plasmablasts were screened using this assay, of which 787 supernatants bound to SARS-CoV-2 spike and/or RBD.
  • SARS-CoV-2-specific antibodies were expressed and characterized from plasmablasts and 47 from MBCs (Fig.6).
  • 59 targeted the RBD
  • 64 targeted the NTD
  • 46 targeted neither (presumably S2- specific), indicating that the primary antibody response to SARS-CoV-2 is distributed along the entire spike protein.
  • VH3-53 was more common among MBCs (4 th most frequent) than plasmablasts (14 th most frequent). It was also found that both plasmablasts and MBCs had similarly low mutation levels ( ⁇ 3%) in their heavy and light chain genes (Fig.2B), consistent with their differentiation from na ⁇ ve B cells without extensive germinal center experience. A minority of antibodies from plasmablasts and MBCs had higher mutation levels ( ⁇ 10%), suggesting that they arose from pre- existing memory B cells.
  • the most potent antibodies performed comparably to the benchmark antibodies across the different assays.
  • the NTD-specific antibodies and C135 (Robbiani et al. Nature.2020 Aug;584(7821):437-442. doi: 10.1038/s41586-020-2456-9. Epub 2020 Jun 18), which targets the RBD but does not block ACE2 binding, did not show efficacy in the pseudovirus assay (Fig.2H).
  • the relative potency of each antibody was reasonably consistent but the absolute IC 50 values differed greatly between assays (Fig.2H), highlighting the benefit of using standardized assays to compare antibodies from various sources.
  • High-throughput surface plasmon resonance (SPR) was performed to determine the affinity of the RBD- and NTD-specific antibodies.
  • RBD-specific antibodies isolated from MBCs and plasmablasts had similar affinities, with a few antibodies reaching sub-nM affinity (Fig.3A, Fig.9).
  • the top four neutralizing antibodies had high affinity ( ⁇ 10nM) to the RBD, but other high-affinity antibodies, including the antibody with the highest affinity in our panel, were non-neutralizing (Fig.3B), suggesting that high affinity for the RBD may be necessary but not sufficient for neutralization.
  • NTD-specific antibodies from plasmablasts generally had higher affinities than those from MBCs (Fig.3C, Fig.9B).
  • the most potent antibodies had relatively low affinity for the NTD, approaching micromolar K D values (Fig. 3D).
  • High-throughput SPR was also used to perform epitope binning of the antibodies, and several antibodies with known binding sites were included as controls.
  • the RBD-specific antibodies fell broadly into four bins (Fig.3E, Fig.10).
  • the three most potent RBD-specific antibodies, CV503, CV664 and CV993, were located in separate bins and did not compete for binding to RBD (Fig.3E and 3F).
  • Most of the other RBD-specific neutralizing antibodies mapped to a bin with ACE2 or a second site containing the benchmark antibody C135.
  • DVD-IG TM bispecific DVD-Immunoglobulin antibodies
  • DiGiammarino, et al. “Design and generation of DVD-Ig molecules for dual-specific targeting,” in Therapeutic Proteins, Methods in Molecular Biology (Methods and Protocols), Voynov and Caravella, Eds. (Humana Press, 2012), vol.899, pp.145–156, incorporated herein by reference
  • This bispecific antibody form is relatively easy to express and has a similar structure to standard IgG, except for an additional antigen-binding site on top of the native site (Fig.4A). Then DVD-IG TM antibodies were expressed, and SDS-PAGE and size exclusion chromatography confirmed that 9 out of 10 antibodies contained a single dominant product with the expected molecular weight (Figs. 13A-13B). It was found that bispecific antibodies that combined an RBD-specific and NTD-specific antibody retained binding to both domains (Fig.4B). Moreover, SPR experiments confirmed that bispecific antibodies containing two different RBD-binding sites were able to utilize both sites (Fig.13C).
  • CV503_664_GS which combined CV503 and CV664, was able to bind to RBD previously attached to either component antibody (Fig.13C).
  • the panel of bispecific antibodies was tested in the authentic SARS-CoV-2 and psuedovirus neutralization assays (Fig.4C).
  • CV1206_521_GS which combined an RBD-specific (CV1206) and NTD-specific (CV521) antibody, was substantially more potent than either component antibody (Fig.4D).
  • a reduced effect was seen with CV1206_521_EL (Fig.14) which combines the same two antibodies but has a different linker, consistent with the lower expression purity of this antibody (Figs.13A-13B).
  • CV664_993_GS was detected over its constituent antibodies with authentic SARS-CoV-2 (Fig.4D).
  • CV1206_521_GS was the most potent neutralizing antibody in the panel (either bispecific or native IgG), a fact that was surprising given the lower potency of the antibody CV1206 compared to some of the other RBD-specific antibodies (Fig.2H, Fig.4C).
  • Negative-stain EM experiments revealed that this antibody was able to use both domains to cross-link different spike proteins using both RBD and NTD specificities (Fig. 4E).
  • CV993 also binds to SARS-CoV-1.
  • An advantage of bispecific antibodies is resistance to current and future viral escape mutants because these antibodies target multiple sites on the spike protein.
  • the bispecific antibodies were tested for binding to spike proteins carrying individual and total mutations encoded by the Alpha and Beta variants. Only the bispecific antibodies whose two components both lost binding to the SARS-CoV-2 variants, such as CV1206_521_GS, were unable to bind and neutralize these variants, suggesting that this antibody form is more resistant to spike mutations than regular mAbs.
  • To confirm the binding results and investigate the full neutralization range of the bispecific antibodies their ability to neutralize SARS-CoV-2 D614G, Alpha, Beta, Gamma, and Delta pseudotyped virus was tested.
  • a bispecific antibody combining CV503 and CV664 (CV503_664_EL) actually improved slightly in potency against the Beta variant, although CV503 and CV664 were both less effective against the variant as individual mAbs.
  • Two bispecific antibodies neutralized the Alpha, Beta, and Gamma variants, as well as the recently ascendant Delta variant, with little loss of potency compared to wild-type virus (Fig.15).
  • the bispecific antibodies were tested for efficacy against SARS-CoV-2 infection in the well- established Syrian hamster model, because this model resembles features of severe COVID-19 in humans (Figs.16A-16D) (Baum et al., Science 370, 1110–1115 (2020); Rogers et al., Science 369, 956–963 (2020)).
  • the bispecific antibodies were delivered intraperitonially at 2.5 or 10 mg/kg, followed by intranasal administration of 105 plaque-forming units (PFU) of SARS-CoV-224 hours later. Change in body weight and a blinded clinical score were used to assess SARS-CoV-2–mediated disease.
  • hamsters injected with phosphate-buffered saline (PBS) as sham treatment had a greater than 10% reduction in body weight through day 6 after infection, followed by a rebound in weight, whereas mock-exposed hamsters had no weight change (Fig.16A) (Andreano et al., Cell 184, 1821– 1835.e16 (2021); Chan et al., Clin. Infect. Dis.71, 2428–2446 (2020); Imai et al., Proc. Natl. Acad. Sci. U.S.A.117, 16587–16595 (2020)).
  • PBS phosphate-buffered saline

