WO2024086566A2 - Pan-sarbecovirus neutralizing antibodies and methods of use thereof - Google Patents

Pan-sarbecovirus neutralizing antibodies and methods of use thereof Download PDF

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Publication number
WO2024086566A2
WO2024086566A2 PCT/US2023/077073 US2023077073W WO2024086566A2 WO 2024086566 A2 WO2024086566 A2 WO 2024086566A2 US 2023077073 W US2023077073 W US 2023077073W WO 2024086566 A2 WO2024086566 A2 WO 2024086566A2
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antibody
seq
chain variable
cov
sars
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PCT/US2023/077073
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French (fr)
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WO2024086566A3 (en
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Bali Pulendran
Yupeng Feng
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The Board Of Trustees Of The Leland Stanford Junior University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—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
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—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

  • Sequence Listing is provided herewith as a Sequence Listing XML, “S22-373_STAN- 2028WO_SEQ_LIST.xml” created on October 6, 2023 and having a size of 202,752 bytes.
  • the contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
  • SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2
  • COVID-19 coronavirus disease 2019
  • SARS-CoV-2 variants of concern VOCs
  • the originally emerged Omicron variant of SARS-CoV-2 harbors 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), raising a great concern about the protective efficacies of current vaccines and therapeutic antibodies.
  • SARS-CoV-2 together with SARS-CoV-1 , which caused a global outbreak in 2002- 2003, are members of the genus Betacoronavirus and subgenus Sarbecoronavirus.
  • bat coronaviruses such as WIV-1 , RaTG13, SCH014, are a probable origin of SARS-CoV and SARS-CoV-2.
  • WIV-1 WIV-1
  • RaTG13 RaTG13
  • SCH014 a probable origin of SARS-CoV and SARS-CoV-2.
  • cross-species transmission of coronaviruses can never be neglected.
  • pan-sarbecovirus neutralizing antibodies targeting cryptic conserved epitopes in the receptor binding domain retained potency against Omicron, but exhibited significant reductions in the neutralizing activity.
  • antibodies and antigen binding fragments that are capable of specifically binding to a betacoronavirus, e.g. a sarbecovirus selected from one or more of SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV-1 , WIV-1 , RaTG13, and SCH014.
  • a sarbecovirus selected from one or more of SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV-1 , WIV-1 , RaTG13, and SCH014.
  • an antibody or antigen binding fragment of the disclosure is capable of specifically binding to a surface glycoprotein of two, three, four, five, or more sarbecoviruses.
  • an antibody or antigen binding fragment of the disclosure is capable of binding to a surface glycoprotein of a sarbecovirus virion and/or a surface glycoprotein expressed on the surface of a cell infected by a sarbecovirus.
  • presently disclosed antibodies and antigen binding fragments can neutralize infection by two, three, four, or five distinct sarbecovirus in an in vitro model of infection and/or in a human subject.
  • the antibodies are humanized or chimeric, e.g. comprising a human Fc region sequence.
  • polynucleotides that encode the antibodies and antigen binding fragments, vectors, host cells, and related compositions, as well as methods of using the antibodies, nucleic acids, vectors, host cells, and related compositions to treat, e.g, reduce, delay, eliminate, or prevent, infection by any two, three, four, or five distinct sarbecoviruses in a subject and/or in the manufacture of a medicament for treating infection in a subject by any two, three, four, or five distinct sarbecoviruses.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3, and is capable of binding to a surface glycoprotein of a sarbecovirus.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the antibody may comprise an Fc region sequence.
  • the antibody or antigen binding fragment thereof is capable of binding to a surface glycoprotein of two or more distinct sarbecoviruses.
  • the antibody or antigen binding fragment thereof is capable of binding to a surface glycoprotein of any two, three, four, or five distinct sarbecoviruses. In some embodiments, the antibody or antigen-binding fragment thereof is capable of binding to a surface glycoprotein of one or more of SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV, WIV-1 , RaTG13, and SCH014.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 20A7, which binds to the spike protein of SARS-CoV-2, at least 5 variants of concern of SARS-CoV-2, and SARS-CoV.
  • 20A7 neutralizes SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV, WIV-1 , RaTG13, and SCH014.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 27A12, which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 27A12 neutralizes all known variants of concern of SARS-CoV-2, WIV-1 , and SCH014.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 27E3, which specifically binds to the spikes of at least 5 variants of concern of SARS-CoV-2.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 27E3 neutralizes at least 5 variants of concern of SARS-CoV-2, and RaTG13.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 21 B6, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 21 B6 neutralizes SARS-CoV-2 for at least 5 variants of concern.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 27E4, which binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2 and SARS-CoV.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 27E4 neutralizes SARS-CoV-2 for at least 5 variants of concern, WIV-1 , RaTG13, and SCH014.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 26C3, which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 26C3 neutralizes SARS- CoV-2 and at least 5 variants of concern, and RaTG13.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 15F1 , which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2, and SARS-CoV.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 15F1 neutralizes SARS-CoV-2 and at least 5 variants of concern, SARS-CoV, WIV-1 , and SCH014.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25C7, which binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2, and SARS-CoV.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 25C7 neutralizes SARS-CoV-2 and all variants of concern.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25F9, which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2, and SARS-CoV.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 25F9 neutralizes SARS-CoV-2 for all variants of concern, SARS-CoV, WIV-1 , RaTG13, and SCH014.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25A10, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 25A10 neutralizes SARS-CoV-2 for all variants of concern.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 20F2, which specifically binds to the spikes of SARS-CoV-2, for at least 5 variants of concern of SARS-CoV-2, and SARS-CoV.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 20F2 neutralizes SARS-CoV-2 and all variants of concern, excepting the beta strain.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 21 F2, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 21 F2 neutralizes SARS- CoV-2 and all variants of concern.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25A1 1 , which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 25A11 neutralizes SARS- CoV-2 and all known variants of concern.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a GDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 26G10, which specifically binds to the spikes of SARS-CoV-2, for at least 5 variants of concern of SARS-CoV-2, and SARS-CoV.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 26G10 neutralizes SARS-CoV-2 and all variants of concern.
  • the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 20C3, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2, and SARS-CoV.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • 20C3 neutralizes SARS-CoV-2 and at least 5 variants of concern, and SARS-CoV.
  • the invention provides a polypeptide comprising or consisting of an epitope that binds to an antibody disclosed herein.
  • epitopes of the invention include, but not limited to, an epitope comprising amino acids from SARS-CoV-2, SARS-CoV-2 beta, SARS-CoV-2 omicron, SARS-CoV, Wl -1 , RaTG13, and SCH014 that are specifically bound by at least one antibody of the disclosure.
  • the invention provides polynucleotides that encode the disclosed antibodies and antigen-binding fragments, vectors, host cells, and related compositions as well as methods of using the antibodies, nucleic acids, vectors, host cells, and related compositions to treat (e.g., reduce, delay, eliminate, or prevent) the infection caused by sarbecoviruses in a subject.
  • FIG. 1 shows schematic representation of the study design.
  • Rhesus macaques received RBD-NP or Hexapro-NP adjuvanted with AS03 on day 0 and day 21 .
  • Blood samples were collected and analysis was performed as illustrated in the diagram.
  • FIG. 2 Gating strategy of sorting and analyzing the spike+ and/or RBD+ B cells in total CD20+ cells. Gating was on singlets that were live CD20+ CD3- CD14- CD16- IgM/D- lgG+ Antigen+.
  • FIG. 3 Diagram depicting the strategy for antigen specific memory B cell sorting, monoclonal antibody isolation and characterization.
  • FIG. 4 The graph shows the binding activity to SARS-CoV-2 spike of all monoclonal antibodies in this study measured by ELISA.
  • 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 Wuhan spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 450 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
  • FIG. 5 The graph shows the half-maximal inhibitory concentrations (IC50s) of all monoclonal antibodies against pseudotyped SARS-CoV-2 Wuhan, BA.1 and BA.4/5 respectively.
  • IC50s half-maximal inhibitory concentrations
  • FIG. 6 shows the neutralizing activities of 15 monoclonal antibodies against pseudotyped SARS-CoV-2 Wuhan strain. IC50s were labeled in parallel with each mAb on the right.
  • FIG. 7 shows the neutralizing activities of 15 monoclonal antibodies against pseudotyped SARS-CoV-2 BA.1 strain. IC50s were labeled in parallel with each mAb on the right.
  • FIG. 8 shows the neutralizing activities of 15 monoclonal antibodies against pseudotyped SARS-CoV-2 BA.4/5 strain. IC50s were labeled in parallel with each mAb on the right.
  • FIG. 9 shows ELISA results of 15 monoclonal antibodies on spike protein of SARS- CoV-2 Wuhan. The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right.
  • 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 Wuhan spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 405 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
  • FIG. 10 shows ELISA results of 15 monoclonal antibodies on spike protein of SARS- CoV-2 BA.1 .
  • concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right.
  • 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 BA.1 spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 405 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
  • FIG. 11 shows ELISA results of 15 monoclonal antibodies on spike protein of SARS- CoV-2 BA.4/5. The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right.
  • 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 BA.4/5 spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 405 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
  • FIG. 12 shows cross-reactive profiles of monoclonal antibodies isolated from RBD-NP vaccinated individuals, as measured by Meso Scale V-PLEX SARS-CoV-2 panel 9.
  • concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. Plates were coated 30 minutes at room temperature with blocking buffer and incubated with serial dilutions of mAbs for 2 hour at room temperature. Subsequently, the plates were washed, and Sulfo-anti-human IgG was added and incubated for 1 hour. After further washing, the read buffer was added and plates were read using MESO QuickPlex SO 120. The data were plotted with Graphpad Prism software.
  • FIG. 13 shows cross- reactive profiles of monoclonal antibodies isolated from Hexapro- NP vaccinated individuals, as measured by Meso Scale V-PLEX SARS-CoV-2 panel 13.
  • concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. Plates were coated 30 minutes at room temperature with blocking buffer and incubated with serial dilutions of mAbs for 2 hour at room temperature. Subsequently, the plates were washed, and Sulfo-anti-human IgG was added and incubated for 1 hour. After further washing, the read buffer was added and plates were read using MESO QuickPlex SQ 120. The data were plotted with Graphpad Prism software.
  • FIG. 14 shows cross-reactivities against SARS-CoV-1 spike protein of exemplary monoclonal antibodies, as measured by Meso Scale V-PLEX coronavirus panel 3.
  • concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. Plates were coated 30 minutes at room temperature with blocking buffer and incubated with serial dilutions of mAbs for 2 hour at room temperature. Subsequently, the plates were washed, and Sulfo-anti-human IgG was added and incubated for 1 hour. After further washing, the read buffer was added and plates were read using MESO QuickPlex SQ 120. The data were plotted with Graphpad Prism software.
  • FIG. 15 shows the binding affinities of exemplary monoclonal antibodies against SARS-CoV RBD, as mearsued by ForteBio’s Octet RH16 system.
  • FIG. 16 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 WA1 strain.
  • the IC50s were labeled in parallel on the right.
  • FIG. 17 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 alpha strain. The IC50s were labeled in parallel on the right.
  • FIG. 18 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 Beta strain. The IC50s were labeled in parallel on the right.
  • FIG. 19 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 Gamma strain.
  • the IC50s were labeled in parallel on the right.
  • FIG. 20 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 Delta strain.
  • the IC50s were labeled in parallel on the right.
  • FIG. 21 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 BA.1 strain.
  • the IC50s were labeled in parallel on the right.
  • FIG. 22 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 BA.2 strain.
  • the IC50s were labeled in parallel on the right.
  • FIG. 23 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 BA.3 strain.
  • the IC50s were labeled in parallel on the right.
  • FIG. 24 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV.
  • the IC50s were labeled in parallel on the right.
  • FIG. 25 shows the neutralizing activities of monoclonal antibodies against pseudotyped WIV-1 .
  • the IC50s were labeled in parallel on the right.
  • FIG. 26 shows the neutralizing activities of monoclonal antibodies against pseudotyped RaTG13.
  • the IC50s were labeled in parallel on the right.
  • FIG. 27 shows the neutralizing activities of monoclonal antibodies against pseudotyped SCH014.
  • the IC50s were labeled in parallel on the right.
  • SARS-CoV-2 is a betacoronavirus that causes a respiratory disease called coronavirus disease 19 (COVID-19).
  • SARS-CoV-2 is in the Sarbecovirus subgenus of Coronaviridae.
  • SARS-CoV-2 also referred to herein as “Wuhan Strain” comprising the amino acid sequence of SARS-CoV-2 isolate Wuhan-Hu-1 (see GenBank QHD43416.1 , Januray 23, 2020), was first known and identified in Wuhan, Hubei province China, in 2019. The virus is thought to spread from person to person through droplets released when an infected person coughs, sneezes, or talks.
  • SARS-CoV-2 infection includes fever, dry cough, dyspnea, fatigue, body aches, headache, new loss of tase or smell, sore throat, congestions or runny nose, nausea or vomiting, diarrhea, persistent pressure or pain n the chest, new confusion, inability to wake or stay awake, and bluish lips or face.
  • variants refers to a viral genome (genetic code) that may contain one or more mutations and is genetically distinct from a main strain.
  • a group of variants with similar genetic changes such as a lineage or group of lineages, may be designated by public health organizations as a Variant Being Monitored (VBM), Variant of Concern (VOC) or a Variant of Interest (VOI) due to shared attributes and characteristics that may require public health action.
  • VBM Variant Being Monitored
  • VOC Variant of Concern
  • VOI Variant of Interest
  • a Variant of Interest or a Variant of Concern may be downgraded to a Variant Being Monitored after a significant and sustained reduction in its national and regional proportions over time, or other evidence indicates that a variant does not pose significant risk to public health in the United States.
  • the Alpha strain also known as the lineage B.1 .1 .7 and Q lineages
  • the Beta strain also know as the lineage B.1.351
  • the Gamma strain also known as the lineage P.1
  • the Delta strain also known as the lineage B.1 .617.2 and AY lineages
  • the Epsilon strain also known as the lineage B.1.426 and the lineage B.1.429.
  • the current SARS-CoV-2 variant of concern contains spike protein substitutions: A67V, del69-70, T95I, dell 42-144, Y145D, del211 , L212I, ins214EPE, G339D, S371 L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501 Y, Y505H, T547K, D614G, H655Y, N679K, P681 H, N764K, D796Y, N856K, Q954H, N969K, L981 F, which has enhanced transmissibility, increased resistentance to the neutralization by some EUA mono
  • SARS-CoV-2 The genomic sequence of SARS-CoV-2 is publicly available, for example at GenBank NC_045512. A database of variants is maintained at NCBI virus, which is publicly available.
  • SARS-CoV-2 comprises a "spike” or surface (“S") type I transmembrane glycoprotein containing a receptor binding domain (RBD).
  • SARS-CoV-2 comprises a "spike” or surface (“S") type I transmembrane glycoprotein containing a receptor binding domain (RBD).
  • SARS-CoV-2 comprises a "spike” or surface (“S") type I transmembrane glycoprotein containing a receptor binding domain (RBD).
  • SARS-CoV-2 comprises a "spike” or surface (“S") type I transmembrane glycoprotein containing a receptor binding domain (RBD).
  • SARS-CoV-2 comprises a "spike” or surface (“S") type I transmembrane glycoprotein containing a receptor binding domain (RBD).
  • sarbecoviruses include, without limitation: SARS-CoV-1 , WIV-1 , RaTG13, and SCH014.
  • SARS-CoV is another betacoronavirus that causes respiratory symptoms in infected individuals.
  • the genomic sequence of SARS-CoV-2 Urbani strain has GenBank accession number AAP13441 .1 .
  • any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms “a” and “an” as used herein refer to “one or more" of the enumerated components.
  • a protein domain, region, or module e.g., a binding domain
  • a protein "consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy - terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phosphoserine.
  • Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • mutation refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively.
  • a mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
  • a “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain.
  • Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3 : Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (lie or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W).
  • Group 1 Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T);
  • Group 2 Aspartic
  • amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing).
  • an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and lie.
  • Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, lie, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
  • protein or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated.
  • variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
  • Nucleic acid molecule or “polynucleotide” or “polynucleic acid” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g ., purine or pyrimidine bases) or non-natural subunits (e.g, morpholine ring).
  • Purine bases include adenine, guanine, hypoxanthine, and xanthine
  • pyrimidine bases include uracil, thymine, and cytosine.
  • Nucleic acid molecules include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense) strand.
  • a nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
  • the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof.
  • the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5-methylcytidine, a 2- thiouridine, or any combination thereof.
  • the pseudouridine comprises N-methylpseudouridine.
  • Variants of nucleic acid molecules of this disclosure are also contemplated.
  • Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein.
  • Nucleic acid molecule variants retain the capacity to encode a binding domain thereof having a functionality described herein, such as binding a target molecule.
  • a “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide.
  • a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant).
  • binding affinity e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant.
  • Percent sequence identity refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g ., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated.
  • nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide.
  • isolated can, in some embodiments, also describe an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition that is outside of a human body.
  • a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function).
  • a biological benefit e.g., effector function
  • a “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity).
  • the term "engineered,” “recombinant,” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous or heterologous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention).
  • Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene, or operon.
  • heterologous or “non-endogenous” or “exogenous” refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered.
  • Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules.
  • heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra- chromosomal genetic material (e.g., as a plasmid or other self-replicating vector).
  • homologous or homolog refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain.
  • a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof.
  • a non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity may be from the same species, a different species, or a combination thereof.
  • endogenous or “native” refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.
  • operably linked refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter).
  • Unlinked means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
  • construct refers to any polynucleotide that contains a recombinant nucleic acid molecule.
  • a (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome.
  • a "vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule.
  • Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules.
  • Vectors of the present disclosure also include transposon systems (e.g ., Sleeping Beauty, see, e.g., Geurts et ah, Mol. Ther. 5:108, 2003: Mates et ah, Nat. Genet. ⁇ 7:753, 2009).
  • Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).
  • expression vector refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host.
  • control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation.
  • the vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert.
  • the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence.
  • plasmid "expression plasmid,” “virus,” and “vector” are often used interchangeably.
  • polynucleotides of the present disclosure may be operatively linked to certain elements of a vector.
  • polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked.
  • Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion.
  • Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
  • the vector comprises a plasmid vector or a viral vector e.g ., a lentiviral vector or a y-retroviral vector.
  • Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g, influenza virus), rhabdovirus (e.g, rabies and vesicular stomatitis virus), paramyxovirus (e.g, measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g, vaccinia, fowlpox, and canarypox).
  • herpesvirus e.g, Herpe
  • viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus.
  • retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus.
  • the term "host” refers to a cell or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure).
  • a host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods.
  • a "host” refers to a cell or a subject infected with a sarbecovirus.
  • Antigen refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells, activation of complement, antibody dependent cytotoxicicity, or any combination thereof.
  • An antigen immunogenic molecule
  • An antigen may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like, for example a sarbecovirus spike protein. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample.
  • Exemplary biological samples that can contain one or more antigens include tissue samples, stool samples, cells, biological fluids, or combinations thereof.
  • Antigens can be produced by cells that have been modified or genetically engineered to express an antigen.
  • Antigens can also be present in a betacoronavirus (e.g, a surface glycoprotein, spike protein, or portion thereof), such as present in a virion, or expressed or presented on the surface of a cell infected by a betacoronavirus.
  • a betacoronavirus e.g, a surface glycoprotein, spike protein, or portion thereof
  • An antigen may be killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites, or polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including bacterial cells), tissues, polysaccharides, or lipids, or fragments thereof, or the like, individually or in any combination thereof.
  • epitope includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein.
  • Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • the epitope can be comprised of consecutive amino acids (e.g, a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g, a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding.
  • antibody refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen.
  • Immunoglobulins are serum proteins composed of "light” and “heavy” polypeptide chains having "constant” and “variable” regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
  • the term “monoclonal antibody” or “mAb” or “monoclonal antibody composition” refers to a population of antibody molecules that contains only one molecular species of antibody molecule containing one unique heavy chain product and one unique light chain product.
  • the antibodies of the invention are “chimeric monoclonal antibodies” or “humanized monoclonal antibodies”, refering to antibodies with reduced immunogenicity in humans built by genetically linking a non-human primate variable region to human constant domains (Fc).
  • neutralizing antibody refers to an antibody, for example, a monoclonal antibody, that can neutralize the ability of a pathogen to initiate and/or perpetuate an infection in a host.
  • neutralizing antibody and “an antibody that neutralizes” or “ neutralization of an antibody” are used interchangeably herein.
  • an antibody or antigen binding fragment of the present disclosure specifically binds to a sarbecovirus surface glycoprotein.
  • “specifically binds” refers to an association or union of an antibody or antigen binding fragment to an antigen with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10' 5 M (which equals the ratio of the on-rate [Kon] to the off rate [Koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample.
  • Ka i.e., an equilibrium association constant of a particular binding interaction with units of 1/M
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 5 M to 10 13 M).
  • Antibodies may be classified as “high- affinity” antibodies or as “low-affinity” antibodies.
  • “High-affinity” antibodies refer to those antibodies having a Ka of less than 10 -7 M, less than 10 -8 M, less than 10 -9 M, less than 10 -10 M, less than 10 -11 M, less than 10' 12 M, or less than 10' 13 M.
  • “Low-affinity” antibodies refer to those antibodies having a Ka of greater than 10‘ 7 M, greater than 10‘ 6 , greater than 10 -5 M.
  • affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10 5 M to 10 13 M).
  • a variety of assays are known for identifying antibodies of the present disclosure that bind a particular target, as well as determining binding domain or binding protein affinities, such as Western blot, ELISA (e.g, direct, indirect, or sandwich), analytical ultracentrifugation, spectroscopy, biolayer interferometry, and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard etal., Ann. N.Y. Acad. Sci. 57:660, 1949; Wilson, Science 295: 2103, 2002).
  • Assays for assessing affinity or apparent affinity or relative affinity are also known.
  • affinity is determined by surface plasmon resonance (SPR), e.g. as used by Biacore systems.
  • the affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g. at 25°C.
  • subject refers to a mammal being assessed for immunization and/or being treated.
  • Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, rhesus macaques etc.
  • isolated antibodies or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a provided CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a GDRL3 sequence, which is capable of binding the surface glycoprotein of SARS-CoV-2.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the invention also provides fragments of these monoclonal antibodies (e.g.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • an antibody or antigen binding fragment of the present disclosure binds to a SARS-CoV-2 (including Wuhan strain and one or more additional variant) surface glycoprotein epitope, while not significantly associating or uniting with any other molecules or components in a sample.
  • an antibody or antigen binding fragment of the present disclosure is reactive and specific for the SARS- CoV-2 and variants.
  • an antibody or antigen binding fragment of the present disclosure binds to a SARS-CoV-2 (including Wuhan strain and one or more additional variant) surface glycoprotein epitope, and can also binds to epitopes from other sarbecovirus present in the sample, but does not significantly bind to or associate with any other molecules or components in the sample.
  • an antibody or antigen binding fragment of the present disclosure is cross-reactive for SARS-CoV-2 and one or more additional sarbecovirus, and is specific for the SARS-CoV-2 and one or more additional coronavirus.
  • an antibody of the present disclosure is capable of preventing and/or neutralizing infection by SARS-CoV-2 (including Wuhan strain and one or more additional variant), but does does not significantly prevent or neutralize infection by other coronavirus.
  • an antibody or antigen binding fragment of the present disclosure is broad SARS-CoV-2 neutralizing antibody.
  • an antibody of the present disclosure is capable of preventing and/or neutralizing infection by SARS-CoV-2 (including Wuhan strain and one or more additional variant), and can also prevent or neutralize infection by one or more additional sarbecovirus.
  • SARS-CoV-2 including Wuhan strain and one or more additional variant
  • an antibody or antigen binding fragment of the present disclosure is pan-sarbecovirus neutralizing antibody.
  • variable binding regions refer to the variable binding region from an antibody heavy chain and an antibody light chain, respectively.
  • a VL is a kappa class (also “VK” herein).
  • a VL is a lambda class (also “V ” herein).
  • the variable binding regions comprise discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs).
  • CDRs complementarity determining regions
  • CDRs complementarity determining regions
  • an antibody VH comprises four FRs and three HCDRs as follows: FR1 -HCDR1 -FR2-HCDR2- FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three LCDRs as follows: FR1 - LCDR1 -FR2-LCDR2-FR3-LCDR3-FR4.
  • the VH and the VL together form the antigen binding site through their respective CDRs.
  • an antibody or antigen binding fragment referred to as 15F1 , 20A7, 20C3, 20F2, 21 B6, 21 F2, 25A10, 25A11 , 25C7, 25F9, 2603, 26G10, 27A12, 27E3, or 27E4 comprising the three CDRs of a VH sequence according to the SEQ ID NO.: 5, 21 , 37, 53, 69, 85, 101 , 117, 133, 149, 165, 181 , 197, 213, 229, respectively and/or the three CDRs of a VL sequence according to the SEQ ID NO.:13, 29, 45, 61 , 77, 93, 109, 125, 141 , 157, 173, 189, 205, 221 , 237, respectively, as determined using IMGT numbering method. Sequences are provided in Table 1.
  • an antibody or fragment thereof of 15F1 comprises the three CDRs of a VH sequence according to the SEQ ID NO:5, i.e. SEQ ID NO:6, 7, 8; and the three CDRs of a VL sequence according to SEQ ID NO:13, i.e. SEQ ID NO:14, 15, 16.
  • an antibody or fragment thereof of 20A7 comprises the three CDRs of a VH sequence according to the SEQ ID NO:21 , i.e. SEQ ID NO:22, 23, 24; and the three CDRs of a VL sequence according to SEQ ID NO:29, i.e. SEQ ID NQ:30, 31 , 32.
  • an antibody or fragment thereof of 20C3 comprises the three CDRs of a VH sequence according to the SEQ ID NO:37, i.e. SEQ ID NO:38, 39, 40; and the three CDRs of a VL sequence according to SEQ ID NO:45, i.e. SEQ ID NO:46, 47, 48.
  • an antibody or fragment thereof of 20F2 comprises the three CDRs of a VH sequence according to the SEQ ID NO:53, i.e. SEQ ID NO:54, 55, 56; and the three CDRs of a VL sequence according to SEQ ID NO:61 , i.e. SEQ ID NO:62, 63, 64.
  • an antibody or fragment thereof of 21 B6 comprises the three CDRs of a VH sequence according to the SEQ ID NO:69, i.e. SEQ ID NOTO, 71 , 72; and the three CDRs of a VL sequence according to SEQ ID NO:77, i.e. SEQ ID NO:78, 79, 80.
  • an antibody or fragment thereof of 21 F2 comprises the three CDRs of a VH sequence according to the SEQ ID NO:85, i.e. SEQ ID NO:86, 87, 88; and the three CDRs of a VL sequence according to SEQ ID NO:93, i.e. SEQ ID NO:94, 95, 96.
  • an antibody or fragment thereof of 25A10 comprises the three CDRs of a VH sequence according to the SEQ ID NQ:101 , i.e. SEQ ID NQ:102, 103, 104; and the three CDRs of a VL sequence according to SEQ ID NQ:109, i.e. SEQ ID NQ:1 10, 11 1 , 1 12.
  • an antibody or fragment thereof of 25A1 1 comprises the three CDRs of a VH sequence according to the SEQ ID NO:1 17, i.e. SEQ ID NO:1 18, 119, 120; and the three CDRs of a VL sequence according to SEQ ID NO:125, i.e. SEQ ID NO:126, 127, 128.
  • an antibody or fragment thereof of 25C7 comprises the three CDRs of a VH sequence according to the SEQ ID NO:133, i.e. SEQ ID NO:134, 135, 136; and the three CDRs of a VL sequence according to SEQ ID NO:141 , i.e. SEQ ID NO:142, 143, 144.
  • an antibody or fragment thereof of 25F9 comprises the three CDRs of a VH sequence according to the SEQ ID NO:149, i.e. SEQ ID NQ:150, 151 , 152; and the three CDRs of a VL sequence according to SEQ ID NO:157, i.e. SEQ ID NO:158, 159, 160.
  • an antibody or fragment thereof of 26C3 comprises the three CDRs of a VH sequence according to the SEQ ID NO:165, i.e. SEQ ID NO:166, 167, 168; and the three CDRs of a VL sequence according to SEQ ID NO:173, i.e. SEQ ID NO:174, 175, 176.
  • an antibody or fragment thereof of 26G10 comprises the three CDRs of a VH sequence according to the SEQ ID NO:181 , i.e. SEQ ID NO:182, 183, 184; and the three CDRs of a VL sequence according to SEQ ID NO:189, i.e. SEQ ID NQ:190, 191 , 192.
  • an antibody or fragment thereof of 27A12 comprises the three CDRs of a VH sequence according to the SEQ ID NO:197, i.e. SEQ ID NO:198, 199, 200; and the three CDRs of a VL sequence according to SEQ ID NQ:205, i.e. SEQ ID NQ:206, 207, 208.
  • an antibody or fragment thereof of 27E3 comprises the three CDRs of a VH sequence according to the SEQ ID NO:213, i.e. SEQ ID NO:214, 215, 216; and the three CDRs of a VL sequence according to SEQ ID NO:221 , i.e. SEQ ID NO:222, 223, 224.
  • an antibody or fragment thereof of 27E4 comprises the three CDRs of a VH sequence according to the SEQ ID NO:229, i.e. SEQ ID NQ:230, 232, 232; and the three CDRs of a VL sequence according to SEQ ID NO:237, i.e. SEQ ID NO:238, 239, 240.
  • Antibodies of the invention may comprise an Fc sequence, e.g. from any of the human antibody isotypes, including the gamma isotypes: lgG1 , lgG2a, lgG2b, lgG3, lgG4; or may comprise an Fc of IgM, IgD, IgA, IgE, etc.
  • the Fc sequence may be other than a naturally occurring Fc sequence of the specific antibody.
  • An antibody comprising a variable region and an Fc region may be referred to as an “entire” antibody.
  • An Fc sequence may provide for enhanced effector functions, e.g. by increasing their binding capacities to FcyRI 11 A and increasing ADCC activity.
  • fucose attached to the AMinked glycan at Asn-297 of Fc sterically hinders the interaction of Fc with FcyRI 11 A, and removal of fucose by glyco-engineering can increase the binding to FcyRI 11 A, which translates into >50-fold higher ADCC activity compared with wild type IgG 1 controls.
  • Protein engineering, through amino acid mutations in the Fc portion of lgG1 has generated multiple variants that increase the affinity of Fc binding to FcyRI I IA.
  • the triple alanine mutant S298A/E333A/K334A displays 2-fold increase binding to FcyRI HA and ADCC function.
  • S239D/I332E (2X) and S239D/I332E/A330L (3X) variants have a significant increase in binding affinity to FcyRI I IA and augmentation of ADCC capacity in vitro and in vivo.
  • Other Fc variants identified by yeast display also showed the improved binding to FcyRIIIA and enhanced tumor cell killing in mouse xenograft models. See, for example Liu et al. (2014) JBC 289(6):3571 -90, herein specifically incorporated by reference.
  • the heavy chain constant region is selected to provide for high ADCC activity, including without limitation human lgG1 .
  • a low ADCC activity may be selected, e.g. with human lgG4, or with a “dead” Fc in which effector functions are absent or reduced.
  • Fc variants are possible, including without limitation one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto.
  • one or more Fc portions of the scFc molecule can comprise one or more mutations in the hinge region to eliminate disulfide bonding.
  • the hinge region of an Fc can be removed entirely.
  • the scFc molecule can comprise an Fc variant.
  • an Fc variant can be constructed to remove or substantially reduce effector functions by substituting, deleting or adding amino acid residues to effect complement binding or Fc receptor binding.
  • a deletion may occur in a complementbinding site, such as a C1 q-binding site.
  • Techniques of preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478.
  • the Fc domain may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
  • Antibodies include antibody fragments.
  • a "Fab" fragment antigen binding
  • Fab fragment antigen binding
  • Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site.
  • Pepsin treatment of an antibody yields a single large F(ab') 2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen binding activity and is still capable of cross-linking antigen.
  • Both the Fab and F(ab’)2 are examples of "antigen binding fragments.”
  • Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region.
  • Fab fragments may be joined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as "scFab.”
  • a single chain Fab also referred to herein as "scFab.”
  • an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain.
  • a heavy chain-derived Fab fragment e.g., comprising, consisting of, or consisting essentially of VH + CHI, or "Fd”
  • a light chain-derived Fab fragment e.g., comprising, consisting of, or consisting essentially of VL + CL
  • a scFab may be arranged, in N-terminal to C-terminal direction, according to (heavy chain Fab fragment - linker - light chain Fab fragment) or (light chain Fab fragment - linker -heavy chain Fab fragment).
  • Fv is a small antibody fragment that contains a complete antigen-recognition and antigen binding site. This fragment generally consists of a dimer of one heavy- and one lightchain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv”
  • sFv single-chain Fv
  • the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for antigen binding.
  • a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art.
  • the antibody or antigen binding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain.
  • a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH- linker-VL orientation or in a VL-linker-VH orientation.
  • Any scFv of the present disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e. , (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C).
  • a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both.
  • the antibody, or the antigen binding fragment comprises a human antibody, a humanized antibody, a human antibody comprising a variant Fc sequence, a monoclonal antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, or a scFab.
  • an antibody or antigen binding fragment of the present disclosure is monospecific (e.g., binds to a single epitope) or is multispecific (e.g., binds to multiple epitopes and/or target molecules).
  • Antibodies and antigen binding fragments may be constructed in various formats. Exemplary antibody formats disclosed in Spiess et al., Mol. Immunol.
  • Bispecific T cell Engagers BiTEs
  • DARTs Knobs-lnto-Holes
  • scFv-CH3-KIH assemblies KIH Common Light-Chain antibodies
  • TandAbs Triple Bodies
  • TriBi Minibodies Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y assemblies, Fcabs, kl-bodies, orthogonal Fabs
  • WuxiBody formats e.g, PCT Publication No. WO 2019/057122
  • In-Elbow-lnsert Ig formats lEI-lg; e.g, PCT Publication Nos. WO 2019/024979 and WO 2019/025391 ).
  • the antibody or antigen binding fragment comprises two or more of VH domains, two or more VL domains, or both (i.e. , two or more VH domains and two or more VL domains).
  • an antigen binding fragment comprises the format (N-terminal to C-terminal direction) VH-linker-VL-linker-VH-linker-VL, wherein the two VH sequences can be the same or different and the two VL sequences can be the same or different.
  • Such linked scFvs can include any combination of VH and VL domains arranged to bind to a given target, and in formats comprising two or more VH and/or two or more VL, one, two, or more different eptiopes or antigens may be bound. It will be appreciated that formats incorporating multiple antigen binding domains may include VH and/or VL sequences in any combination or orientation.
  • the antigen binding fragment can comprise the format VL4 linker-VH4 linker-VL-linker-VH, VH-linker-VL-linker-VL4 linker-VH, or VL4 linker-VH4 linker-VH4 linker-VL.
  • Monospecific or multispecific antibodies or antigen binding fragments of the present disclosure constructed comprise any combination of the VH and VL sequences and/or any combination of the CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 sequences disclosed herein.
  • a bispecific or multispecific antibody or antigen binding fragment may, in some embodiments, comprise one, two, or more antigen binding domains (e.g ., a VH and a VL) of the instant disclosure.
  • Two or more binding domains may be present that bind to the same or a different betacoronavirus epitope, and a bispecific or multispecific antibody or antigen binding fragment as provided herein can, in some embodiments, comprise a further SARS-CoV-2 binding domain, and/or can comprise a binding domain that binds to a different antigen or pathogen altogether.
  • the antibody or antigen binding fragment can be multispecific; e.g., bispecific, trispecific, or the like.
  • the antibody or antigen binding fragment can be monoclonal.
  • the term "monoclonal antibody” (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present, in some cases in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The term "monoclonal” is not to be construed as requiring production of the antibody by any particular method.
  • Antibodies and antigen binding fragments of the present disclosure include "chimeric antibodies" in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.
  • chimeric antibodies may comprise human and non human residues.
  • chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
  • a "humanized antibody” is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These nonhuman amino acid residues are typically taken from a variable domain. Humanization may be performed following the method of Winter and co-workers (Jones et al, Nature, 321 :522-525 (1986); Reichmann et al., Nature , 332:323-327 (1988); Verhoeyen et al., Science , 239: 1534- 1536 (1988)), by substituting non-human variable sequences for the corresponding sequences of a human antibody.