Abstract

L'invention concerne des anticorps monoclonaux, des fragments de liaison à l'antigène et des anticorps bispécifiques qui se lient spécifiquement à une protéine de spicule de coronavirus, telle que le SARS-CoV-2. L'invention concerne également l'utilisation de ces anticorps et d'anticorps bispécifiques pour inhiber une infection à coronavirus, telle qu'une infection par le SARS-CoV et/ou une infection par le SARS-CoV-2. De plus, l'invention concerne des procédés de détection d'un coronavirus, tel que le SARS-CoV-2, dans un échantillon biologique, à l'aide des anticorps décrits et des anticorps bispécifiques.
PCT/US2021/063525 2020-12-17 2021-12-15 Anticorps monoclonaux humains ciblant le sars-cov-2 WO2022132904A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202063127077P 2020-12-17 2020-12-17
US63/127,077 2020-12-17

Publications (1)

Publication Number Publication Date
WO2022132904A1 true WO2022132904A1 (fr) 2022-06-23

Family

ID=80001354

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2021/063525 WO2022132904A1 (fr) 2020-12-17 2021-12-15 Anticorps monoclonaux humains ciblant le sars-cov-2

Country Status (1)

Country Link
WO (1) WO2022132904A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116218789A (zh) * 2023-01-06 2023-06-06 扬州大学 一株杂交瘤细胞株、抗PDCoV NS6蛋白的单克隆抗体及其应用