  • polynucleotides that encode any of the presently disclosed antibodies or an antigen binding fragment thereof, or a portion thereof (e.g., a CDR, a VH, a VL, a heavy chain, or a light chain).
  • the polynucleotide is codon-optimized for expression in a host cell. Once a coding sequence is known or identified, codon optimization can be performed usng known techniques and tools.
  • Codon-optimized sequences include sequences that are partially codon-optimized (i.e., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.
  • Polynucleotides may be provided in a vector, e.g. an expression vector as disclosed herein. Such vectors may comprise or contain a polynucleotide as disclosed herein.
  • a vector can comprise any one or more of the vectors disclosed herein.
  • a vector is provided that comprises a DNA plasmid construct encoding the antibody or antigen binding fragment, or a portion thereof.
  • a DNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigen binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide.
  • the substituent components of the antibody or antigen binding fragment are encoded by a polynucleotide comprised in a single plasmid.
  • the substituent components of the antibody or antigen binding fragment are encoded by a polynucleotide comprised in two or more plasmids (e.g, a first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL).
  • a single plasmid comprises a polynucleotide encoding a heavy chain and/or a light chain from two or more antibodies or antigen binding fragments of the present disclosure.
  • An exemplary expression vector is pVaxI, available from Invitrogen®.
  • a DNA plasmid of the present disclosure can be delivered to a subject by, for example, electroporation (e.g, intramuscular electroporation), or with an appropriate formulation (e.g, hyaluronidase).
  • polynucleotides encoding antibodies and antigen binding fragments of the present disclosure may possess different nucleotide sequences while still encoding a same antibody or antigen binding fragment due to, for example, the degeneracy of the genetic code, splicing, and the like.
  • the polynucleotide can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA).
  • the RNA comprises messenger RNA (mRNA). Table 1 . Sequences
  • the present disclosure also provides a host cell expressing an antibody or antigen binding fragment according to the present disclosure; or comprising or containing a vector or polynucleotide according the present disclosure.
  • a host cell expressing an antibody or antigen binding fragment according to the present disclosure; or comprising or containing a vector or polynucleotide according the present disclosure.
  • examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli.
  • the cells are mammalian cells.
  • the cells are a mammalian cell line such as CHO cells (e.g, DHFR- CHO cells, human embryonic kidney cells (e.g, HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells.
  • human liver cells e.g. Hepa RG cells, myeloma cells or hybridoma cells.
  • mammalian host cell lines include mouse sertoli cells (e.g, TM4 cells); monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1 ); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells.
  • mouse sertoli cells e.g, TM4 cells
  • COS-7 monkey kidney CV1 line transformed by SV40
  • BHK baby hamster kidney cells
  • African green monkey kidney cells VEO-76
  • monkey kidney cells CV1
  • human cervical carcinoma cells HELA
  • human lung cells W138
  • human liver cells Hep G2
  • a host cell is a prokaryotic cell, such as an E. coli.
  • antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed.
  • the cell may be transfected with a vector according to the present description with an expression vector.
  • Host cells of the present disclosure may be transfected stably or transiently with a vector according to the present disclosure, e.g. for expressing an antibody, or an antigen binding fragment thereof, according to the present disclosure.
  • the cell may be of a species that is different to the species from which the antibody was fully or partially obtained (e.g ., CHO cells expressing a human antibody or an engineered human antibody).
  • the cell type of the host cell does not express the antibody or antigen binding fragment in nature.
  • the host cell may impart a post-translational modification (PTM; e.g., glysocylation or fucosylation) on the antibody or antigen binding fragment that is not present in a native state of the antibody or antigen binding fragment (or in a native state of a parent antibody from which the antibody or antigen binding fragment was engineered or derived).
  • PTM post-translational modification
  • Such a PTM may result in a functional difference (e.g, reduced immunogenicity).
  • an antibody or antigen binding fragment of the present disclosure that is produced by a host cell as disclosed herein may include one or more post-translational modification that is distinct from the antibody (or parent antibody) in its native state (e.g, a human antibody produced by a CHO cell can comprise a more post-translational modification that is distinct from the antibody when isolated from the human and/or produced by the native human B cell or plasma cell).
  • the present disclosure provides methods for producing an antibody, or antigen binding fragment, wherein the methods comprise culturing a host cell of the present disclosure under conditions and for a time sufficient to produce the antibody, or the antigen binding fragment.
  • Methods useful for isolating and purifying recombinantly produced antibodies may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant antibody into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin.
  • One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant antibody described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of soluble antibodies may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.
  • compositions that comprise any one or more of the presently disclosed antibodies, antigen binding fragments, polynucleotides, vectors, or host cells, singly or in any combination, and can further comprise a pharmaceutically acceptable carrier, excipient, or diluent. Carriers, excipients, and diluents are discussed in further detail herein.
  • a composition comprises two or more different antibodies or antigen binding fragments according to the present disclosure.
  • antibodies or antigen binding fragments to be used in a combination each independently have one or more of the following characteristics: neutralize one, two, three, four, five, or more naturally occurring sarbecovirus variants; do not compete with one another for Spike protein binding; bind distinct sarbecovirus Spike protein epitopes; have a reduced formation of resistance to sarbecovirus; when in a combination, have a reduced formation of resistance to sarbecovirus; potently neutralize one, two, three, four, five or more live betacoronaviruses; exhibit additive or synergistic effects on neutralization of one, two, three, four, five or more or more live sarbecoviruses when used in combination; exhibit effector functions; are protective in relevant animal model(s) of An active agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and in
  • Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration.
  • An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.
  • an antibody can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application).
  • a suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art.
  • An "effective amount” refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
  • An antibody can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient.
  • the preferred form depends on the intended mode of administration and therapeutic application.
  • the compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration.
  • the diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution.
  • the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
  • compounds which are "commercially available” may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc.
  • the antibodies are incorporated into a variety of formulations for therapeutic administration.
  • the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols.
  • administration of the active agents and/or other compounds can be achieved in various ways.
  • the active agents and/or other compounds may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.
  • the antibodies may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds.
  • the agents may be combined, as previously described, to provide a cocktail of activities.
  • the following methods and excipients are exemplary and are not to be construed as limiting the invention.
  • Formulations may be typically provided in a unit dosage form, where the term "unit dosage form,” refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle.
  • unit dosage forms of the present invention depend on the particular antibodies employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.
  • the pharmaceutically acceptable excipients such as vehicles, adjuvants, carriers or diluents, are commercially available.
  • pharmaceutically acceptable auxiliary substances such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available.
  • Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt.
  • “Pharmaceutically acceptable base addition salt” refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid.
  • Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like.
  • Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts.
  • Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like.
  • Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
  • the antibodies may be administered in dosages of 0.01 mg to 500 mg /kg body weight per day, e.g. from 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg; or more. Dosages will be appropriately adjusted for pediatric formulation.
  • a therapeutically effective daily dose of an antibody or antigen binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1 .75 g).
  • a therapeutically effective dose may be different than for an antibody or antigen binding fragment.
  • compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized SepharoseTM, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
  • a carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations.
  • Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties.
  • the nature of the carrier can be either soluble or insoluble for purposes of the invention.
  • Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, his
  • compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • the pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index.
  • the data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans).
  • the exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
  • Methods for use of an antibody or antigen binding fragment, nucleic acid, vector, cell, or composition of the present disclosure include the diagnosis and treatment of a sarbecovirus infection (e.g, in a human subject, or in a sample obtained from a human subject).
  • Methods of diagnosis may include contacting an antibody, antibody fragment (e.g., antigen binding fragment) with a sample.
  • samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood.
  • the methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody or antibody fragment with a sample.
  • a detection step can be performed at the bench, i.e. without any contact to the human or animal body. Examples of detection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay), including direct, indirect, and sandwich ELISA.
  • Treatment refers to medical management of a disease, disorder, or condition of a subject (e.g, a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat).
  • a subject e.g, a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat.
  • an appropriate dose or treatment regimen comprising an antibody or composition of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit.
  • Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease- free status; diminishment of extent of disease, stabilization of disease state; delay or prevention of disease progression; remission; survival; prolonged survival; or any combination thereof.
  • therapeutic or prophylactic/preventive benefit includes reduction or prevention of hospitalization for treatment of a sarbecovirus infection (i.e., in a statistically significant manner). In certain embodiments, therapeutic or prophylactic/preventive benefit includes a reduced duration of hospitalization for treatment of a sarbecovirus infection (i.e., in a statistically significant manner). In certain embodiments, therapeutic or prophylactic/preventive benefit includes a reduced or abrogated need for respiratory intervention, such as intubation and/or the use of a respirator device. In certain embodiments, therapeutic or prophylactic/preventive benefit includes reversing a late-stage disease pathology and/or reducing mortality.
  • a "therapeutically effective amount” or “effective amount” of an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition of this disclosure refers to an amount of the composition or molecule sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner.
  • a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone.
  • a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially, sequentially, or simultaneously.
  • a combination may comprise, for example, two different antibodies that specifically bind sarbecovirus antigens, which in certain embodiments, may be the same or different sarbecovirus antigens, and/or can comprise the same or different epitopes.
  • methods for treating a sarbecovirus infection in a subject, wherein the methods comprise administering to the subject an effective amount of an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition as disclosed herein.
  • Subjects that can be treated by the present disclosure are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes. Other model organisms, such as mice and rats, may also be treated according to the present disclosure.
  • the subject may be a human subject.
  • the subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects.
  • a number of criteria are believed to contribute to high risk for severe symptoms or death associated with a sarbecovirus infection. These include, but are not limited to, age, occupation, general health, pre-existing health conditions, obesity, and lifestyle habits.
  • a subject treated according to the present disclosure comprises one or more risk factors.
  • a human subject treated according to the present disclosure is an infant, a child, a young adult, an adult of middle age, or an elderly person. In certain embodiments, a human subject treated according to the present disclosure is less than 1 year old, or is 1 to 5 years old, or is between 5 and 125 years old ( e.g ., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 1 15, or 125 years old, including any and all ages therein or therebetween).
  • a human subject treated according to the present disclosure is 0-19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older. Persons of middle, and especially of elderly age are believed to be at particular risk.
  • the human subject is 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older.
  • the human subject is male.
  • the human subject is female.
  • a human subject treated according to the present disclosure is a resident of a nursing home or a long-term care facility, is a hospice care worker, is a healthcare provider or healthcare worker, is a first responder, is a family member or other close contact of a subject diagnosed with or suspected of having a sarbecovirus infection, is overweight or clinically obese, is or has been a smoker, has or had chronic obstructive pulmonary disease (COPD), is asthmatic (e.g., having moderate to severe asthma), has an autoimmune disease or condition (e.g., diabetes), and/or has a compromised or depleted immune system (e.g, due to AIDS/HIV infection, a cancer such as a blood cancer, a lymphodepleting therapy such as a chemotherapy, a bone marrow or organ transplantation, or a genetic immune condition), has chronic liver disease, has cardiovascular disease, has a pulmonary or heart defect, works or otherwise spends time in close proximity with others, such as in a factory
  • a subject treated according to the present disclosure has received a vaccine for a sarbecovirus and the vaccine is determined to be ineffective, e.g, by post-vaccine infection or symptoms in the subject, by clinical diagnosis or scientific or regulatory criteria.
  • treatment is administered as peri-exposure prophylaxis.
  • treatment is administered to a subject with mild-to-moderate disease, which may be in an outpatient setting.
  • treatment is administered to a subject with moderate-to-severe disease, such as requiring hospitalization.
  • an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome (e.g ., a decrease in frequency, duration, or severity of diarrhea or associated dehydration, or inflammation, or longer disease-free and/or overall survival, or a lessening of symptom severity).
  • a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.
  • Prophylactic benefit of the compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
  • compositions are administered in an effective amount (e.g., to treat a sarbecovirus infection), which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy.
  • an effective amount e.g., to treat a sarbecovirus infection
  • test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated as compared to placebo-treated or other suitable control subjects.
  • a method comprises administering the antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition to the subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more.
  • a method comprises administering the antibody, antigen binding fragment, or composition to the subject a plurality of times, wherein a second or successive administration is performed at about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 24, about 48, about 74, about 96 hours, or more, following a first or prior administration, respectively.
  • a method comprises administering the antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition at least one time prior to the subject being infected by a sarbecovirus.
  • compositions comprising an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents.
  • combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising an antibody or antigen binding fragment of the disclosure and each active agent in its own separate dosage formulation.
  • an antibody or antigen binding fragment thereof as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations.
  • an antibody or antigen binding fragment as described herein and the other active agent can be administered to the subject together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations.
  • a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations.
  • the compositions comprising an antibody or antigen binding fragment and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
  • a combination therapy comprises one or more anti-sarbecovirus antibody (or one or more nucleic acid, host cell, vector, or composition) of the present disclosure and one or more anti-inflammatory agent and/or one or more anti-viral agent.
  • the one or more anti-inflammatory agent comprises a corticosteroid such as, for example, dexamethasone, prednisone, or the like.
  • the one or more anti-inflammatory agents comprise a cytokine antagonist such as, for example, an antibody that binds to IL6 (such as siltuximab), or to IL-6R (such as tocilizumab), or to IL-lp, IL-7, IL-8, IL-9, IL-10, FGF, G-CSF, GM-CSF, IFN-y, IP-10, MCP-1 , MIP-1 A, MIP1 -B, PDGR, TNF-a, or VEGF.
  • a cytokine antagonist such as, for example, an antibody that binds to IL6 (such as siltuximab), or to IL-6R (such as tocilizumab), or to IL-lp, IL-7, IL-8, IL-9, IL-10, FGF, G-CSF, GM-CSF, IFN-y, IP-10, MCP-1 , MIP-1 A, MIP1 -B, PDGR, T
  • the one or more anti-viral agents comprise nucleotide analogs or nucelotide analog prodrugs such as, for example, remdesivir, sofosbuvir, acyclovir, and zidovudine.
  • an anti-viral agent comprises lopinavir, ritonavir, favipiravir, or any combination thereof.
  • Other anti-inflammatory agents for use in a combination therapy of the present disclosure include non-steroidal anti-inflammatory drugs (NSAIDS).
  • NSAIDS non-steroidal anti-inflammatory drugs
  • the one or more antibody (or one or more nucleic acid, host cell, vector, or composition) and the one or more anti-inflammatory agent and/or one or the more antiviral agent can be administered in any order and any sequence, or together.
  • an antibody (or one or more nucleic acid, host cell, vector, or composition) is administered to a subject who has previously received one or more anti inflammatory agent and/or one or more antiviral agent.
  • one or more anti-inflammatory agent and/or one or more antiviral agent is administered to a subject who has previously received an antibody (or one or more nucleic acid, host cell, vector, or composition).
  • a combination therapy is provided that comprises two or more anti-sarbecovirus antibodies of the present disclosure.
  • a method can comprise administering a first antibody to a subject who has received a second antibody, or can comprise administering two or more antibodies together.
  • a method comprises administering to the subject (a) a first antibody or antigen binding fragment, when the subject has received a second antibody or antigen binding fragment; (b) the second antibody or antigen binding fragment, when the subject has received the first antibody or antigen binding fragment; or (c) the first antibody or antigen binding fragment, and the second antibody or antigen binding fragment.
  • an antibody, antigen binding fragment, or composition is provided for use in a method of manufacturing or preparing a medicament for treating a sarbecovirus infection in a subject.
  • 25F9 and 20A7 neutralize authentic clade I sarbecoviruses beyond SARS-CoV-2 with ultrapotentcy (IC50s against SARS-CoV: 0.00085 ug/ml, 0.013 ug/ml; WIV-1 : 0.003 ug/ml, 0.002 ug/ml; SHC014: 0.006 ug/ml, 0.018 ug/ml, Pangolin: 0.006 ug/ml, 0.345 ug/ml, respectively).
  • IC50s against SARS-CoV 0.00085 ug/ml, 0.013 ug/ml
  • WIV-1 0.003 ug/ml, 0.002 ug/ml
  • SHC014 0.006 ug/ml, 0.018 ug/ml
  • Pangolin 0.006 ug/ml, 0.345 ug/ml, respectively).
  • IgD PE Pacificn Biotech, 2030-09
  • IgM PerCP-Cy5.5 BioLegend, 314512
  • CD20 APC-H7 BD Biosciences, 560734
  • CD27 PE-Cy7 BioLegend, 302838
  • CD14 BV650 BioLegend, 301836)
  • CD16 BV650 BioLegend, 302042
  • IgG BUV496 BD Biosciences, 741 172
  • CD3 BV650 BD Biosciences, 563916
  • CD21 PE- CF594 BD Biosciences, 563474
  • Alexa Fluor 488-labeled Beta spike antibodies-online, ABIN6963740
  • Alexa Fluor 647 labeled Omicron spike SinoBiological, 40589-V08H26
  • BV421 labeled Wuhan Spike TinoBiological, 40589-V27B-B
  • the 96- well plates will go through a quick freeze-thaw cycle, and 0.5 ug Oligo(dT)18 (Thermo Scientific), 0.5 mM dNTP mix (Invitrogen), and 200 U Superscript IV (Invitrogen) will be added in a total volume of 4 ul followed by thorough mixing and spinning.
  • the reverse transcription will be performed as follows: 10 min at 42 e C, 10 min at 23 e C, 20 min at 50 S C, 5 min at 55 S C, 10 min at 80 a C and finally cooling to 4 a C.
  • Ig heavy chain and light chain (kappa/lambda) rearrangements will be amplified by nested PCR (HotStarTaq DNA Polymerase, QIAGEN) using primer cocktails specific for all V gene families and constant domains at a concentration of 250 nM per primer.
  • the PCR mix will consist of 2.5 ul 10x PCR buffer, 0.5 ul 10 mM dNTP mix (Invitrogen), 0.5 ul 25 mM MgCI2 (only added in the first round PCR), 5 ul Q-solution, 1 U HotStarTaq, 0.5 ul 5’ and 3’ primers. Water will be added up to a total volume of 25 ul.
  • the primer list and PCR program were shown in Extended Fig 8.
  • the 2nd round PCR products will be evaluated on 2% agarose gels, purified using QIAquick spin columns (Qiagen) and sequenced using 2nd round PCR reverse primers. The sequences were analyzed using the online IMGT/HighV-QUEST tool. The productive heavy-light paired Ig genes will be used for antibody production (Sino Biological).