Citations (58)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US5593972A (en) 1993-01-26 1997-01-14 The Wistar Institute Genetic immunization
US5643578A (en) 1992-03-23 1997-07-01 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
WO1997030087A1 (fr) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation d'anticorps glycosyles
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
WO1998058964A1 (fr) 1997-06-24 1998-12-30 Genentech, Inc. Procedes et compositions concernant des glycoproteines galactosylees
US5880103A (en) 1992-08-11 1999-03-09 President And Fellows Of Harvard College Immunomodulatory peptides
WO1999022764A1 (fr) 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
WO1999054440A1 (fr) 1998-04-21 1999-10-28 Micromet Gesellschaft Für Biomedizinische Forschung Mbh Polypeptides specifiques a cd19 et cd3 et leurs utilisations
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2000061739A1 (fr) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
WO2001029246A1 (fr) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'un polypeptide
WO2002031140A1 (fr) 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cellules produisant des compositions d'anticorps
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
WO2003011878A2 (fr) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
WO2003085119A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia
WO2003084570A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia
WO2003085107A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cellules à génome modifié
US6723538B2 (en) 1999-03-11 2004-04-20 Micromet Ag Bispecific antibody and chemokine receptor constructs
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
WO2004056312A2 (fr) 2002-12-16 2004-07-08 Genentech, Inc. Variants d'immunoglobuline et utilisations
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
US20050079574A1 (en) 2003-01-16 2005-04-14 Genentech, Inc. Synthetic antibody phage libraries
WO2005035778A1 (fr) 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase
WO2005035586A1 (fr) 2003-10-08 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Composition proteique hybride
US20050119455A1 (en) 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005053742A1 (fr) 2003-12-04 2005-06-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition a base d'anticorps
US20050266000A1 (en) 2004-04-09 2005-12-01 Genentech, Inc. Variable domain library and uses
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US20070117126A1 (en) 1999-12-15 2007-05-24 Genentech, Inc. Shotgun scanning
US7229760B2 (en) 2000-03-24 2007-06-12 Micromet Ag mRNA amplification
US7235641B2 (en) 2003-12-22 2007-06-26 Micromet Ag Bispecific antibodies
US20070160598A1 (en) 2005-11-07 2007-07-12 Dennis Mark S Binding polypeptides with diversified and consensus vh/vl hypervariable sequences
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
US20070292936A1 (en) 2006-05-09 2007-12-20 Genentech, Inc. Binding polypeptides with optimized scaffolds
US7323440B2 (en) 2002-02-13 2008-01-29 Micromet Ag De-immunized MOG (poly)peptide constructs
US7332168B2 (en) 2000-08-22 2008-02-19 Micromet Ag Composition for the elimination of autoreactive B-cells
WO2008077546A1 (fr) 2006-12-22 2008-07-03 F. Hoffmann-La Roche Ag Anticorps contre le récepteur du facteur de croissance i de type insuline et leurs utilisations
US7435549B1 (en) 1997-11-17 2008-10-14 Micromet Ag Method of identifying binding site domains that retain the capacity of binding to an epitope
US20090002360A1 (en) 2007-05-25 2009-01-01 Innolux Display Corp. Liquid crystal display device and method for driving same
US7635472B2 (en) 2003-05-31 2009-12-22 Micromet Ag Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
US7820166B2 (en) 2002-10-11 2010-10-26 Micromet Ag Potent T cell modulating molecules
US7919089B2 (en) 2003-05-31 2011-04-05 Micromet Ag Pharmaceutical composition comprising a bispecific antibody for EpCAM
US8007796B2 (en) 2005-12-16 2011-08-30 Micromet Ag Means and methods for the treatment of tumorous diseases
US8017748B2 (en) 2005-04-18 2011-09-13 Micromet Ag Antibody neutralizers of human granulocyte macrophage colony stimulating factor
US8076459B2 (en) 2003-10-16 2011-12-13 Micromet Ag Multispecfic deimmunized CD3-binders
WO2013163427A1 (fr) 2012-04-25 2013-10-31 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Anticorps pour traiter les infections par le vih-1
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments

Patent Citations (62)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5589466A (en) 1989-03-21 1996-12-31 Vical Incorporated Induction of a protective immune response in a mammal by injecting a DNA sequence
US6075181A (en) 1990-01-12 2000-06-13 Abgenix, Inc. Human antibodies derived from immunized xenomice
US6150584A (en) 1990-01-12 2000-11-21 Abgenix, Inc. Human antibodies derived from immunized xenomice
US5770429A (en) 1990-08-29 1998-06-23 Genpharm International, Inc. Transgenic non-human animals capable of producing heterologous antibodies
US5750373A (en) 1990-12-03 1998-05-12 Genentech, Inc. Enrichment method for variant proteins having altered binding properties, M13 phagemids, and growth hormone variants
US5643578A (en) 1992-03-23 1997-07-01 University Of Massachusetts Medical Center Immunization by inoculation of DNA transcription unit
US5880103A (en) 1992-08-11 1999-03-09 President And Fellows Of Harvard College Immunomodulatory peptides
US5817637A (en) 1993-01-26 1998-10-06 The Trustees Of The University Of Pennsylvania Genetic immunization
US5593972A (en) 1993-01-26 1997-01-14 The Wistar Institute Genetic immunization
WO1997030087A1 (fr) 1996-02-16 1997-08-21 Glaxo Group Limited Preparation d'anticorps glycosyles
WO1998058964A1 (fr) 1997-06-24 1998-12-30 Genentech, Inc. Procedes et compositions concernant des glycoproteines galactosylees
WO1999022764A1 (fr) 1997-10-31 1999-05-14 Genentech, Inc. Compositions renfermant des glycoformes de glycoproteine et methodes afferentes
US7435549B1 (en) 1997-11-17 2008-10-14 Micromet Ag Method of identifying binding site domains that retain the capacity of binding to an epitope
US7189826B2 (en) 1997-11-24 2007-03-13 Institute For Human Genetics And Biochemistry Monoclonal human natural antibodies
US6602684B1 (en) 1998-04-20 2003-08-05 Glycart Biotechnology Ag Glycosylation engineering of antibodies for improving antibody-dependent cellular cytotoxicity
US7575923B2 (en) 1998-04-21 2009-08-18 Micromet Ag CD19xCD3 specific polypeptides and uses thereof
WO1999054440A1 (fr) 1998-04-21 1999-10-28 Micromet Gesellschaft Für Biomedizinische Forschung Mbh Polypeptides specifiques a cd19 et cd3 et leurs utilisations
US7112324B1 (en) 1998-04-21 2006-09-26 Micromet Ag CD 19×CD3 specific polypeptides and uses thereof
US6723538B2 (en) 1999-03-11 2004-04-20 Micromet Ag Bispecific antibody and chemokine receptor constructs
WO2000061739A1 (fr) 1999-04-09 2000-10-19 Kyowa Hakko Kogyo Co., Ltd. Methode de regulation de l'activite d'une molecule immunologiquement fonctionnelle
WO2001029246A1 (fr) 1999-10-19 2001-04-26 Kyowa Hakko Kogyo Co., Ltd. Procede de production d'un polypeptide
US20070117126A1 (en) 1999-12-15 2007-05-24 Genentech, Inc. Shotgun scanning
US7229760B2 (en) 2000-03-24 2007-06-12 Micromet Ag mRNA amplification
US7332168B2 (en) 2000-08-22 2008-02-19 Micromet Ag Composition for the elimination of autoreactive B-cells
US20030115614A1 (en) 2000-10-06 2003-06-19 Yutaka Kanda Antibody composition-producing cell
US20020164328A1 (en) 2000-10-06 2002-11-07 Toyohide Shinkawa Process for purifying antibody
WO2002031140A1 (fr) 2000-10-06 2002-04-18 Kyowa Hakko Kogyo Co., Ltd. Cellules produisant des compositions d'anticorps
US20070061900A1 (en) 2000-10-31 2007-03-15 Murphy Andrew J Methods of modifying eukaryotic cells
US7041870B2 (en) 2000-11-30 2006-05-09 Medarex, Inc. Transgenic transchromosomal rodents for making human antibodies
WO2003011878A2 (fr) 2001-08-03 2003-02-13 Glycart Biotechnology Ag Variants de glycosylation d'anticorps presentant une cytotoxicite cellulaire accrue dependante des anticorps
US20030157108A1 (en) 2001-10-25 2003-08-21 Genentech, Inc. Glycoprotein compositions
US20040093621A1 (en) 2001-12-25 2004-05-13 Kyowa Hakko Kogyo Co., Ltd Antibody composition which specifically binds to CD20
US7323440B2 (en) 2002-02-13 2008-01-29 Micromet Ag De-immunized MOG (poly)peptide constructs
US20040132140A1 (en) 2002-04-09 2004-07-08 Kyowa Hakko Kogyo Co., Ltd. Production process for antibody composition
US20040110282A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells in which activity of the protein involved in transportation of GDP-fucose is reduced or lost
US20040109865A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Antibody composition-containing medicament
US20040110704A1 (en) 2002-04-09 2004-06-10 Kyowa Hakko Kogyo Co., Ltd. Cells of which genome is modified
WO2003085107A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Cellules à génome modifié
WO2003084570A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition d'anticorps appropriee au patient souffrant de polymorphisme fc$g(g)riiia
WO2003085119A1 (fr) 2002-04-09 2003-10-16 Kyowa Hakko Kogyo Co., Ltd. Procede d'amelioration de l'activite d'une composition d'anticorps de liaison avec le recepteur fc$g(g) iiia
US20050119455A1 (en) 2002-06-03 2005-06-02 Genentech, Inc. Synthetic antibody phage libraries
US7820166B2 (en) 2002-10-11 2010-10-26 Micromet Ag Potent T cell modulating molecules
WO2004056312A2 (fr) 2002-12-16 2004-07-08 Genentech, Inc. Variants d'immunoglobuline et utilisations
US20050079574A1 (en) 2003-01-16 2005-04-14 Genentech, Inc. Synthetic antibody phage libraries
US7635472B2 (en) 2003-05-31 2009-12-22 Micromet Ag Pharmaceutical compositions comprising bispecific anti-cd3, anti-cd19 antibody constructs for the treatment of b-cell related disorders
US7919089B2 (en) 2003-05-31 2011-04-05 Micromet Ag Pharmaceutical composition comprising a bispecific antibody for EpCAM
WO2005035586A1 (fr) 2003-10-08 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Composition proteique hybride
WO2005035778A1 (fr) 2003-10-09 2005-04-21 Kyowa Hakko Kogyo Co., Ltd. Procede permettant de produire une composition d'anticorps par inhibition par l'arn de la fonction de $g(a)1,6-fucosyltransferase
US8076459B2 (en) 2003-10-16 2011-12-13 Micromet Ag Multispecfic deimmunized CD3-binders
US20050123546A1 (en) 2003-11-05 2005-06-09 Glycart Biotechnology Ag Antigen binding molecules with increased Fc receptor binding affinity and effector function
WO2005053742A1 (fr) 2003-12-04 2005-06-16 Kyowa Hakko Kogyo Co., Ltd. Medicament contenant une composition a base d'anticorps
US7235641B2 (en) 2003-12-22 2007-06-26 Micromet Ag Bispecific antibodies
US20050266000A1 (en) 2004-04-09 2005-12-01 Genentech, Inc. Variable domain library and uses
US8017748B2 (en) 2005-04-18 2011-09-13 Micromet Ag Antibody neutralizers of human granulocyte macrophage colony stimulating factor
US20070160598A1 (en) 2005-11-07 2007-07-12 Dennis Mark S Binding polypeptides with diversified and consensus vh/vl hypervariable sequences
US20070237764A1 (en) 2005-12-02 2007-10-11 Genentech, Inc. Binding polypeptides with restricted diversity sequences
US8007796B2 (en) 2005-12-16 2011-08-30 Micromet Ag Means and methods for the treatment of tumorous diseases
US20070292936A1 (en) 2006-05-09 2007-12-20 Genentech, Inc. Binding polypeptides with optimized scaffolds
WO2008077546A1 (fr) 2006-12-22 2008-07-03 F. Hoffmann-La Roche Ag Anticorps contre le récepteur du facteur de croissance i de type insuline et leurs utilisations
US20090002360A1 (en) 2007-05-25 2009-01-01 Innolux Display Corp. Liquid crystal display device and method for driving same
WO2013163427A1 (fr) 2012-04-25 2013-10-31 The United States Of America, As Represented By The Secretary, Department Of Health & Human Services Anticorps pour traiter les infections par le vih-1
US10787501B1 (en) * 2020-04-02 2020-09-29 Regeneron Pharmaceuticals, Inc. Anti-SARS-CoV-2-spike glycoprotein antibodies and antigen-binding fragments