  • Monoclonal antibodies were assayed at 10 ug/ml starting concentration and 1 1 additional 4-fold serial dilutions. Plates were washed and 50 ul of Sulfo-tag conjugated anti-IgG was added, and the plates were incubated at room temperature for 1 h. After incubation, the plates were washed and 0.15 ml of MSD-Gold read buffer was added. The plates were immediately read using the MSD instrument. The binding capacities of plasma or monoclonal antibodies were presented as MSD area under curve (AUC), calculated using Prism v9.3.1 (GraphPad).
  • AUC MSD area under curve
  • ELISA All monoclonal antibodies generated in this study were screened for their binding to the spikes of SARS-CoV-2 Wuhan strain, BA.1 strain and BA.4/5 by ELISA. Briefly, 96-well plates were coated with 50 pl per well of a 1 pg ml-1 protein solution in PBS overnight at 4 °C. Plates were washed 3 times with washing buffer (1 x PBS with 0.05% Tween-20 (MP Biomedicals)) and incubated with 200 pl per well blocking buffer (1 x PBS with 5% skim milk powder (Bio-Rad) and 0.05% Tween-20 (MP Biomedicals)) for 1 h at room temperature.
  • HRP horseradish peroxidase
  • the reaction was developed for 4 min and stopped by adding 100 ul per well of 450 nm stop solution (Abeam).
  • the plates were immediately read using an ELISA microplate reader (Bio-Rad).
  • the binding capacities of monoclonal antibodies were presented as ELISA area under curve (AUG), calculated using Prism v9.3.1 (GraphPad).
  • Pseudovirion neutralization assay For initial screening, neutralization assay using pseudotyped viruses (using HIV backbone) carrying spikes of SARS-CoV-2 wild type strain, BA.1 strain and BA.4/5 strain respectively was performed by Sino Biological Inc. Briefly, 293T cells overexpressing ACE2 were seeded in 96-well plates (Costar, 3955) at 3 x 10 4 cells/well. Fifty microlitre serially diluted mAbs or a control monoclonal antibody (S2H97) were mixed with 50 pL pseudovirus and then they were added into plates seeded with 293T-ACE2 cells (1 x 10 4 TCID50/mL).
  • SARS-CoV- 2 pseudovirion neutralization assay PsVNA
  • SARS-CoV-2 neutralizing activity measured by PsVNA correlates with PRNT (plaque reduction neutralization test with authentic SARS-CoV-2 virus) in previous studies (Neerukonda et al., 2021 ; Ravichandran et al., 2020; Tang et al., 2021 ).
  • Pseudovirions were produced as previously described (Neerukonda et al., 2021 ). Briefly, human codon-optimized cDNA encoding SARS-CoV-2 spike glycoprotein of the WA- 1 /2020 and variants were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and Xhol sites. Pseudovirions were produced by cotransfection Lenti - X 293T cells with psPAX2(gag/pol), pTrip-luc lentiviral vector and pcDNA 3.1 SARS-CoV-2-spike-deltaC19, using Lipofectamine 3000. The supernatants were harvested at 48h post transfection and filtered through 0.45pm membranes and titrated using 293T-ACE2-TMPRSS2 cells (HEK 293T cells that express ACE2 and TMPRSS2 proteins).
  • Neutralization assays were performed as previously described (Ravichandran et al., 2020; Tang et al., 2021 ).
  • 50 pL of SARS-CoV-2 S pseudovirions (counting -200,000 relative light units) were pre-incubated with an equal volume of medium containing serial dilutions of MAbs at room temperature for 1 h.
  • 50 pL of virus-antibody mixtures were added to 293T-ACE2-TMPRSS2 cells (10 4 cells/50 pL) in a 96-well plate.
  • the input virus with all SARS-CoV-2 strains used in the current study were the same (2 x 10 5 relative light units/50 pL/well).
  • Vero E6 cells were seeded into black-walled, clear-bottom 96-well plates at 2 x 10 4 cells/well and cultured overnight at 37°C. The next day, 9-point 4-fold serial dilutions of mAbs were prepared in infection media (DMEM + 10% FBS). SARS-CoV-2-Nluc was diluted in infection media at a final MOI of 0.1 PFU/cell, added to the mAb dilutions and incubated for 30 minutes at 37”C. Media was removed from the Vero E6 cells, mAb-virus complexes were added and incubated at 37°C for 6 hours.
  • Nano-Gio luciferase substrate Promega was added according to the manufacturer’s recommendations, incubated for 10 minutes at room temperature and the luciferase signal was quantified on a VICTOR Nivo plate reader (Perkin Elmer).
  • mAbs 27E4, 26G10, 20A7, 25F9, 15F1 , 25C7, 20F2, 20C3 displayed extraordinary high affinity against SARS-CoV with Kds of 12.4 nM, 0.57 nM, 9.39 nM, 0.015 nM, 6.38 nM, 1.62 nM, 3.35 nM, 1 nM, respectively (Figure 15).
  • bnAbs 20A7, 25F9, 26C3, 27E3, 27E4 can potently neutralize RaTG13 with IC50s of 0.147 ug/ml, 0.021 ug/ml, 0.166 ug/ml, 0.014 ug/ml, 0.577 ug/ml, respectively ( Figure 26).
  • bnAbs 15F1 , 20A7, 25F9, 27A12, 27E4 neutralized SHC014 with IC50s of 0.039 ug/ml, 0.014 ug/ml, 0.014 ug/ml, 0.079 ug/ml, 0.014 ug/ml, respectively (Figure 27).
  • 25F9 and 20A7 neutralize authentic clade I sarbecoviruses beyond SARS-CoV-2 with ultrapotentcy (IC50s against SARS-CoV: 0.00085 ug/ml, 0.013 ug/ml; WIV-1 : 0.003 ug/ml, 0.002 ug/ml; SHC014: 0.006 ug/ml, 0.018 ug/ml, Pangolin: 0.006 ug/ml, 0.345 ug/ml, respectively) (Figure 28).
  • IC50s against SARS-CoV 0.00085 ug/ml, 0.013 ug/ml
  • WIV-1 0.003 ug/ml, 0.002 ug/ml
  • SHC014 0.006 ug/ml, 0.018 ug/ml
  • Pangolin 0.006 ug/ml, 0.345 ug/ml, respectively

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Abstract

Provided herein are antibodies and antigen binding fragments that are capable of binding to a betacoronavirus, e.g. a sarbecovirus selected from one or more of SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV-1, WIV-1, RaTG13, and SCH014.

Description

PAN-SARBECOVIRUS NEUTRALIZING ANTIBODIES AND METHODS OF USE THEREOF
CROSS REFERENCE TO OTHER APPLICATIONS
[0001 ] This application claims the benefit of U.S. Provisional Application No. 63/380,204, filed October 19, 2022, the contents of which are hereby incorporated by reference in its entirety.
INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS AN XML FILE
[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “S22-373_STAN- 2028WO_SEQ_LIST.xml” created on October 6, 2023 and having a size of 202,752 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety.
BACKGROUND
[0003] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in Wuhan China, in December 2019, leading to an ongoing global pandemic, also referred to as coronavirus disease 2019 (COVID-19). As of 27 September 2023, there had been 770,875,433 confirmed cases of COVID-19, including 6,959,316 deaths, reported to WHO. Previously emerged SARS-CoV-2 variants of concern (VOCs) have developed higher transmissibility, virulence and resistance to current vaccines and therapeutics antibodies. For example, the originally emerged Omicron variant of SARS-CoV-2 harbors 37 amino acid substitutions in the spike protein, 15 of which are in the receptor-binding domain (RBD), raising a great concern about the protective efficacies of current vaccines and therapeutic antibodies.
[0004] SARS-CoV-2, together with SARS-CoV-1 , which caused a global outbreak in 2002- 2003, are members of the genus Betacoronavirus and subgenus Sarbecoronavirus. Recent studies proposed that bat coronaviruses, such as WIV-1 , RaTG13, SCH014, are a probable origin of SARS-CoV and SARS-CoV-2. As such, in addition to the ongoing antigen drift of SARS-CoV-2, cross-species transmission of coronaviruses can never be neglected. Recent studies showed that some pan-sarbecovirus neutralizing antibodies targeting cryptic conserved epitopes in the receptor binding domain retained potency against Omicron, but exhibited significant reductions in the neutralizing activity.
[0005] Ultrapotent broadly neutralizing antibodies for preventing or treating sarbecoronavirus infection are needed.
SUMMARY
[0006] Provided herein are antibodies and antigen binding fragments that are capable of specifically binding to a betacoronavirus, e.g. a sarbecovirus selected from one or more of SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV-1 , WIV-1 , RaTG13, and SCH014. In some embodiments, an antibody or antigen binding fragment of the disclosure is capable of specifically binding to a surface glycoprotein of two, three, four, five, or more sarbecoviruses. In some embodiments, an antibody or antigen binding fragment of the disclosure is capable of binding to a surface glycoprotein of a sarbecovirus virion and/or a surface glycoprotein expressed on the surface of a cell infected by a sarbecovirus. In certain embodiments, presently disclosed antibodies and antigen binding fragments can neutralize infection by two, three, four, or five distinct sarbecovirus in an in vitro model of infection and/or in a human subject. In some embodiments the antibodies are humanized or chimeric, e.g. comprising a human Fc region sequence. Also provided are polynucleotides that encode the antibodies and antigen binding fragments, vectors, host cells, and related compositions, as well as methods of using the antibodies, nucleic acids, vectors, host cells, and related compositions to treat, e.g, reduce, delay, eliminate, or prevent, infection by any two, three, four, or five distinct sarbecoviruses in a subject and/or in the manufacture of a medicament for treating infection in a subject by any two, three, four, or five distinct sarbecoviruses.
[0007] In an embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3, and is capable of binding to a surface glycoprotein of a sarbecovirus. The antibody may comprise an Fc region sequence. In some embodiments, the antibody or antigen binding fragment thereof is capable of binding to a surface glycoprotein of two or more distinct sarbecoviruses. In some embodiment, the antibody or antigen binding fragment thereof is capable of binding to a surface glycoprotein of any two, three, four, or five distinct sarbecoviruses. In some embodiments, the antibody or antigen-binding fragment thereof is capable of binding to a surface glycoprotein of one or more of SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV, WIV-1 , RaTG13, and SCH014.
[0008] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 20A7, which binds to the spike protein of SARS-CoV-2, at least 5 variants of concern of SARS-CoV-2, and SARS-CoV. In certain embodiments, 20A7 neutralizes SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV, WIV-1 , RaTG13, and SCH014.
[0009] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 27A12, which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2. In certain embodiments, 27A12 neutralizes all known variants of concern of SARS-CoV-2, WIV-1 , and SCH014.
[0010] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 27E3, which specifically binds to the spikes of at least 5 variants of concern of SARS-CoV-2. In certain embodiments, 27E3 neutralizes at least 5 variants of concern of SARS-CoV-2, and RaTG13.
[001 1 ] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 21 B6, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern. In certain embodiments, 21 B6 neutralizes SARS-CoV-2 for at least 5 variants of concern.
[0012] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 27E4, which binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2 and SARS-CoV. In certain embodiments, 27E4 neutralizes SARS-CoV-2 for at least 5 variants of concern, WIV-1 , RaTG13, and SCH014.
[0013] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 26C3, which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2. In certain embodiments, 26C3 neutralizes SARS- CoV-2 and at least 5 variants of concern, and RaTG13.
[0014] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 15F1 , which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2, and SARS-CoV. In certain embodiments, 15F1 neutralizes SARS-CoV-2 and at least 5 variants of concern, SARS-CoV, WIV-1 , and SCH014.
[0015] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25C7, which binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2, and SARS-CoV. In certain embodiments, 25C7 neutralizes SARS-CoV-2 and all variants of concern.
[0016] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25F9, which specifically binds to the spikes of SARS-CoV-2, and at least 5 variants of concern of SARS-CoV-2, and SARS-CoV. In certain embodiments, 25F9 neutralizes SARS-CoV-2 for all variants of concern, SARS-CoV, WIV-1 , RaTG13, and SCH014.
[0017] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25A10, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern. In certain embodiments, 25A10 neutralizes SARS-CoV-2 for all variants of concern.
[0018] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 20F2, which specifically binds to the spikes of SARS-CoV-2, for at least 5 variants of concern of SARS-CoV-2, and SARS-CoV. In certain embodiments, 20F2 neutralizes SARS-CoV-2 and all variants of concern, excepting the beta strain.
[0019] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 21 F2, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2. In certain embodiments, 21 F2 neutralizes SARS- CoV-2 and all variants of concern.
[0020] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 25A1 1 , which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2. In certain embodiments, 25A11 neutralizes SARS- CoV-2 and all known variants of concern.
[0021 ] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a GDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 26G10, which specifically binds to the spikes of SARS-CoV-2, for at least 5 variants of concern of SARS-CoV-2, and SARS-CoV. In certain embodiments, 26G10 neutralizes SARS-CoV-2 and all variants of concern.
[0022] In one embodiment, the disclosure provides an isolated antibody, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a CDRL3 of antibody 20C3, which specifically binds to the spikes of SARS-CoV-2 for at least 5 variants of concern of SARS-CoV-2, and SARS-CoV. In certain embodiments, 20C3 neutralizes SARS-CoV-2 and at least 5 variants of concern, and SARS-CoV.
[0023] In yet another embodiment, the invention provides a polypeptide comprising or consisting of an epitope that binds to an antibody disclosed herein. Exemplary epitopes of the invention include, but not limited to, an epitope comprising amino acids from SARS-CoV-2, SARS-CoV-2 beta, SARS-CoV-2 omicron, SARS-CoV, Wl -1 , RaTG13, and SCH014 that are specifically bound by at least one antibody of the disclosure.
[0024] In yet another embodiment, the invention provides polynucleotides that encode the disclosed antibodies and antigen-binding fragments, vectors, host cells, and related compositions as well as methods of using the antibodies, nucleic acids, vectors, host cells, and related compositions to treat (e.g., reduce, delay, eliminate, or prevent) the infection caused by sarbecoviruses in a subject.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.
[0026] FIG. 1 shows schematic representation of the study design. Rhesus macaques received RBD-NP or Hexapro-NP adjuvanted with AS03 on day 0 and day 21 . Blood samples were collected and analysis was performed as illustrated in the diagram.
[0027] FIG. 2 Gating strategy of sorting and analyzing the spike+ and/or RBD+ B cells in total CD20+ cells. Gating was on singlets that were live CD20+ CD3- CD14- CD16- IgM/D- lgG+ Antigen+.
[0028] FIG. 3 Diagram depicting the strategy for antigen specific memory B cell sorting, monoclonal antibody isolation and characterization.
[0029] FIG. 4 The graph shows the binding activity to SARS-CoV-2 spike of all monoclonal antibodies in this study measured by ELISA. 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 Wuhan spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 450 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
[0030] FIG. 5 The graph shows the half-maximal inhibitory concentrations (IC50s) of all monoclonal antibodies against pseudotyped SARS-CoV-2 Wuhan, BA.1 and BA.4/5 respectively. The geometric mean valuves were labeld on top of each strain. The statistical differences between the groups were measured by non-parametric Friedman test.
[0031 ] FIG. 6 shows the neutralizing activities of 15 monoclonal antibodies against pseudotyped SARS-CoV-2 Wuhan strain. IC50s were labeled in parallel with each mAb on the right.
[0032] FIG. 7 shows the neutralizing activities of 15 monoclonal antibodies against pseudotyped SARS-CoV-2 BA.1 strain. IC50s were labeled in parallel with each mAb on the right.
[0033] FIG. 8 shows the neutralizing activities of 15 monoclonal antibodies against pseudotyped SARS-CoV-2 BA.4/5 strain. IC50s were labeled in parallel with each mAb on the right.
[0034] FIG. 9 shows ELISA results of 15 monoclonal antibodies on spike protein of SARS- CoV-2 Wuhan. The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 Wuhan spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 405 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
[0035] FIG. 10 shows ELISA results of 15 monoclonal antibodies on spike protein of SARS- CoV-2 BA.1 . The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 BA.1 spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 405 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
[0036] FIG. 11 shows ELISA results of 15 monoclonal antibodies on spike protein of SARS- CoV-2 BA.4/5. The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. 96-well ELISA plates were coated overnight at 4°C with 1 pg/ml of SARS-CoV-2 BA.4/5 spikes in PBS. Plates were blocked with a 5% w/v skim milk powder (Biorad) in PBST and incubated with serial dilutions of mAbs for 1 hour at room temperature. Subsequently, the plates were washed, and anti-human IgG coupled to HRP (EMD Millipore) was added and incubated for 1 hour. After further washing, the substrate (TMB, EMD Millipore) was added and plates were read at 405 nm using a microplate reader (Biotek). The data have been plotted with Graphpad Prism software.
[0037] FIG. 12 shows cross-reactive profiles of monoclonal antibodies isolated from RBD-NP vaccinated individuals, as measured by Meso Scale V-PLEX SARS-CoV-2 panel 9. The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. Plates were coated 30 minutes at room temperature with blocking buffer and incubated with serial dilutions of mAbs for 2 hour at room temperature. Subsequently, the plates were washed, and Sulfo-anti-human IgG was added and incubated for 1 hour. After further washing, the read buffer was added and plates were read using MESO QuickPlex SO 120. The data were plotted with Graphpad Prism software.
[0038] FIG. 13 shows cross- reactive profiles of monoclonal antibodies isolated from Hexapro- NP vaccinated individuals, as measured by Meso Scale V-PLEX SARS-CoV-2 panel 13. The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. Plates were coated 30 minutes at room temperature with blocking buffer and incubated with serial dilutions of mAbs for 2 hour at room temperature. Subsequently, the plates were washed, and Sulfo-anti-human IgG was added and incubated for 1 hour. After further washing, the read buffer was added and plates were read using MESO QuickPlex SQ 120. The data were plotted with Graphpad Prism software.
[0039] FIG. 14 shows cross-reactivities against SARS-CoV-1 spike protein of exemplary monoclonal antibodies, as measured by Meso Scale V-PLEX coronavirus panel 3. The concentrations of antibody that gives half-maximal binding (EC50s) were labeled in parallel on the right. Plates were coated 30 minutes at room temperature with blocking buffer and incubated with serial dilutions of mAbs for 2 hour at room temperature. Subsequently, the plates were washed, and Sulfo-anti-human IgG was added and incubated for 1 hour. After further washing, the read buffer was added and plates were read using MESO QuickPlex SQ 120. The data were plotted with Graphpad Prism software.