Non-Patent Citations (78)

* Cited by examiner, † Cited by third party
Title
"Antibody Engineering", 2010, SPRINGER-VERLAG
"Antibody Expression and Production", 2011, SPRINGER
"Current Protocols in Molecular Biology", 2017, JOHN WILEY AND SONS
AHMAD ET AL., CLIN. DEV. IMMUNOL., 2012
AL-LAZIKANI ET AL.: "Standard conformations for the canonical structures of immunoglobulins", J. MOL. BIO., vol. 273, no. 4, 1997, pages 927 - 948, XP004461383, DOI: 10.1006/jmbi.1997.1354
ALTSCHUL ET AL., J. MOL. BIOL., vol. 215, no. 3, 1990, pages 403 - 410
ANDREANO ET AL., CELL, vol. 184, 2021, pages 1821 - 1835
BAROUCH ET AL., J. VIROL., vol. 79, no. 14, 2005, pages 8828 - 8834
BIRD ET AL., SCIENCE, vol. 242, no. 4877, 1988, pages 423 - 426
BOERNER ET AL., J. IMMUNOL., vol. 147, 1991, pages 86 - 3242
BRACKEN COLTON J ET AL: "Bi-paratopic and multivalent VH domains block ACE2 binding and neutralize SARS-CoV-2", NATURE CHEMICAL BIOLOGY, vol. 17, no. 1, 20 October 2020 (2020-10-20), pages 113 - 121, XP037317215, ISSN: 1552-4450, DOI: 10.1038/S41589-020-00679-1 *
BRODEUR ET AL.: "Monoclonal Antibody Production Techniques and Applications", 1987, MARCEL DEKKER, INC., pages: 51 - 63
BROUWER ET AL., SCIENCE, vol. 369, no. 6504, 15 June 2020 (2020-06-15), pages 643 - 650
BRUHL ET AL., J. IMMUNOL., vol. 166, no. 4, 2001, pages 2420 - 2426
CHAN ET AL., GIN. INFECT DIS., vol. 71, 2020, pages 2428 - 2446
CHEN ET AL., J. MOL. BIOL., vol. 293, no. 4, 1999, pages 865 - 881
CHO HYESEON ET AL: "Ultrapotent bispecific antibodies neutralize emerging SARS-CoV-2 variants", BIORXIV, 1 April 2021 (2021-04-01), United States, XP055852588, Retrieved from the Internet <URL:https://www.biorxiv.org/content/10.1101/2021.04.01.437942v1.full.pdf> [retrieved on 20211019], DOI: 10.1101/2021.04.01.437942 *
CORPET, NUCLEIC ACIDS RES., vol. 16, no. 22, 1988, pages 10881 - 10890
DALL'ACQUA ET AL., J. BIOL. CHEM., vol. 281, no. 33, 2006, pages 23514 - 23524
DIGIAMMARINO ET AL.: "Therapeutic Proteins, Methods in Molecular Biology (Methods and Protocols", vol. 899, 2012, HUMANA PRESS, article "Design and generation of DVD-Ig molecules for dual-specific targeting", pages: 145 - 156
FELLOUSE, PROC. NATL. ACAD. SCI. USA, vol. 101, no. 34, 2004, pages 12467 - 12472
GARDNER ET AL., NATURE, vol. 519, no. 7541, 2015, pages 87 - 91
GREENFIELD: "Antibodies: A Laboratory Manual", 2014, COLD SPRING HARBOR LABORATORY PRESS
GRIFFITHS ET AL., EMBO J, vol. 12, 1993, pages 725 - 734
HARLOWLANE: "Remington: The Science and Practice of Pharmacy", 2013, COLD SPRING HARBOR PUBLICATIONS
HIGGINSSHARP, BIOINFORMATICS, vol. 5, no. 2, 1989, pages 151 - 3
HIGGINSSHARP, GENE, vol. 73, no. 1, 1988, pages 237 - 244
HINTON ET AL., J IMMUNOL., vol. 176, no. 1, 2006, pages 346 - 356
HOOGENBOOMWINTER, J. MOL. BIOL., vol. 227, 1992, pages 381 - 388
HUANG ET AL., BIOINFORMATICS, vol. 8, no. 2, 1992, pages 155 - 165
HUSTON ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 85, no. 16, 1988, pages 5879 - 5883
IMAI ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 117, 2020, pages 16587 - 16595
JAKOB ET AL., MABS, vol. 5, 2013, pages 358 - 363
JOHNSON ET AL., NAT. MED., vol. 15, no. 8, 2009, pages 901 - 906
KANDA ET AL., BIOTECHNOL. BIOENG., vol. 94, no. 4, 2006, pages 680 - 688
KOZBOR, J. IMMUNOL., vol. 133, 1984, pages 3001
KREYE ET AL., CELL, vol. 183, no. 4, 4 September 2020 (2020-09-04), pages 1058 - 1069
KUFER ET AL., CANCER IMMUNOL. IMMUNOTHER., vol. 45, 1997, pages 193 - 197
LEE ET AL., J. IMMUNOL. METHODS, vol. 284, 2004, pages 119 - 132
LEFRANC ET AL.: "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains", DEV. COMP. IMMUNOL., vol. 27, no. 1, 2003, pages 55 - 77, XP055585227, DOI: 10.1016/S0145-305X(02)00039-3
LI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 103, 2006, pages 3557 - 3562