[0040] FIG. 15 shows the binding affinities of exemplary monoclonal antibodies against SARS-CoV RBD, as mearsued by ForteBio’s Octet RH16 system.
[0041 ] FIG. 16 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 WA1 strain. The IC50s were labeled in parallel on the right.
[0042] FIG. 17 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 alpha strain. The IC50s were labeled in parallel on the right. [0043] FIG. 18 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 Beta strain. The IC50s were labeled in parallel on the right.
[0044] FIG. 19 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 Gamma strain. The IC50s were labeled in parallel on the right.
[0045] FIG. 20 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 Delta strain. The IC50s were labeled in parallel on the right.
[0046] FIG. 21 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 BA.1 strain. The IC50s were labeled in parallel on the right.
[0047] FIG. 22 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 BA.2 strain. The IC50s were labeled in parallel on the right.
[0048] FIG. 23 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV-2 BA.3 strain. The IC50s were labeled in parallel on the right.
[0049] FIG. 24 shows the neutralizing activities of monoclonal antibodies against pseudotyped SARS-CoV. The IC50s were labeled in parallel on the right.
[0050] FIG. 25 shows the neutralizing activities of monoclonal antibodies against pseudotyped WIV-1 . The IC50s were labeled in parallel on the right.
[0051] FIG. 26 shows the neutralizing activities of monoclonal antibodies against pseudotyped RaTG13. The IC50s were labeled in parallel on the right.
[0052] FIG. 27 shows the neutralizing activities of monoclonal antibodies against pseudotyped SCH014. The IC50s were labeled in parallel on the right.
[0053] FIG. 28 shows the neutralization of authentic SARS-CoV-2 D614, SARS-CoV-2 BA.1 , Pangolin-GD, SARS-CoV, SHC014, and WIV-1 by indicated antibodies. Symbols are means ± SD. Dotted lines indicate IC50 values, n = 4.
DETAILED DESCRIPTION
[0054] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.
[0055] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described below. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.
[0056] As used herein, the term “SARS-CoV-2” is a betacoronavirus that causes a respiratory disease called coronavirus disease 19 (COVID-19). SARS-CoV-2 is in the Sarbecovirus subgenus of Coronaviridae. SARS-CoV-2, also referred to herein as “Wuhan Strain” comprising the amino acid sequence of SARS-CoV-2 isolate Wuhan-Hu-1 (see GenBank QHD43416.1 , Januray 23, 2020), was first known and identified in Wuhan, Hubei province China, in 2019. The virus is thought to spread from person to person through droplets released when an infected person coughs, sneezes, or talks. It may also be spread by touching a surface with the virus on it and then touching one’s mouth, nose, or eyes, but this is less common. Symptoms of SARS-CoV-2 infection include fever, dry cough, dyspnea, fatigue, body aches, headache, new loss of tase or smell, sore throat, congestions or runny nose, nausea or vomiting, diarrhea, persistent pressure or pain n the chest, new confusion, inability to wake or stay awake, and bluish lips or face.
[0057] There have been a number of emerging SARS-CoV-2 variants. As used herein, the term “variant” refers to a viral genome (genetic code) that may contain one or more mutations and is genetically distinct from a main strain. In some cases, a group of variants with similar genetic changes, such as a lineage or group of lineages, may be designated by public health organizations as a Variant Being Monitored (VBM), Variant of Concern (VOC) or a Variant of Interest (VOI) due to shared attributes and characteristics that may require public health action. There have been a number of emerging SARS-CoV-2 variants. A Variant of Interest or a Variant of Concern may be downgraded to a Variant Being Monitored after a significant and sustained reduction in its national and regional proportions over time, or other evidence indicates that a variant does not pose significant risk to public health in the United States. Based on the CDC update on April 26, 2022, the Alpha strain (also known as the lineage B.1 .1 .7 and Q lineages), the Beta strain (also know as the lineage B.1.351 ), the Gamma strain (also known as the lineage P.1 ), the Delta strain (also known as the lineage B.1 .617.2 and AY lineages), the Epsilon strain (also known as the lineage B.1.426 and the lineage B.1.429).
[0058] The current SARS-CoV-2 variant of concern, the Omicron strain (also known as B.1.1.529, BA.1 , BA.1 .1 , BA.2, BA.3, BA.4 and BA.5 lineages), contains spike protein substitutions: A67V, del69-70, T95I, dell 42-144, Y145D, del211 , L212I, ins214EPE, G339D, S371 L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501 Y, Y505H, T547K, D614G, H655Y, N679K, P681 H, N764K, D796Y, N856K, Q954H, N969K, L981 F, which has enhanced transmissibility, increased resistentance to the neutralization by some EUA monoclonal antbody treatments and to the neutralization by postvaccination sera. [0059] The genomic sequence of SARS-CoV-2 is publicly available, for example at GenBank NC_045512. A database of variants is maintained at NCBI virus, which is publicly available. Like other coronaviruses (e.g., SARS- CoV-1), SARS-CoV-2 comprises a "spike" or surface ("S") type I transmembrane glycoprotein containing a receptor binding domain (RBD). The RBD is believed to mediate entry of the lineage B SARS coronavirus to respiratory epithelial cells by binding to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). In particular, a receptor binding motif (RBM) in the virus RBD is believed to interact with ACE2.
[0060] In addition to SARS-CoV-2 and variants, sarbecoviruses include, without limitation: SARS-CoV-1 , WIV-1 , RaTG13, and SCH014. SARS-CoV is another betacoronavirus that causes respiratory symptoms in infected individuals. The genomic sequence of SARS-CoV-2 Urbani strain has GenBank accession number AAP13441 .1 .
[0061] In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative ( e.g ., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have," and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
[0062] The term "consisting essentially of is not equivalent to "comprising" and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain) or a protein "consists essentially of a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy - terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (i.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).
[0063] "Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.
[0064] Individual constructs, or groups of constructs, derived from the various combinations of the structures and subunits described herein, are disclosed by the present application to the same extent as if each construct or group of constructs was set forth individually. Thus, selection of particular structures or particular subunits is within the scope of the present disclosure.
[0065] As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, g- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g, norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
[0066] As used herein, "mutation" refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).
[0067] A "conservative substitution" refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3 : Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (lie or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and lie. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, lie, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.
[0068] As used herein, "protein" or "polypeptide" refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.
[0069] "Nucleic acid molecule" or "polynucleotide" or "polynucleic acid" refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits ( e.g ., purine or pyrimidine bases) or non-natural subunits (e.g, morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense) strand. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.
[0070] In some embodiments, the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof. In certain embodiments, the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5-methylcytidine, a 2- thiouridine, or any combination thereof. In some embodiments, the pseudouridine comprises N-methylpseudouridine.
[0071 ] Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein. Nucleic acid molecule variants retain the capacity to encode a binding domain thereof having a functionality described herein, such as binding a target molecule. A "functional variant" refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has "similar binding," "similar affinity" or "similar activity" when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant).
[0072] "Percent sequence identity" refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes ( e.g ., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs.
[0073] The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. "Isolated" can, in some embodiments, also describe an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition that is outside of a human body.
[0074] As used herein, a "functional portion" or "functional fragment" refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A "functional portion" or "functional fragment" of a polypeptide or encoded polypeptide of this disclosure has "similar binding" or "similar activity" when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity).
[0075] As used herein, the term "engineered," "recombinant," or "non-natural" refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous or heterologous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene, or operon.
[0076] As used herein, "heterologous" or "non-endogenous" or "exogenous" refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules ( e.g ., receptors, ligands, etc.) may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra- chromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term "homologous" or "homolog" refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof.
[0077] As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.
[0078] The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., the coding sequence is under the transcriptional control of the promoter). "Unlinked" means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.
[0079] The term "construct" refers to any polynucleotide that contains a recombinant nucleic acid molecule. A (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon systems ( e.g ., Sleeping Beauty, see, e.g., Geurts et ah, Mol. Ther. 5:108, 2003: Mates et ah, Nat. Genet. ¥7:753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g., viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).
[0080] As used herein, "expression vector" or "vector" refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence. In the present specification, "plasmid," "expression plasmid," "virus," and "vector" are often used interchangeably.
[0081 ] In certain embodiments, polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
[0082] In certain embodiments, the vector comprises a plasmid vector or a viral vector e.g ., a lentiviral vector or a y-retroviral vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g, influenza virus), rhabdovirus (e.g, rabies and vesicular stomatitis virus), paramyxovirus (e.g, measles and Sendai), positive strand RNA viruses such as picomavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g, Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g, vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus.
[0083] As used herein, the term "host" refers to a cell or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest ( e.g., an antibody of the present disclosure). A host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods.
[0084] In the context of a sarbecovirus infection, a "host" refers to a cell or a subject infected with a sarbecovirus.
[0085] "Antigen" or "Ag", as used herein, refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells, activation of complement, antibody dependent cytotoxicicity, or any combination thereof. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like, for example a sarbecovirus spike protein. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, stool samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen. Antigens can also be present in a betacoronavirus (e.g, a surface glycoprotein, spike protein, or portion thereof), such as present in a virion, or expressed or presented on the surface of a cell infected by a betacoronavirus. An antigen may be killed, inactivated, attenuated, or modified live bacteria, viruses, or parasites, or polynucleotides, polypeptides, recombinant proteins, synthetic peptides, protein extract, cells (including bacterial cells), tissues, polysaccharides, or lipids, or fragments thereof, or the like, individually or in any combination thereof.
[0086] The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Where an antigen is or comprises a peptide or protein, the epitope can be comprised of consecutive amino acids (e.g, a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g, a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding.
[0087] As used herein, the term "antibody" refers to an immunoglobulin molecule that can bind to a specific antigen as the result of an immune response to that antigen. Immunoglobulins are serum proteins composed of "light" and "heavy" polypeptide chains having "constant" and "variable" regions and are divided into classes (e.g., IgA, IgD, IgE, IgG, and IgM) based on the composition of the constant regions.
[0088] As used herein, the term “monoclonal antibody” or “mAb” or “monoclonal antibody composition” refers to a population of antibody molecules that contains only one molecular species of antibody molecule containing one unique heavy chain product and one unique light chain product. The antibodies of the invention are “chimeric monoclonal antibodies” or “humanized monoclonal antibodies”, refering to antibodies with reduced immunogenicity in humans built by genetically linking a non-human primate variable region to human constant domains (Fc).
[0089] As used herein, the term “neutralizing antibody” refers to an antibody, for example, a monoclonal antibody, that can neutralize the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms “neutralizing antibody” and “an antibody that neutralizes” or “ neutralization of an antibody” are used interchangeably herein.
[0090] In certain embodiments, an antibody or antigen binding fragment of the present disclosure specifically binds to a sarbecovirus surface glycoprotein. As used herein, "specifically binds" refers to an association or union of an antibody or antigen binding fragment to an antigen with an affinity or Ka (i.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10'5 M (which equals the ratio of the on-rate [Kon] to the off rate [Koff] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M ( e.g., 105 M to 1013 M). Antibodies may be classified as "high- affinity" antibodies or as "low-affinity" antibodies. "High-affinity" antibodies refer to those antibodies having a Ka of less than 10-7 M, less than 10-8 M, less than 10-9 M, less than 10-10 M, less than 10-11 M, less than 10'12 M, or less than 10'13 M. "Low-affinity" antibodies refer to those antibodies having a Ka of greater than 10‘7 M, greater than 10‘6, greater than 10-5 M. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 105 M to 1013 M).
[0091 ] A variety of assays are known for identifying antibodies of the present disclosure that bind a particular target, as well as determining binding domain or binding protein affinities, such as Western blot, ELISA (e.g, direct, indirect, or sandwich), analytical ultracentrifugation, spectroscopy, biolayer interferometry, and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard etal., Ann. N.Y. Acad. Sci. 57:660, 1949; Wilson, Science 295: 2103, 2002). Assays for assessing affinity or apparent affinity or relative affinity are also known. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g. as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g. at 25°C.
[0092] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for immunization and/or being treated. Subjects may be human, but also include other mammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, rhesus macaques etc.
Antibodies
[0093] Provided herein are isolated antibodies, or an antigen binding fragment thereof, that comprises a heavy chain variable domain (VH) comprising a provided CDRH1 , a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1 , a CDRL2, and a GDRL3 sequence, which is capable of binding the surface glycoprotein of SARS-CoV-2. The invention also provides fragments of these monoclonal antibodies (e.g. antigen binding fragments) thereof, that comprise the heavy chain variable domain (VH) comprising CDRH1 , CDRH2, CDRH3, and the light chain variable domain (VL) comprising CDRL1 , CDRL2, CDRL3, and is capable of binding to the surface glycoprotein of SARS-CoV-2.
[0094] In certain embodiments, an antibody or antigen binding fragment of the present disclosure binds to a SARS-CoV-2 (including Wuhan strain and one or more additional variant) surface glycoprotein epitope, while not significantly associating or uniting with any other molecules or components in a sample. In other words, in certain embodiments, an antibody or antigen binding fragment of the present disclosure is reactive and specific for the SARS- CoV-2 and variants.
[0095] In certain embodiments, an antibody or antigen binding fragment of the present disclosure binds to a SARS-CoV-2 (including Wuhan strain and one or more additional variant) surface glycoprotein epitope, and can also binds to epitopes from other sarbecovirus present in the sample, but does not significantly bind to or associate with any other molecules or components in the sample. In other words, in certain embodiments, an antibody or antigen binding fragment of the present disclosure is cross-reactive for SARS-CoV-2 and one or more additional sarbecovirus, and is specific for the SARS-CoV-2 and one or more additional coronavirus.
[0096] In certain embodiments, an antibody of the present disclosure is capable of preventing and/or neutralizing infection by SARS-CoV-2 (including Wuhan strain and one or more additional variant), but does does not significantly prevent or neutralize infection by other coronavirus. In other words, an antibody or antigen binding fragment of the present disclosure is broad SARS-CoV-2 neutralizing antibody.
[0097] In certain embodiments, an antibody of the present disclosure is capable of preventing and/or neutralizing infection by SARS-CoV-2 (including Wuhan strain and one or more additional variant), and can also prevent or neutralize infection by one or more additional sarbecovirus. I n other words, an antibody or antigen binding fragment of the present disclosure is pan-sarbecovirus neutralizing antibody.
[0098] As used herein, the terms “VH” and “VL” refer to the variable binding region from an antibody heavy chain and an antibody light chain, respectively. In certan embodiments, a VL is a kappa class (also “VK” herein). In certain embodiments, a VL is a lambda class (also “V ” herein). The variable binding regions comprise discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs). The terms “complementarity determining regions” and “CDRs” or “CDR” refer to sequences of amino acids within antibody variable regions, which, in general, together confer the antigen specificity and/or binding affinity of the antibody, wherein consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from one another in primary structure by a framework region. There are three CDRs in each variable region (HCDR1 , HCDR2, HCDR3; LCDR1 , LCDR2, LCDR3; also referred to as CDRHs and CDRLs, respectively). In certain embodiments, an antibody VH comprises four FRs and three HCDRs as follows: FR1 -HCDR1 -FR2-HCDR2- FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three LCDRs as follows: FR1 - LCDR1 -FR2-LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen binding site through their respective CDRs.
[0099] In certain embodiments, provided herein is an antibody or antigen binding fragment referred to as 15F1 , 20A7, 20C3, 20F2, 21 B6, 21 F2, 25A10, 25A11 , 25C7, 25F9, 2603, 26G10, 27A12, 27E3, or 27E4 comprising the three CDRs of a VH sequence according to the SEQ ID NO.: 5, 21 , 37, 53, 69, 85, 101 , 117, 133, 149, 165, 181 , 197, 213, 229, respectively and/or the three CDRs of a VL sequence according to the SEQ ID NO.:13, 29, 45, 61 , 77, 93, 109, 125, 141 , 157, 173, 189, 205, 221 , 237, respectively, as determined using IMGT numbering method. Sequences are provided in Table 1.
[00100] In some embodiments, an antibody or fragment thereof of 15F1 comprises the three CDRs of a VH sequence according to the SEQ ID NO:5, i.e. SEQ ID NO:6, 7, 8; and the three CDRs of a VL sequence according to SEQ ID NO:13, i.e. SEQ ID NO:14, 15, 16.
[00101 ] In some embodiments, an antibody or fragment thereof of 20A7 comprises the three CDRs of a VH sequence according to the SEQ ID NO:21 , i.e. SEQ ID NO:22, 23, 24; and the three CDRs of a VL sequence according to SEQ ID NO:29, i.e. SEQ ID NQ:30, 31 , 32.
[00102] In some embodiments, an antibody or fragment thereof of 20C3 comprises the three CDRs of a VH sequence according to the SEQ ID NO:37, i.e. SEQ ID NO:38, 39, 40; and the three CDRs of a VL sequence according to SEQ ID NO:45, i.e. SEQ ID NO:46, 47, 48.
[00103] In some embodiments, an antibody or fragment thereof of 20F2 comprises the three CDRs of a VH sequence according to the SEQ ID NO:53, i.e. SEQ ID NO:54, 55, 56; and the three CDRs of a VL sequence according to SEQ ID NO:61 , i.e. SEQ ID NO:62, 63, 64.
[00104] In some embodiments, an antibody or fragment thereof of 21 B6 comprises the three CDRs of a VH sequence according to the SEQ ID NO:69, i.e. SEQ ID NOTO, 71 , 72; and the three CDRs of a VL sequence according to SEQ ID NO:77, i.e. SEQ ID NO:78, 79, 80.
[00105] In some embodiments, an antibody or fragment thereof of 21 F2 comprises the three CDRs of a VH sequence according to the SEQ ID NO:85, i.e. SEQ ID NO:86, 87, 88; and the three CDRs of a VL sequence according to SEQ ID NO:93, i.e. SEQ ID NO:94, 95, 96.
[00106] In some embodiments, an antibody or fragment thereof of 25A10 comprises the three CDRs of a VH sequence according to the SEQ ID NQ:101 , i.e. SEQ ID NQ:102, 103, 104; and the three CDRs of a VL sequence according to SEQ ID NQ:109, i.e. SEQ ID NQ:1 10, 11 1 , 1 12.
[00107] In some embodiments, an antibody or fragment thereof of 25A1 1 comprises the three CDRs of a VH sequence according to the SEQ ID NO:1 17, i.e. SEQ ID NO:1 18, 119, 120; and the three CDRs of a VL sequence according to SEQ ID NO:125, i.e. SEQ ID NO:126, 127, 128.
[00108] In some embodiments, an antibody or fragment thereof of 25C7 comprises the three CDRs of a VH sequence according to the SEQ ID NO:133, i.e. SEQ ID NO:134, 135, 136; and the three CDRs of a VL sequence according to SEQ ID NO:141 , i.e. SEQ ID NO:142, 143, 144.
[00109] In some embodiments, an antibody or fragment thereof of 25F9 comprises the three CDRs of a VH sequence according to the SEQ ID NO:149, i.e. SEQ ID NQ:150, 151 , 152; and the three CDRs of a VL sequence according to SEQ ID NO:157, i.e. SEQ ID NO:158, 159, 160.
[001 10] In some embodiments, an antibody or fragment thereof of 26C3 comprises the three CDRs of a VH sequence according to the SEQ ID NO:165, i.e. SEQ ID NO:166, 167, 168; and the three CDRs of a VL sequence according to SEQ ID NO:173, i.e. SEQ ID NO:174, 175, 176.
[001 11 ] In some embodiments, an antibody or fragment thereof of 26G10 comprises the three CDRs of a VH sequence according to the SEQ ID NO:181 , i.e. SEQ ID NO:182, 183, 184; and the three CDRs of a VL sequence according to SEQ ID NO:189, i.e. SEQ ID NQ:190, 191 , 192.
[001 12] In some embodiments, an antibody or fragment thereof of 27A12 comprises the three CDRs of a VH sequence according to the SEQ ID NO:197, i.e. SEQ ID NO:198, 199, 200; and the three CDRs of a VL sequence according to SEQ ID NQ:205, i.e. SEQ ID NQ:206, 207, 208.
[001 13] In some embodiments, an antibody or fragment thereof of 27E3 comprises the three CDRs of a VH sequence according to the SEQ ID NO:213, i.e. SEQ ID NO:214, 215, 216; and the three CDRs of a VL sequence according to SEQ ID NO:221 , i.e. SEQ ID NO:222, 223, 224.
[001 14] In some embodiments, an antibody or fragment thereof of 27E4 comprises the three CDRs of a VH sequence according to the SEQ ID NO:229, i.e. SEQ ID NQ:230, 232, 232; and the three CDRs of a VL sequence according to SEQ ID NO:237, i.e. SEQ ID NO:238, 239, 240.
[001 15] Antibodies of the invention may comprise an Fc sequence, e.g. from any of the human antibody isotypes, including the gamma isotypes: lgG1 , lgG2a, lgG2b, lgG3, lgG4; or may comprise an Fc of IgM, IgD, IgA, IgE, etc. The Fc sequence may be other than a naturally occurring Fc sequence of the specific antibody. An antibody comprising a variable region and an Fc region may be referred to as an “entire” antibody.
[001 16] An Fc sequence may provide for enhanced effector functions, e.g. by increasing their binding capacities to FcyRI 11 A and increasing ADCC activity. For example, fucose attached to the AMinked glycan at Asn-297 of Fc sterically hinders the interaction of Fc with FcyRI 11 A, and removal of fucose by glyco-engineering can increase the binding to FcyRI 11 A, which translates into >50-fold higher ADCC activity compared with wild type IgG 1 controls. Protein engineering, through amino acid mutations in the Fc portion of lgG1 , has generated multiple variants that increase the affinity of Fc binding to FcyRI I IA. Notably, the triple alanine mutant S298A/E333A/K334A displays 2-fold increase binding to FcyRI HA and ADCC function. S239D/I332E (2X) and S239D/I332E/A330L (3X) variants have a significant increase in binding affinity to FcyRI I IA and augmentation of ADCC capacity in vitro and in vivo. Other Fc variants identified by yeast display also showed the improved binding to FcyRIIIA and enhanced tumor cell killing in mouse xenograft models. See, for example Liu et al. (2014) JBC 289(6):3571 -90, herein specifically incorporated by reference.
[001 17] In some embodiments of the invention, the heavy chain constant region is selected to provide for high ADCC activity, including without limitation human lgG1 . In other embodiments a low ADCC activity may be selected, e.g. with human lgG4, or with a “dead” Fc in which effector functions are absent or reduced.
[001 18] Other Fc variants are possible, including without limitation one in which a region capable of forming a disulfide bond is deleted, or in which certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto. Thus, in one embodiment of the invention, one or more Fc portions of the scFc molecule can comprise one or more mutations in the hinge region to eliminate disulfide bonding. In yet another embodiment, the hinge region of an Fc can be removed entirely. In still another embodiment, the scFc molecule can comprise an Fc variant.
[001 19] Further, an Fc variant can be constructed to remove or substantially reduce effector functions by substituting, deleting or adding amino acid residues to effect complement binding or Fc receptor binding. For example, and not limitation, a deletion may occur in a complementbinding site, such as a C1 q-binding site. Techniques of preparing such sequence derivatives of the immunoglobulin Fc fragment are disclosed in International Patent Publication Nos. WO 97/34631 and WO 96/32478. In addition, the Fc domain may be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, acetylation, amidation, and the like.
[00120] Antibodies include antibody fragments. For example, a "Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, i.e., it has a single antigen binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen binding activity and is still capable of cross-linking antigen. Both the Fab and F(ab’)2 are examples of "antigen binding fragments." Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody hinge region.
[00121 ] Fab fragments may be joined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as "scFab." In these embodiments, an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain. A heavy chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VH + CHI, or "Fd") and a light chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VL + CL) may be linked in any arrangement to form a scFab. For example, a scFab may be arranged, in N-terminal to C-terminal direction, according to (heavy chain Fab fragment - linker - light chain Fab fragment) or (light chain Fab fragment - linker -heavy chain Fab fragment).
[00122] "Fv" is a small antibody fragment that contains a complete antigen-recognition and antigen binding site. This fragment generally consists of a dimer of one heavy- and one lightchain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site.
[00123] "Single-chain Fv" also abbreviated as "sFv" or "scFv", are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for antigen binding. Such a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer- Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In certain embodiments, the antibody or antigen binding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain. In particular embodiments, a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH- linker-VL orientation or in a VL-linker-VH orientation. Any scFv of the present disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (i.e. , (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some embodiments, a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both.
[00124] In some embodiments, the antibody, or the antigen binding fragment, comprises a human antibody, a humanized antibody, a human antibody comprising a variant Fc sequence, a monoclonal antibody, a single chain antibody, a Fab, a Fab’, a F(ab’)2, a Fv, a scFv, or a scFab.
[00125] In certain embodiments, an antibody or antigen binding fragment of the present disclosure is monospecific (e.g., binds to a single epitope) or is multispecific (e.g., binds to multiple epitopes and/or target molecules). Antibodies and antigen binding fragments may be constructed in various formats. Exemplary antibody formats disclosed in Spiess et al., Mol. Immunol. 67(2):95 (2015), and in Brinkmann and Kontermann, mAbs 9(2): 182-212 (2017), which formats and methods of making the same are incorporated herein by reference and include, for example, Bispecific T cell Engagers (BiTEs), DARTs, Knobs-lnto-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH Common Light-Chain antibodies, TandAbs, Triple Bodies, TriBi Minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y assemblies, Fcabs, kl-bodies, orthogonal Fabs, DVD-lgs (e.g., US Patent No. 8,258,268, which formats are incorporated herein by reference in their entirety), lgG(H)-scFv, scFv- (H)lgG, lgG(L)-scFv, scFv-(L)lgG, lgG(L,H)-Fv, lgG(H)-V, V(H)-lgG, lgG(L)-V, V(L)-lgG, KIH IgG-scFab, 2scFv-lgG, lgG-2scFv, scFv4-lg, Zybody, and DVI-IgG (four-in-one), as well as FIT-lg (e.g, PCT Publication No. WO 2015/10307), WuxiBody formats (e.g, PCT Publication No. WO 2019/057122), and In-Elbow-lnsert Ig formats (lEI-lg; e.g, PCT Publication Nos. WO 2019/024979 and WO 2019/025391 ).
[00126] In certain embodiments, the antibody or antigen binding fragment comprises two or more of VH domains, two or more VL domains, or both (i.e. , two or more VH domains and two or more VL domains). In particular embodiments, an antigen binding fragment comprises the format (N-terminal to C-terminal direction) VH-linker-VL-linker-VH-linker-VL, wherein the two VH sequences can be the same or different and the two VL sequences can be the same or different. Such linked scFvs can include any combination of VH and VL domains arranged to bind to a given target, and in formats comprising two or more VH and/or two or more VL, one, two, or more different eptiopes or antigens may be bound. It will be appreciated that formats incorporating multiple antigen binding domains may include VH and/or VL sequences in any combination or orientation. For example, the antigen binding fragment can comprise the format VL4 linker-VH4 linker-VL-linker-VH, VH-linker-VL-linker-VL4 linker-VH, or VL4 linker-VH4 linker-VH4 linker-VL.
[00127] Monospecific or multispecific antibodies or antigen binding fragments of the present disclosure constructed comprise any combination of the VH and VL sequences and/or any combination of the CDRH1 , CDRH2, CDRH3, CDRL1 , CDRL2, and CDRL3 sequences disclosed herein. A bispecific or multispecific antibody or antigen binding fragment may, in some embodiments, comprise one, two, or more antigen binding domains ( e.g ., a VH and a VL) of the instant disclosure. Two or more binding domains may be present that bind to the same or a different betacoronavirus epitope, and a bispecific or multispecific antibody or antigen binding fragment as provided herein can, in some embodiments, comprise a further SARS-CoV-2 binding domain, and/or can comprise a binding domain that binds to a different antigen or pathogen altogether.
[00128] In any of the presently disclosed embodiments, the antibody or antigen binding fragment can be multispecific; e.g., bispecific, trispecific, or the like.
[00129] In any of the presently disclosed embodiments, the antibody or antigen binding fragment can be monoclonal. The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present, in some cases in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The term "monoclonal" is not to be construed as requiring production of the antibody by any particular method.
[00130] Antibodies and antigen binding fragments of the present disclosure include "chimeric antibodies" in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity. For example, chimeric antibodies may comprise human and non human residues. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance.
[00131 ] A "humanized antibody" is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These nonhuman amino acid residues are typically taken from a variable domain. Humanization may be performed following the method of Winter and co-workers (Jones et al, Nature, 321 :522-525 (1986); Reichmann et al., Nature , 332:323-327 (1988); Verhoeyen et al., Science , 239: 1534- 1536 (1988)), by substituting non-human variable sequences for the corresponding sequences of a human antibody.
Polynucleotides [00132] In another aspect, the present disclosure provides isolated polynucleotides that encode any of the presently disclosed antibodies or an antigen binding fragment thereof, or a portion thereof (e.g., a CDR, a VH, a VL, a heavy chain, or a light chain). In certain embodiments, the polynucleotide is codon-optimized for expression in a host cell. Once a coding sequence is known or identified, codon optimization can be performed usng known techniques and tools.
[00133] Codon-optimized sequences include sequences that are partially codon-optimized (i.e., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.
[00134] Polynucleotides may be provided in a vector, e.g. an expression vector as disclosed herein. Such vectors may comprise or contain a polynucleotide as disclosed herein. A vector can comprise any one or more of the vectors disclosed herein. In particular embodiments, a vector is provided that comprises a DNA plasmid construct encoding the antibody or antigen binding fragment, or a portion thereof. In certain embodiments, a DNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigen binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide. In some embodiments, the substituent components of the antibody or antigen binding fragment are encoded by a polynucleotide comprised in a single plasmid. In other embodiments, the substituent components of the antibody or antigen binding fragment are encoded by a polynucleotide comprised in two or more plasmids (e.g, a first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL). In certain embodiments, a single plasmid comprises a polynucleotide encoding a heavy chain and/or a light chain from two or more antibodies or antigen binding fragments of the present disclosure. An exemplary expression vector is pVaxI, available from Invitrogen®. A DNA plasmid of the present disclosure can be delivered to a subject by, for example, electroporation (e.g, intramuscular electroporation), or with an appropriate formulation (e.g, hyaluronidase).
[00135] It will be also appreciated that polynucleotides encoding antibodies and antigen binding fragments of the present disclosure may possess different nucleotide sequences while still encoding a same antibody or antigen binding fragment due to, for example, the degeneracy of the genetic code, splicing, and the like.
[00136] In any of the presently disclosed embodiments, the polynucleotide can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the RNA comprises messenger RNA (mRNA). Table 1 . Sequences
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Figure imgf000033_0001
Figure imgf000034_0001
Figure imgf000035_0001
Figure imgf000036_0001
Figure imgf000037_0001
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Table 2
Figure imgf000043_0002
Figure imgf000044_0001
[00137] In a further aspect, the present disclosure also provides a host cell expressing an antibody or antigen binding fragment according to the present disclosure; or comprising or containing a vector or polynucleotide according the present disclosure. Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In certain such embodiments, the cells are a mammalian cell line such as CHO cells (e.g, DHFR- CHO cells, human embryonic kidney cells (e.g, HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells. NSO cells, human liver cells, e.g. Hepa RG cells, myeloma cells or hybridoma cells. Other examples of mammalian host cell lines include mouse sertoli cells (e.g, TM4 cells); monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1 ); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. In certain embodiments, a host cell is a prokaryotic cell, such as an E. coli. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. In particular embodiments, the cell may be transfected with a vector according to the present description with an expression vector. Host cells of the present disclosure may be transfected stably or transiently with a vector according to the present disclosure, e.g. for expressing an antibody, or an antigen binding fragment thereof, according to the present disclosure.
[00138] Also provided are recombinant host cells that heterologously express an antibody or antigen binding fragment of the present disclosure. For example, the cell may be of a species that is different to the species from which the antibody was fully or partially obtained ( e.g ., CHO cells expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not express the antibody or antigen binding fragment in nature. Moreover, the host cell may impart a post-translational modification (PTM; e.g., glysocylation or fucosylation) on the antibody or antigen binding fragment that is not present in a native state of the antibody or antigen binding fragment (or in a native state of a parent antibody from which the antibody or antigen binding fragment was engineered or derived). Such a PTM may result in a functional difference (e.g, reduced immunogenicity). Accordingly, an antibody or antigen binding fragment of the present disclosure that is produced by a host cell as disclosed herein may include one or more post-translational modification that is distinct from the antibody (or parent antibody) in its native state (e.g, a human antibody produced by a CHO cell can comprise a more post-translational modification that is distinct from the antibody when isolated from the human and/or produced by the native human B cell or plasma cell).
[00139] In a related aspect, the present disclosure provides methods for producing an antibody, or antigen binding fragment, wherein the methods comprise culturing a host cell of the present disclosure under conditions and for a time sufficient to produce the antibody, or the antigen binding fragment. Methods useful for isolating and purifying recombinantly produced antibodies, by way of example, may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant antibody into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant antibody described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of soluble antibodies may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.
Compositions [00140] Also provided herein are compositions that comprise any one or more of the presently disclosed antibodies, antigen binding fragments, polynucleotides, vectors, or host cells, singly or in any combination, and can further comprise a pharmaceutically acceptable carrier, excipient, or diluent. Carriers, excipients, and diluents are discussed in further detail herein.
[00141] In certain embodiments, a composition comprises two or more different antibodies or antigen binding fragments according to the present disclosure. In certain embodiments, antibodies or antigen binding fragments to be used in a combination each independently have one or more of the following characteristics: neutralize one, two, three, four, five, or more naturally occurring sarbecovirus variants; do not compete with one another for Spike protein binding; bind distinct sarbecovirus Spike protein epitopes; have a reduced formation of resistance to sarbecovirus; when in a combination, have a reduced formation of resistance to sarbecovirus; potently neutralize one, two, three, four, five or more live betacoronaviruses; exhibit additive or synergistic effects on neutralization of one, two, three, four, five or more or more live sarbecoviruses when used in combination; exhibit effector functions; are protective in relevant animal model(s) of An active agent can be administered by any suitable means, including topical, oral, parenteral, intrapulmonary, and intranasal. Parenteral infusions include intramuscular, intravenous (bolus or slow drip), intraarterial, intraperitoneal, intrathecal or subcutaneous administration. An agent can be administered in any manner which is medically acceptable. This may include injections, by parenteral routes such as intravenous, intravascular, intraarterial, subcutaneous, intramuscular, intratumor, intraperitoneal, intraventricular, intraepidural, or others as well as oral, nasal, ophthalmic, rectal, or topical. Sustained release administration is also specifically included in the disclosure, by such means as depot injections or erodible implants.
[00142] As noted above, an antibody can be formulated with an a pharmaceutically acceptable carrier (one or more organic or inorganic ingredients, natural or synthetic, with which a subject agent is combined to facilitate its application). A suitable carrier includes sterile saline although other aqueous and non-aqueous isotonic sterile solutions and sterile suspensions known to be pharmaceutically acceptable are known to those of ordinary skill in the art. An "effective amount" refers to that amount which is capable of ameliorating or delaying progression of the diseased, degenerative or damaged condition. An effective amount can be determined on an individual basis and will be based, in part, on consideration of the symptoms to be treated and results sought. An effective amount can be determined by one of ordinary skill in the art employing such factors and using no more than routine experimentation.
[00143] An antibody can be administered as a pharmaceutical composition comprising a pharmaceutically acceptable excipient. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically-acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.
[00144] As used herein, compounds which are "commercially available" may be obtained from commercial sources including but not limited to Acros Organics (Pittsburgh PA), Aldrich Chemical (Milwaukee Wl, including Sigma Chemical and Fluka), Apin Chemicals Ltd. (Milton Park UK), Avocado Research (Lancashire U.K.), BDH Inc. (Toronto, Canada), Bionet (Cornwall, U.K.), Chemservice Inc. (West Chester PA), Crescent Chemical Co. (Hauppauge NY), Eastman Organic Chemicals, Eastman Kodak Company (Rochester NY), Fisher Scientific Co. (Pittsburgh PA), Fisons Chemicals (Leicestershire UK), Frontier Scientific (Logan UT), ICN Biomedicals, Inc. (Costa Mesa CA), Key Organics (Cornwall U.K.), Lancaster Synthesis (Windham NH), Maybridge Chemical Co. Ltd. (Cornwall U.K.), Parish Chemical Co. (Orem UT), Pfaltz & Bauer, Inc. (Waterbury CN), Polyorganix (Houston TX), Pierce Chemical Co. (Rockford IL), Riedel de Haen AG (Hannover, Germany), Spectrum Quality Product, Inc. (New Brunswick, NJ), TCI America (Portland OR), Trans World Chemicals, Inc. (Rockville MD), Wako Chemicals USA, Inc. (Richmond VA), Novabiochem and Argonaut Technology.