LIU ET AL., NATURE, vol. 584, no. 7821, 22 July 2020 (2020-07-22), pages 450 - 456
LIU LIHONG ET AL: "Potent neutralizing antibodies against multiple epitopes on SARS-CoV-2 spike", NATURE, NATURE PUBLISHING GROUP UK, LONDON, vol. 584, no. 7821, 22 July 2020 (2020-07-22), pages 450 - 456, XP037223585, ISSN: 0028-0836, [retrieved on 20200722], DOI: 10.1038/S41586-020-2571-7 *
LOFFLER ET AL., BLOOD, vol. 95, no. 6, 2000, pages 2098 - 2103
LONBERG, NAT. BIOTECH., vol. 23, 2005, pages 1117 - 1125
LONENBERG, CURR. OPIN. IMMUNOL., vol. 20, 2008, pages 450 - 459
MACK ET AL., J. IMMUNOL., vol. 158, no. 8, 1997, pages 3965 - 3970
MACK ET AL., PROC. NATL. ACAD. SCI. U.S.A., vol. 92, no. 15, 1995, pages 7021 - 7025
MARBRYSNAVELY, IDRUGS, vol. 13, no. 8, 2010, pages 543 - 549
MCCAFFERTY ET AL., NATURE, vol. 348, 1990, pages 552 - 554
MCCAFFERTY ET AL., NATURE, vol. 352, 1991, pages 624 - 628
NAT BIOTECHNOL., vol. 25, no. 11, 14 October 2007 (2007-10-14), pages 1290 - 7
NEEDLEMANWUNSCH, J. MOL. BIOL., vol. 48, no. 3, 1970, pages 443 - 453
NI, XIANDAI MIANYIXUE, vol. 26, no. 4, 2006, pages 265 - 268
OKAZAKI ET AL., J. MOL. BIOL., vol. 336, no. 5, 2004, pages 1239 - 1249
PEARSON, METHODS MOL. BIOL., vol. 24, 1994, pages 307 - 331
PETKOVA ET AL., INT. IMMUNOL., vol. 18, no. 12, 2006, pages 1759 - 1769
PRESTA ET AL., CANCER RES., vol. 57, no. 20, 1997, pages 4593 - 4599
RIPKA ET AL., ARCH. BIOCHEM. BIOPHYS., vol. 249, no. 2, 1986, pages 533 - 545
ROBBIANI ET AL., NATURE, vol. 584, no. 7821, 18 June 2020 (2020-06-18), pages 437 - 442
ROGERS ET AL., SCIENCE, vol. 369, 2020, pages 1110 - 1115
SCHOONJANS ET AL., J. IMMUNOL., vol. 165, no. 12, 2000, pages 7050 - 7057
SMITHWATERMAN, ADV. APPL. MATH., vol. 2, no. 4, 1981, pages 482 - 489
VOLLMERSBRANDLEIN, HISTOLOGY AND HISTOPATHOLOGY, vol. 20, no. 3, 2005, pages 927 - 937
VOLLMERSBRANDLEIN, METHODS AND FINDINGS IN EXPERIMENTAL AND CLINICAL PHARMACOLOGY, vol. 27, no. 3, 2005, pages 185 - 91
WANG ET AL., NATURE, vol. 593, 2021, pages 130 - 135
WARD ET AL., NATURE, vol. 341, no. 6242, 1989, pages 544 - 546
WIBMER ET AL., NAT. MED., vol. 27, 2021, pages 622 - 625
WILLEMS ET AL., J. CHROMATOGR. B ANALYT. TECHNOL. BIOMED LIFE SCI., vol. 786, 2003, pages 161 - 176
WINTER ET AL., ANN. REV. IMMUNOL., vol. 12, 1994, pages 433 - 455
WRIGHT ET AL., TRENDS BIOTECHNOL., vol. 15, no. 1, 1997, pages 26 - 32
WU ET AL., MABS, vol. 1, 2009, pages 339 - 47
WU YAN ET AL: "Supplementary Material - A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2", SCIENCE, vol. 368, no. 6496, 13 May 2020 (2020-05-13), US, pages 1274 - 1278, XP055799109, ISSN: 0036-8075, Retrieved from the Internet <URL:https://science.sciencemag.org/highwire/filestream/744452/field_highwire_adjunct_files/1/abc2241_Wu_SM.pdf> DOI: 10.1126/science.abc2241 *
XIANG YUFEI ET AL: "Versatile and multivalent nanobodies efficiently neutralize SARS-CoV-2", SCIENCE (AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE), 5 November 2020 (2020-11-05), United States, pages 1479 - 1484, XP055852797, Retrieved from the Internet <URL:https://www.science.org/doi/pdf/10.1126/science.abe4747> [retrieved on 20211019], DOI: 10.1126/science.abe4747 *
YAMANE-OHNUKI ET AL., BIOTECHNOL. BIOENG., vol. 87, no. 5, 2004, pages 614 - 622
YAN WU ET AL: "A noncompeting pair of human neutralizing antibodies block COVID-19 virus binding to its receptor ACE2", SCIENCE (AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE), 13 May 2020 (2020-05-13), United States, pages 1274 - 1278, XP055758869, Retrieved from the Internet <URL:https://science.sciencemag.org/content/sci/368/6496/1274.full.pdf> DOI: 10.1126/science.abc2241 *
YUAN ET AL., SCIENCE, vol. 369, no. 6507, 13 July 2020 (2020-07-13), pages 1119 - 1123
ZALEVSKY ET AL., NATURE BIOTECHNOL., vol. 28, no. 2, 2010, pages 157 - 159

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116218789A (zh) * 2023-01-06 2023-06-06 扬州大学 一株杂交瘤细胞株、抗PDCoV NS6蛋白的单克隆抗体及其应用
CN116218789B (zh) * 2023-01-06 2023-09-22 扬州大学 一株杂交瘤细胞株、抗PDCoV NS6蛋白的单克隆抗体及其应用

Similar Documents

Publication Publication Date Title
CN108137676B (zh) gp120中和抗体及其用途
EP3580235B1 (fr) Anticorps neutralisants dirigés contre la protéine circumsporozoïte de plasmodium falciparum et leur utilisation
US10273288B2 (en) Neutralizing antibodies to Ebola virus glycoprotein and their use
US20220227853A1 (en) Neutralizing antibodies to plasmodium falciparum circumsporozoite protein and their use
US20240117011A1 (en) Antibodies targeting the spike protein of coronaviruses
WO2020236974A1 (fr) Anticorps se liant à la protéine de fusion du métapneumovirus humain et leur utilisation
US20230272048A1 (en) Hiv-1 antibodies
US20130084301A1 (en) Cluster of Neutralizing Antibodies to Hepatitis C Virus
WO2023154824A1 (fr) Anticorps monoclonaux humains ciblant largement les coronavirus
WO2022132904A1 (fr) Anticorps monoclonaux humains ciblant le sars-cov-2
US20210079067A1 (en) Neutralizing antibodies to ebola virus glycoprotein and their use
WO2021257665A1 (fr) Anticorps monoclonal humain ciblant un site conservé sur la protéine circumsporozoïte de plasmodium falciparum
WO2024054822A1 (fr) Anticorps du sars-cov -2 modifiés ayant une largeur de neutralisation accrue
WO2023240246A1 (fr) Anticorps monocolonaux modifiés par calcul informatique et fragments de liaison à l&#39;antigène spécifiques de protéines de spicule du sars-cov-2 et leurs utilisations
US20240043506A1 (en) Sars-cov-2 antibodies
WO2023172881A1 (fr) Anticorps anti-hmpv et leur utilisation
WO2024030829A1 (fr) Anticorps monoclonaux se liant à la face inférieure de la neuraminidase virale de la grippe
WO2024064826A1 (fr) Anticorps neutralisants dirigés contre la protéine plasmodium falciparum circumsporozoïte et leur utilisation
CA3232223A1 (fr) Banque de nanocorps llama humanise synthetique et son utilisation pour identifier des anticorps neutralisant le sars-cov-2
AU2022220611A1 (en) Human monoclonal antibodies against pneumococcal antigens

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21841130

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21841130

Country of ref document: EP

Kind code of ref document: A1