[00145] The antibodies are incorporated into a variety of formulations for therapeutic administration. In one aspect, the agents are formulated into pharmaceutical compositions by combination with appropriate, pharmaceutically acceptable carriers or diluents, and are formulated into preparations in solid, semi-solid, liquid or gaseous forms, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres, and aerosols. As such, administration of the active agents and/or other compounds can be achieved in various ways. The active agents and/or other compounds may be systemic after administration or may be localized by virtue of the formulation, or by the use of an implant that acts to retain the active dose at the site of implantation.
[00146] In pharmaceutical dosage forms, the antibodies may be administered in the form of their pharmaceutically acceptable salts, or they may also be used alone or in appropriate association, as well as in combination with other pharmaceutically active compounds. The agents may be combined, as previously described, to provide a cocktail of activities. The following methods and excipients are exemplary and are not to be construed as limiting the invention.
[00147] Formulations may be typically provided in a unit dosage form, where the term "unit dosage form," refers to physically discrete units suitable as unitary dosages for human subjects, each unit containing a predetermined quantity of active agent in an amount calculated sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms of the present invention depend on the particular antibodies employed and the effect to be achieved, and the pharmacodynamics associated with each complex in the host.
[00148] The pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, are commercially available. Moreover, pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are commercially available. Any compound useful in the methods and compositions of the invention can be provided as a pharmaceutically acceptable base addition salt. "Pharmaceutically acceptable base addition salt" refers to those salts which retain the biological effectiveness and properties of the free acids, which are not biologically or otherwise undesirable. These salts are prepared from addition of an inorganic base or an organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, the sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Preferred inorganic salts are the ammonium, sodium, potassium, calcium, and magnesium salts. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins and the like. Particularly preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.
[00149] Depending on the patient and condition being treated and on the administration route, the antibodies may be administered in dosages of 0.01 mg to 500 mg /kg body weight per day, e.g. from 0.5 mg/kg, 1 mg/kg, 2.5 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 15 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 40 mg/kg, 50 mg/kg; or more. Dosages will be appropriately adjusted for pediatric formulation.
[00150] Generally, a therapeutically effective daily dose of an antibody or antigen binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.e., 1 .75 g). For polynucleotides, vectors, host cells, and related compositions of the present disclosure, a therapeutically effective dose may be different than for an antibody or antigen binding fragment.
[00151] In some embodiments, pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes).
[00152] A carrier may bear the agents in a variety of ways, including covalent bonding either directly or via a linker group, and non-covalent associations. Suitable covalent-bond carriers include proteins such as albumins, peptides, and polysaccharides such as aminodextran, each of which have multiple sites for the attachment of moieties. The nature of the carrier can be either soluble or insoluble for purposes of the invention.
[00153] Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyidimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEENTM, PLURONICSTM or polyethylene glycol (PEG). Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes.
[00154] Compositions can be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-1 19, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. [00155] Toxicity of the active agents can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD100 (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in further optimizing and/or defining a therapeutic dosage range and/or a sub-therapeutic dosage range (e.g., for use in humans). The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition.
Methods of Use
[00156] Methods for use of an antibody or antigen binding fragment, nucleic acid, vector, cell, or composition of the present disclosure include the diagnosis and treatment of a sarbecovirus infection (e.g, in a human subject, or in a sample obtained from a human subject).
[00157] Methods of diagnosis (e.g, in vitro, ex vivo ) may include contacting an antibody, antibody fragment (e.g., antigen binding fragment) with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood. The methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody or antibody fragment with a sample. Such a detection step can be performed at the bench, i.e. without any contact to the human or animal body. Examples of detection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay), including direct, indirect, and sandwich ELISA.
[00158] Also provided are methods of treating a subject using an antibody or antigen binding fragment of the present disclosure, or a composition comprising the same, wherein the subject has, is believed to have, or is at risk for having an infection by a sarbecovirus, including, for example, a variant of SARS-CoV-2.
[00159] "Treat," "treatment," or "ameliorate" refers to medical management of a disease, disorder, or condition of a subject (e.g, a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). In general, an appropriate dose or treatment regimen comprising an antibody or composition of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease- free status; diminishment of extent of disease, stabilization of disease state; delay or prevention of disease progression; remission; survival; prolonged survival; or any combination thereof.
[00160] In certain embodiments, therapeutic or prophylactic/preventive benefit includes reduction or prevention of hospitalization for treatment of a sarbecovirus infection (i.e., in a statistically significant manner). In certain embodiments, therapeutic or prophylactic/preventive benefit includes a reduced duration of hospitalization for treatment of a sarbecovirus infection (i.e., in a statistically significant manner). In certain embodiments, therapeutic or prophylactic/preventive benefit includes a reduced or abrogated need for respiratory intervention, such as intubation and/or the use of a respirator device. In certain embodiments, therapeutic or prophylactic/preventive benefit includes reversing a late-stage disease pathology and/or reducing mortality.
[00161 ] A "therapeutically effective amount" or "effective amount" of an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition of this disclosure refers to an amount of the composition or molecule sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner. When referring to an individual active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially, sequentially, or simultaneously. A combination may comprise, for example, two different antibodies that specifically bind sarbecovirus antigens, which in certain embodiments, may be the same or different sarbecovirus antigens, and/or can comprise the same or different epitopes.
[00162] Accordingly, in certain embodiments, methods are provided for treating a sarbecovirus infection in a subject, wherein the methods comprise administering to the subject an effective amount of an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition as disclosed herein.
[00163] Subjects that can be treated by the present disclosure are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes. Other model organisms, such as mice and rats, may also be treated according to the present disclosure. In any of the aforementioned embodiments, the subject may be a human subject. The subjects can be male or female and can be any suitable age, including infant, juvenile, adolescent, adult, and geriatric subjects. [00164] A number of criteria are believed to contribute to high risk for severe symptoms or death associated with a sarbecovirus infection. These include, but are not limited to, age, occupation, general health, pre-existing health conditions, obesity, and lifestyle habits. In some embodiments, a subject treated according to the present disclosure comprises one or more risk factors.
[00165] In certain embodiments, a human subject treated according to the present disclosure is an infant, a child, a young adult, an adult of middle age, or an elderly person. In certain embodiments, a human subject treated according to the present disclosure is less than 1 year old, or is 1 to 5 years old, or is between 5 and 125 years old ( e.g ., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 1 15, or 125 years old, including any and all ages therein or therebetween). In certain embodiments, a human subject treated according to the present disclosure is 0-19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older. Persons of middle, and especially of elderly age are believed to be at particular risk. In particular embodiments, the human subject is 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older. In some embodiments, the human subject is male. In some embodiments, the human subject is female.
[00166] In certain embodiments, a human subject treated according to the present disclosure is a resident of a nursing home or a long-term care facility, is a hospice care worker, is a healthcare provider or healthcare worker, is a first responder, is a family member or other close contact of a subject diagnosed with or suspected of having a sarbecovirus infection, is overweight or clinically obese, is or has been a smoker, has or had chronic obstructive pulmonary disease (COPD), is asthmatic (e.g., having moderate to severe asthma), has an autoimmune disease or condition (e.g., diabetes), and/or has a compromised or depleted immune system (e.g, due to AIDS/HIV infection, a cancer such as a blood cancer, a lymphodepleting therapy such as a chemotherapy, a bone marrow or organ transplantation, or a genetic immune condition), has chronic liver disease, has cardiovascular disease, has a pulmonary or heart defect, works or otherwise spends time in close proximity with others, such as in a factory, shipping center, hospital setting, or the like.
[00167] In certain embodiments, a subject treated according to the present disclosure has received a vaccine for a sarbecovirus and the vaccine is determined to be ineffective, e.g, by post-vaccine infection or symptoms in the subject, by clinical diagnosis or scientific or regulatory criteria.
[00168] In certain embodiments, treatment is administered as peri-exposure prophylaxis. In certain embodiments, treatment is administered to a subject with mild-to-moderate disease, which may be in an outpatient setting. In certain embodiments, treatment is administered to a subject with moderate-to-severe disease, such as requiring hospitalization. [00169] In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome ( e.g ., a decrease in frequency, duration, or severity of diarrhea or associated dehydration, or inflammation, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder. Prophylactic benefit of the compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.
[00170] Compositions are administered in an effective amount (e.g., to treat a sarbecovirus infection), which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. In certain embodiments, tollowing administration of therapies according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated as compared to placebo-treated or other suitable control subjects.
[00171 ] In certain embodiments, a method comprises administering the antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition to the subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. In certain embodiments, a method comprises administering the antibody, antigen binding fragment, or composition to the subject a plurality of times, wherein a second or successive administration is performed at about 6, about 7, about 8, about 9, about 10, about 1 1 , about 12, about 24, about 48, about 74, about 96 hours, or more, following a first or prior administration, respectively. In certain embodiments, a method comprises administering the antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition at least one time prior to the subject being infected by a sarbecovirus.
[00172] Compositions comprising an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising an antibody or antigen binding fragment of the disclosure and each active agent in its own separate dosage formulation. For example, an antibody or antigen binding fragment thereof as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, an antibody or antigen binding fragment as described herein and the other active agent can be administered to the subject together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions comprising an antibody or antigen binding fragment and one or more additional active agents can be administered at essentially the same time, i.e., concurrently, or at separately staggered times, i.e., sequentially and in any order; combination therapy is understood to include all these regimens.
[00173] In certain embodiments, a combination therapy is provided that comprises one or more anti-sarbecovirus antibody (or one or more nucleic acid, host cell, vector, or composition) of the present disclosure and one or more anti-inflammatory agent and/or one or more anti-viral agent. In particular embodiments, the one or more anti-inflammatory agent comprises a corticosteroid such as, for example, dexamethasone, prednisone, or the like. In some embodiments, the one or more anti-inflammatory agents comprise a cytokine antagonist such as, for example, an antibody that binds to IL6 (such as siltuximab), or to IL-6R (such as tocilizumab), or to IL-lp, IL-7, IL-8, IL-9, IL-10, FGF, G-CSF, GM-CSF, IFN-y, IP-10, MCP-1 , MIP-1 A, MIP1 -B, PDGR, TNF-a, or VEGF. In some embodiments, anti-inflammatory agents such as leronlimab, ruxolitinib and/or anakinra are used. In some embodiments, the one or more anti-viral agents comprise nucleotide analogs or nucelotide analog prodrugs such as, for example, remdesivir, sofosbuvir, acyclovir, and zidovudine.
[00174] In particular embodiments, an anti-viral agent comprises lopinavir, ritonavir, favipiravir, or any combination thereof. Other anti-inflammatory agents for use in a combination therapy of the present disclosure include non-steroidal anti-inflammatory drugs (NSAIDS). It will be appreciated that in such a combination therapy, the one or more antibody (or one or more nucleic acid, host cell, vector, or composition) and the one or more anti-inflammatory agent and/or one or the more antiviral agent can be administered in any order and any sequence, or together.
[00175] In some embodiments, an antibody (or one or more nucleic acid, host cell, vector, or composition) is administered to a subject who has previously received one or more anti inflammatory agent and/or one or more antiviral agent. In some embodiments, one or more anti-inflammatory agent and/or one or more antiviral agent is administered to a subject who has previously received an antibody (or one or more nucleic acid, host cell, vector, or composition). [00176] In certain embodiments, a combination therapy is provided that comprises two or more anti-sarbecovirus antibodies of the present disclosure. A method can comprise administering a first antibody to a subject who has received a second antibody, or can comprise administering two or more antibodies together. For example, in particular embodiments, a method is provided that comprises administering to the subject (a) a first antibody or antigen binding fragment, when the subject has received a second antibody or antigen binding fragment; (b) the second antibody or antigen binding fragment, when the subject has received the first antibody or antigen binding fragment; or (c) the first antibody or antigen binding fragment, and the second antibody or antigen binding fragment.
[00177] In certain embodiments, an antibody, antigen binding fragment, or composition is provided for use in a method of manufacturing or preparing a medicament for treating a sarbecovirus infection in a subject.
EXAMPLES
[00178] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.
[00179] In post-pandemic era, countermeasures of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants and potential future spillover of zoonotic coronavirus into human beings are rare and urgently needed. Herein, we defined the molecular basis of the potent, heterotypic, and durable immune protection of an AS03-adjuvanted subunit vaccine and identified monoclonal antibodies (mAbs), that can recognize diverse conserved cryptic epitopes of the receptor binding domain (RBD) and neutralize potently a broad panel of clade I sarbecoviruses. Longitudinal analysis revealed that there was a continuing memory B cell evolution within one year after a 2-dose primary vaccination, evidenced by increasing somatic hypermutation and amino acid length of the complementarity-determining region 3 (CDR3), while a booster did not drive a further evolution. Accompanied with the memory B cell evolution, mAbs matured over time at a similar pace and potent broad neutralizing monoclonal antibodies (bnAbs) were identified early at 5~6 months, underlying the cellular and molecular basis of the rapid recall of broad antibody response to the booster vaccination. 21 B6 and 27A12 potently neutralize SARS-CoV-2 and all variants of concern, including Omicron subvariants BA.1 , BA.2, BA.3, BA.4/5 and BA.2.75. Remarkably, 25F9 and 20A7 neutralize authentic clade I sarbecoviruses beyond SARS-CoV-2 with ultrapotentcy (IC50s against SARS-CoV: 0.00085 ug/ml, 0.013 ug/ml; WIV-1 : 0.003 ug/ml, 0.002 ug/ml; SHC014: 0.006 ug/ml, 0.018 ug/ml, Pangolin: 0.006 ug/ml, 0.345 ug/ml, respectively). Altogether, our data filled a knowledge gap of antibody evolution to the AS03 adjuvanted subunit COVID-19 vaccine and we generated potent broad sarbecovirus neutralizing antibodies with great potential for translational application and guidance for the next-generation pan-sarbecovirus vaccine design.
General methods employed in assays
[00180] Antigen-specific memory B cell staining and single-cell sorting. Cryopreserved PBMCs were thawed and washed twice with 10 mL of FACS buffer (1 x PBS containing 2% FBS and 1 mM EDTA) and resuspended in 100 uL of 1 x PBS containing Zombie UV live/dead dye at 1 :200 dilution (BioLegend, 423108) and incubate at room temperature for 15 minutes. Following washing, cells were incubated with an antibody cocktail for 1 hour protected from light on ice. The following antibodies were used: IgD PE (Southern Biotech, 2030-09), IgM PerCP-Cy5.5 (BioLegend, 314512), CD20 APC-H7 (BD Biosciences, 560734), CD27 PE-Cy7 (BioLegend, 302838), CD14 BV650 (BioLegend, 301836), CD16 BV650 (BioLegend, 302042), IgG BUV496 (BD Biosciences, 741 172), CD3 BV650 (BD Biosciences, 563916), CD21 PE- CF594 (BD Biosciences, 563474) and Alexa Fluor 488-labeled Beta spike (antibodies-online, ABIN6963740), Alexa Fluor 647 labeled Omicron spike (SinoBiological, 40589-V08H26) and BV421 labeled Wuhan Spike (SinoBiological, 40589-V27B-B). All antibodies were used as the manufacturer's instruction and the final concentration of each probe was 0.1 ug/ml. Single CD3- CD14 CD16- CD20+ IgM- IgD- lgG+ Probe-i- B cells were sorted into individual wells of 96-well plates containing 16 pl of lysis buffer per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis. The sorted cells were immediately frozen on dry ice, and immediately used for subsequent RNA reverse transcription.
[00181 ] Generation of recombinant human monoclonal antibodies. Monoclonal antibodies were generated following established protocols. In brief, single memory B cells will be sorted with BD FACS Aria II into 96-well plates containing 16 ul of lysis buffer. The lysis buffer is composed of 20 U RNAse inhibitor (Invitrogen), 5 mM DTT (Invitrogen), 4 ul 5x RT buffer (Invitrogen), 0.0625 ul Igepal (Sigma), 10 ug/ml Carrier RNA (Applied Biosystems). The 96- well plates will go through a quick freeze-thaw cycle, and 0.5 ug Oligo(dT)18 (Thermo Scientific), 0.5 mM dNTP mix (Invitrogen), and 200 U Superscript IV (Invitrogen) will be added in a total volume of 4 ul followed by thorough mixing and spinning. The reverse transcription will be performed as follows: 10 min at 42 eC, 10 min at 23 eC, 20 min at 50 SC, 5 min at 55 SC, 10 min at 80 aC and finally cooling to 4 aC. Ig heavy chain and light chain (kappa/lambda) rearrangements will be amplified by nested PCR (HotStarTaq DNA Polymerase, QIAGEN) using primer cocktails specific for all V gene families and constant domains at a concentration of 250 nM per primer. The PCR mix will consist of 2.5 ul 10x PCR buffer, 0.5 ul 10 mM dNTP mix (Invitrogen), 0.5 ul 25 mM MgCI2 (only added in the first round PCR), 5 ul Q-solution, 1 U HotStarTaq, 0.5 ul 5’ and 3’ primers. Water will be added up to a total volume of 25 ul. The primer list and PCR program were shown in Extended Fig 8. The 2nd round PCR products will be evaluated on 2% agarose gels, purified using QIAquick spin columns (Qiagen) and sequenced using 2nd round PCR reverse primers. The sequences were analyzed using the online IMGT/HighV-QUEST tool. The productive heavy-light paired Ig genes will be used for antibody production (Sino Biological).
[00182] Meso-scale electrochemiluminescence immunoassay (ECLIA). V-plex COVID-19 panels from Mesoscale Discovery were used to evaluate antibodies binding to spikes of SARS-CoV-2, variants and other coronaviruses. The assay was performed as per the manufacturer’s instructions. Briefly, the multi-spot 96 well plates were blocked in 0.15 ml of blocking solution with shaking at 700 rpm at room temperature. Immediately after 30 min of blocking, 50 ul of monoclonal antibodies or plasma samples were added to each plate in the designated wells and incubated at room temperature for 2 h with shaking. Plasma samples were assayed at a 1 :100 starting dilution and 7 additional 5-fold serial dilutions. Monoclonal antibodies were assayed at 10 ug/ml starting concentration and 1 1 additional 4-fold serial dilutions. Plates were washed and 50 ul of Sulfo-tag conjugated anti-IgG was added, and the plates were incubated at room temperature for 1 h. After incubation, the plates were washed and 0.15 ml of MSD-Gold read buffer was added. The plates were immediately read using the MSD instrument. The binding capacities of plasma or monoclonal antibodies were presented as MSD area under curve (AUC), calculated using Prism v9.3.1 (GraphPad).
[00183] ELISA. All monoclonal antibodies generated in this study were screened for their binding to the spikes of SARS-CoV-2 Wuhan strain, BA.1 strain and BA.4/5 by ELISA. Briefly, 96-well plates were coated with 50 pl per well of a 1 pg ml-1 protein solution in PBS overnight at 4 °C. Plates were washed 3 times with washing buffer (1 x PBS with 0.05% Tween-20 (MP Biomedicals)) and incubated with 200 pl per well blocking buffer (1 x PBS with 5% skim milk powder (Bio-Rad) and 0.05% Tween-20 (MP Biomedicals)) for 1 h at room temperature. Immediately after blocking, 100 ul of monoclonal antibodies was added to each plate in the designated wells and incubated at room temperature for 1 h. Monoclonal antibodies were assayed at 1 ug/ml starting concentration and 5 additional 5-fold serial dilutions. Plates were washed and 100 ul of horseradish peroxidase (HRP) conjugated anti-human IgG (Sigma) in blocking buffer at a 1 :5000 dilution was added, and the plates were incubated at room temperature for 1 h. After incubation, the plates were washed three times and 100 ul per well of TMB (Sigma) was added. The reaction was developed for 4 min and stopped by adding 100 ul per well of 450 nm stop solution (Abeam). The plates were immediately read using an ELISA microplate reader (Bio-Rad). The binding capacities of monoclonal antibodies were presented as ELISA area under curve (AUG), calculated using Prism v9.3.1 (GraphPad).
[00184] Detection of the affinity of SARS-CoV spike/RBD with monoclonal antibodies by ForteBio’s Octet RH16 system. Monoclonal antibodies were diluted 2 pg/mL with PBST buffer and SARS-CoV spike/RBD protein were diluted to 377.4 nM, 188.7 nM, 94.3 nM, 47.2 nM, 23.6nM and 0 nM with PBST buffer respectively. After all mAbs were captured by proA biosensor in PBST buffer SARS-CoV spike/RBD protein was incubated with each mAbs respectively. The fully reacted solid phase conjugate was dissociated in PBST buffer. The results were analyzed by Data Analysis 12.0 software to get association rate constants, dissociation rate constants and affinity constants.
[00185] Pseudovirion neutralization assay (PsVNA). For initial screening, neutralization assay using pseudotyped viruses (using HIV backbone) carrying spikes of SARS-CoV-2 wild type strain, BA.1 strain and BA.4/5 strain respectively was performed by Sino Biological Inc. Briefly, 293T cells overexpressing ACE2 were seeded in 96-well plates (Costar, 3955) at 3 x 104 cells/well. Fifty microlitre serially diluted mAbs or a control monoclonal antibody (S2H97) were mixed with 50 pL pseudovirus and then they were added into plates seeded with 293T-ACE2 cells (1 x 104 TCID50/mL). Positive control was set up using a mixture of 50 pL DMEM medium and 50 pL pseudovirus. 100 pL DMEM was used as a negative control. Then cells were cultured at 37 °C for 64 h. Luminous value was detected by Luminometer (Berthold Technologies, Centro LB 960) and the inhibitory rate was calculated by (1 - (mean RLU of sample - RLU of negative control)/(RLU of positive control - RLU of negative control)*100%). Experiments were performed in duplicate, value = mean ± SD.
[00186] The 15 most potent BA.1 neutralizing antibodies were evaluated in a qualified SARS- CoV- 2 pseudovirion neutralization assay (PsVNA). SARS-CoV-2 neutralizing activity measured by PsVNA correlates with PRNT (plaque reduction neutralization test with authentic SARS-CoV-2 virus) in previous studies (Neerukonda et al., 2021 ; Ravichandran et al., 2020; Tang et al., 2021 ).
[00187] Pseudovirions were produced as previously described (Neerukonda et al., 2021 ). Briefly, human codon-optimized cDNA encoding SARS-CoV-2 spike glycoprotein of the WA- 1 /2020 and variants were synthesized by GenScript and cloned into eukaryotic cell expression vector pcDNA 3.1 between the BamHI and Xhol sites. Pseudovirions were produced by cotransfection Lenti - X 293T cells with psPAX2(gag/pol), pTrip-luc lentiviral vector and pcDNA 3.1 SARS-CoV-2-spike-deltaC19, using Lipofectamine 3000. The supernatants were harvested at 48h post transfection and filtered through 0.45pm membranes and titrated using 293T-ACE2-TMPRSS2 cells (HEK 293T cells that express ACE2 and TMPRSS2 proteins).
[00188] Neutralization assays were performed as previously described (Ravichandran et al., 2020; Tang et al., 2021 ). For the neutralization assay, 50 pL of SARS-CoV-2 S pseudovirions (counting -200,000 relative light units) were pre-incubated with an equal volume of medium containing serial dilutions of MAbs at room temperature for 1 h. Then 50 pL of virus-antibody mixtures were added to 293T-ACE2-TMPRSS2 cells (104 cells/50 pL) in a 96-well plate. The input virus with all SARS-CoV-2 strains used in the current study were the same (2 x 105 relative light units/50 pL/well). After a 3 h incubation, fresh medium was added to the wells. Cells were lysed 24 h later, and luciferase activity was measured using One-Gio luciferase assay system (Promega, Cat# E6130). The assay of each serum was performed in duplicate, and the 50% neutralization titer was calculated using Prism 9 (GraphPad Software). Controls included cells only, virus without any antibody. Neutralization was determined using SARS- CoV-2-Nluc, an infectious clone of SARS-CoV-2 (based on strain 2019- nCoV/USA_WA1/2020) which encodes nanoluciferase in place of the viral ORF7 and demonstrated comparable growth kinetics to wildtype virus (35). Vero E6 cells were seeded into black-walled, clear-bottom 96-well plates at 2 x 104 cells/well and cultured overnight at 37°C. The next day, 9-point 4-fold serial dilutions of mAbs were prepared in infection media (DMEM + 10% FBS). SARS-CoV-2-Nluc was diluted in infection media at a final MOI of 0.1 PFU/cell, added to the mAb dilutions and incubated for 30 minutes at 37”C. Media was removed from the Vero E6 cells, mAb-virus complexes were added and incubated at 37°C for 6 hours. Media was removed from the cells, Nano-Gio luciferase substrate (Promega) was added according to the manufacturer’s recommendations, incubated for 10 minutes at room temperature and the luciferase signal was quantified on a VICTOR Nivo plate reader (Perkin Elmer).
Results
[00189] Isolation of monoclonal antibodies from vaccinated rhesus macaque. Rhesus macaques received two doses of AS03 adjuvanted RBD-NP (N=5) or Hexapro-NP (N=6) and got a booster with AS03 adjuvanted RBD (beta)-NP one year later after the primary vaccination and blood samples were collected at regular intervals (Figure 1 ). We used flow cytometry to sort antigen specific lgG+ memory B cells (MBCs) (Figure 2). We generated 514 monoclonal antibodies (mAbs) from single sorted antigen specific lgG+ MBCs at indicated timepoints and carried out functional characterization in a pyramid structure (Figure 3). In total, -83.1% mAbs bound to the SARS-CoV-2 Wuhan spike measured by enzyme-linked immunosorbent assay (ELISA) (Figure 4). To determine the neutralization, the top 100 Omicron cross-reactive mAbs were scoped out for neutralization screening against pseudotyped SARS-CoV-2 Wuhan, BA.1 , and BA.4/5 strain, respectively (Figure 5). [00190] Potent broad sarbecovirus neutralizing antibodies. Currently, only one authorized or approved therapeutic mAb, Bebtelovimab (LY-CoV14041), remains effective against Omicron subvariants, BA.1 , BA.2, BA.3, BA.2.13, BA.2.12.1 , BA.4/5, which, however, cannot prevent potential spill-over from other coronaviruses, like SARS-CoV.2 To identify neutralizing antibodies with both high potency and pan-sarbecovirus breadth, we scrutinized the neutralizing antibodies isolated on 1.4 months and 5~6 months. We selected 15 mAbs showing better potency against SARS-CoV-2 Wuhan (Figure 6) and BA.1 (Figure 7) than that of a control antibody, S2H973, in a side-by-side comparison. Interestingly, only two mAbs 20F2 and 27E3 lost neutralization against the current dominant SARS-CoV-2 BA.4/5 strain (Figure 8).
[00191 ] We next determined the binding capacities and cross-reactivities of these 15 mAbs by ELISA and ECLIA. To test whether these 15 mAbs bind to SARS-CoV-2 Omicron variants, we carried out ELISA against the spikes of SARS.CoV-2 Wuhan, BA.1 and BA.4/5 strain, respectively. All 15 mAbs, 20A7, 27A12, 27E3, 21 B6, 27E4, 26C3, 15F1 , 25C7, 25F9, 25A10, 20F2, 21 F2, 25A1 1 , 26G10, 20C3 showed remarkable low EC50s (Figure 9). Notably, these mAbs bound to SARS-CoV-2 Omircon BA.1 spike with EC50s as comparable as those to SARS-CoV-2 Wuhan spike (Figure 10). And only two mAbs, 26C3 and 27E3, showed reduced and completely loss of binding against the spike of SARS-CoV-2 Omicron BA.4/5 strain (Figure 11 ). Intrestingly, even though 26C3 showed a weak binding, it can neutralize BA.4/5 with a IC50 of 31 .14 ug/ml. We next applied meso scale ECLIA asaays to test the crossreactivities. 4 mAbs, 20A7, 20F2, 20C3, 21 B6, that were isolated from RBD-NP vaccaniated animals displayed extremely high binding against RBDs of SARS-CoV-2 Wuhan RBD and 9 other different variants in a MSD SARS-CoV-2 RBD variants panel 9 (Figure 12). 1 1 mAbs, 15F1 , 21 F2, 25A10, 25A1 1 , 25C7, 25F9, 26C3, 26G10, 27A12, 27E3, 27E4, that were isolated from Hexapro-NP vaccaniated animals using a MSD SARS-CoV-2 spike variants panel 13. Strinkly, 15F1 , 21 F2, 25A10, 25A11 , 25C7, 25F9, 26C3, 26G10, 27A12, 27E3, 27E4 displayed extremely high binding against spikes of SARS-CoV-2 Wuhan spike and 9 other different variants (Figure 13). Remarkably, we found 8 mAbs, 27E4, 26G10, 20A7, 25F9, 15F1 , 25C7, 20F2, 20C3, bound to SARS-CoV spike efficiently (Figure 14), suggesting these mAbs could be pan-sarbecovirus neutralizing antibodies. To further confirm the binding of these 8 mAbs against SARS-CoV, we carried out BLI assay against SARS-CoV RBD. Consistently, mAbs 27E4, 26G10, 20A7, 25F9, 15F1 , 25C7, 20F2, 20C3 displayed extraordinary high affinity against SARS-CoV with Kds of 12.4 nM, 0.57 nM, 9.39 nM, 0.015 nM, 6.38 nM, 1.62 nM, 3.35 nM, 1 nM, respectively (Figure 15).
[00192] The potency and breadth of these 15 mAbs were further confirmed by a panel of 11 pseudoviruses carrying spikes of SARS-CoV-2 WA1 , 9 SARS-CoV-2 variants of concern, and SARS-CoV. Consistent with our first screening of neutralization assay, all 15 mAbs displayed high potency against SARS-CoV-2 WA1 strain, especially 27E3, 21 B6, 27A12, 27E4, 26C3, 25F9, 25C7, 20A7, 15F1 (Figure 16). In the neutralization against SARS-CoV-2 Alpha strain, all 15 mAbs showed comparable potency compared with those against WA1 strain (Figure 17). In the neutralization against SARS-CoV-2 Beta strain, all 15 mAbs showed comparable potency compared with those against WA1 strain, except 20F2 showed (Figure 18). In the neutralization against SARS-CoV-2 gamma strain, all 15 mAbs showed comparable potency compared with those against WA1 strain (Figure 19). In the neutralization against SARS-CoV- 2 Delta strain, all 15 mAbs showed comparable potency compared with those against WA1 strain, except 20F2 (Figure 20). In the neutralization against SARS-CoV-2 BA.1 strain, all 15 mAbs showed comparable potency compared with those against WA1 strain (Figure 21 ). In the neutralization against SARS-CoV-2 BA.2 strain, all 15 mAbs showed comparable potency compared with those against WA1 strain, except 15F1 and 20F2 (Figure 22). In the neutralization against SARS-CoV-2 BA.3 strain, all 15 mAbs showed comparable potency compared with those against WA1 strain, except 15F1 , 25A1 1 and 27E4 (Figure 23). In the neutralization against SARS-CoV, five mAbs 15F1 , 20A7, 20C3, 25F9, 26G10 showed neutralization with IC50s of 0.837 ug/ml, 1.874 ug/ml, 5.245 ug/ml, 0.108 ug/ml, 5.682 ug/ml, respectively (Figure 24).
[00193] To further examine breadth of the pan-sarbecovirus neutralization of those mAbs, we extended our pseudovirues panel with another 3 clade 1 sarbecoviruses, WIV-1 , RaTg13, SHC014. Remarkablely, 4 bnAbs, 15F1 , 20A7, 25F9, 27A12 neutralized WIV-1 with IC50s of 0.014 ug/ml, 0.035 ug/ml, 0.041 ug/ml, 1.958 ug/ml, respectively (Figure 25). bnAbs 20A7, 25F9, 26C3, 27E3, 27E4 can potently neutralize RaTG13 with IC50s of 0.147 ug/ml, 0.021 ug/ml, 0.166 ug/ml, 0.014 ug/ml, 0.577 ug/ml, respectively (Figure 26). bnAbs 15F1 , 20A7, 25F9, 27A12, 27E4 neutralized SHC014 with IC50s of 0.039 ug/ml, 0.014 ug/ml, 0.014 ug/ml, 0.079 ug/ml, 0.014 ug/ml, respectively (Figure 27). Remarkably, 25F9 and 20A7 neutralize authentic clade I sarbecoviruses beyond SARS-CoV-2 with ultrapotentcy (IC50s against SARS-CoV: 0.00085 ug/ml, 0.013 ug/ml; WIV-1 : 0.003 ug/ml, 0.002 ug/ml; SHC014: 0.006 ug/ml, 0.018 ug/ml, Pangolin: 0.006 ug/ml, 0.345 ug/ml, respectively) (Figure 28). In summary, we identified 15 potent broad sarbecovirus neutralizing monoclonal antibodies, which represented the top bnAbs up to date.
References
1 Westendorf, K. et al. LY-CoV1404 (bebtelovimab) potently neutralizes SARS-CoV-2 variants. Cell Rep 39 (2022). 110812
10.1016/j.celrep.2022.110812
2 Cao, Y. L. et al. BA.2.12.1 , BA.4 and BA.5 escape antibodies elicited by Omicron infection. Nature 608, 593-+ (2022).
3 Starr, T. et al. SARS-CoV-2 RBD antibodies that maximize breadth and resistance to escape. Nature 597, 97-+ (2021 ).

Claims

T HAT WHICH IS CLAIMED IS:
1 . An isolated antibody capable of binding to a surface glycoprotein of at least two sarbecoviruses selected from SARS-CoV-2, SARS-CoV-2 omicron, SARS-CoV-2 beta, SARS-CoV, WIV-1 , RaTG13, and SCH014.
2. The antibody of claim 1 , comprising the three CDRs of a VH sequence provided in one of SEQ ID NO: 5, 21 , 37, 53, 69, 85, 101 , 117, 133, 149, 165, 181 , 197, 213, 229, respectively and/or the three CDRs of a VL sequence provided in one of SEQ ID NO.:13, 29, 45, 61 , 77, 93, 109, 125, 141 , 157, 173, 189, 205, 221 , 237, respectively.
3. The antibody of claim 1 or claim 2, comprising an Fc sequence other than a native Fc sequence.
4. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:6, 7, 8 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:14, 15, 16 in a light chain variable sequence.
5. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO: 22, 23, 24 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO: 30, 31 , 32 in a light chain variable sequence.
6. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO: 38, 39, 40 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO: 46, 47, 48 in a light chain variable sequence.
7. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:54, 55, 56 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:62, 63, 64 in a light chain variable sequence.
8. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NQ:70, 71 , 72 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:78, 79, 80 in a light chain variable sequence.
9. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:86, 87, 88 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:94, 95, 96 in a light chain variable sequence.
10. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:102, 103, 104 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:110, 111 , 112 in a light chain variable sequence.
11 . The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:118, 119, 120 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:126, 127, 128 in a light chain variable sequence.
12. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:134, 135, 136 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:142, 143, 144 in a light chain variable sequence.
13. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NQ:150, 151 , 152 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:158, 159, 160 in a light chain variable sequence.
14. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:166, 167, 168 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:174, 175, 176 in a light chain variable sequence.
15. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:182, 183, 184 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NQ:190, 191 , 192 in a light chain variable sequence.
16. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:198, 199, 200 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NQ:206, 207, 208 in a light chain variable sequence.
17. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NO:214, 215, 216 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:222, 223, 224 in a light chain variable sequence.
18. The antibody of any of claims 1 -3, comprising the three CDR sequences of SEQ ID NQ:230, 232, 232 in a heavy chain variable region sequence; and the three CDR sequences of SEQ ID NO:238, 239, 240 in a light chain variable sequence.
19. The antibody of any of claims 1-18, wherein the antibody is a chimeric or humanized antibody.
20. The antibody of claim 19, wherein the antibody is a humanized monoclonal antibody.
21 . A polynucleotide encoding an antibody set forth in any of claims 1 -20.
22. A cell that produces an antibody set forth in any of claims 1 -20.
23. A pharmaceutical composition comprising an antibody set forth in any of claims 1- 20.
24. The pharmaceutical composition of claim 23, comprising a pharmaceutically acceptable excipient.
25. A method of preventing or treating a sarbecovirus infection in an individual, the method comprising: administering to an individual a composition comprising an antibody set forth in any of claims 1 -20 in a dose effective to prevent or treat infection.
26. The method according to claim 25, wherein the individual is human.
27. A method of detecting the presence of a sarbecovirus spike protein in a biological sample or tissue, the method comprising: contacting the biological sample or tissue with the antibody set forth in any of claims 1 -20, and determining the presence or absence of antibody bound to the biological sample or tissue.
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