WO2021254403A1 - Methods and compositions related to neutralizing antibodies against human coronavirus - Google Patents

Methods and compositions related to neutralizing antibodies against human coronavirus Download PDF

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Publication number
WO2021254403A1
WO2021254403A1 PCT/CN2021/100414 CN2021100414W WO2021254403A1 WO 2021254403 A1 WO2021254403 A1 WO 2021254403A1 CN 2021100414 W CN2021100414 W CN 2021100414W WO 2021254403 A1 WO2021254403 A1 WO 2021254403A1
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seq
sequence
hcvr
lcvr
antigen
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PCT/CN2021/100414
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French (fr)
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Liang SCHWEIZER
Kun Sun
Lisu HUANG
Bingqing Shen
Mingjie Chen
Yun-Yueh LU
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Hifibio (Hangzhou) Co., Ltd.
Hifibio (Hk) Limited
Xinhua Hospital Affiliated To Shanghai Jiaotong University School Of Medicine
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Publication of WO2021254403A1 publication Critical patent/WO2021254403A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • 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
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • Coronavirus Disease 2019 2019 (COVID-19) , caused by the novel SARS-CoV-2 coronavirus, has quickly grown into a global pandemic and a major public health crisis. Little is known about the immune response generated against this human coronavirus by infected patients. Thus, a better understanding of the pathological mechanisms caused by the virus, and development of new therapeutic agents against it are urgently needed.
  • the immune system generates a vast repertoire of antibodies in response to infection or immunization that can potentially be explored for diagnostic, therapeutic or research applications.
  • IgGs are secreted by plasmablasts and plasma cells.
  • the target-specific IgGs are matured during ongoing immune responses facilitating a rapid recall of previously-elicited protective immunity in response to repeated pathogen or immunogen exposure.
  • the phenotypic diversity of the target-specific IgG repertoires underlies protection following vaccination or infection, and results from the diversity of IgG sequences in the repertoires. Analyzing the activity and sequence of IgGs secreted by plasmablasts and plasma cells is hence of great interest both to investigate their pivotal role in the humoral immune response and to obtain antibodies for therapeutic or other applications.
  • FACS fluorescence-activated cell sorting
  • a system that couples high-throughput single-cell phenotypic screening with paired V H -V L sequencing of IgG secreting primary cells for both soluble and membrane-bound antigens in a flexible format enables direct screening for desired functional activities.
  • One aspect of the invention provides an isolated or recombinantly produced monoclonal antibody, or an antigen-binding fragment thereof, wherein said monoclonal antibody or antigen-binding fragment thereof is specific for an antigen of SARS-CoV-2, and wherein said monoclonal antibody comprises: (1a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 1, a HCVR CDR2 sequence of SEQ ID NO: 2, and a HCVR CDR3 sequence of SEQ ID NO: 3; and, (1b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 4, a LCVR CDR2 sequence of SEQ ID NO: 5, and a LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 11, a HCVR CDR2 sequence of S
  • the HCVR sequence is SEQ ID NO: 7; and/or, (1B) the LCVR sequence is SEQ ID NO: 8, or, (2A) the HCVR sequence is SEQ ID NO: 17; and/or, (2B) the LCVR sequence is SEQ ID NO: 18, or, (3A) the HCVR sequence is SEQ ID NO: 27; and/or, (3B) the LCVR sequence is SEQ ID NO: 28, or, (4A) the HCVR sequence is SEQ ID NO: 37; and/or, (4B) the LCVR sequence is SEQ ID NO: 38; (5A) the HCVR sequence is SEQ ID NO: 57; and/or, (5B) the LCVR sequence is SEQ ID NO: 58, or, (6A) the HCVR sequence is SEQ ID NO: 67; and/or, (6B) the LCVR sequence is SEQ ID NO: 68,
  • the monoclonal antibody has: (1a) a heavy chain sequence of SEQ ID NO: 9; and/or, (1b) a light chain sequence of SEQ ID NO: 10, or, (2a) a heavy chain sequence of SEQ ID NO: 19; and/or, (2b) a light chain sequence of SEQ ID NO: 20, or, (3a) a heavy chain sequence of SEQ ID NO: 29; and/or, (3b) a light chain sequence of SEQ ID NO: 30, or, (4a) a heavy chain sequence of SEQ ID NO: 39; and/or, (4b) a light chain sequence of SEQ ID NO: 40; (5a) a heavy chain sequence of SEQ ID NO: 59; and/or, (5b) a light chain sequence of SEQ ID NO: 60, or, (6a) a heavy chain sequence of SEQ ID NO: 69; and/or, (6b) a light chain sequence of SEQ ID NO: 70, or, (7a) a heavy chain sequence of SEQ ID NO: 79;
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is a human antibody, a CDR-grafted antibody, or a resurfaced antibody.
  • the antigen-binding fragment thereof is an Fab, Fab’, F (ab’) 2 , F d , single chain Fv or scFv, disulfide linked F v , V-NAR domain, IgNar, intrabody, IgG ⁇ CH 2 , minibody, F (ab’) 3 , tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv) 2 , or scFv-Fc.
  • the monoclonal antibody or antigen-binding fragment thereof binds to the S1 glycoprotein of SARS-CoV-2.
  • the monoclonal antibody or antigen-binding fragment thereof binds to the S2 glycoprotein of SARS-CoV-2.
  • the monoclonal antibody or antigen-binding fragment thereof binds the SARS-CoV-2 antigen with a K d of less than about 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or 0.05 nM.
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2.
  • SARS-CoV-2 antigen e.g., the S1 glycoprotein
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 immobilized on a solid support (such as in ELISA assay) , or inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 expressed on the surface of a cell (such as Vero E6 cell) .
  • SARS-CoV-2 antigen e.g., the S1 glycoprotein
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 10 nM, 8 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 0.6 nM or less than 0.5 nM.
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, less than 0.02 nM, or less than 0.01 nM.
  • a target cell such as Vero E6 cell
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 500 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 80 pM, less than 50 pM, less than 30 pM, less than 10 pM, or less than 5 pM.
  • a target cell such as Vero E6 cell
  • Another aspect of the invention provides an isolated or recombinantly produced monoclonal antibody or an antigen-binding fragment thereof, which competes with the isolated monoclonal antibody or antigen-binding fragment thereof of for binding to the same epitope.
  • the invention provides a non-naturally occurring or existing therapeutic antibody based on the antigen-binding sequences of an antibody isolated from the patient using the method of the invention.
  • Such therapeutic antibody may share one or more CDRs, such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences, with the antibody isolated from the patient using the method of the invention.
  • HC-CDR3 heavy chain CDR3 sequences of certain isolated antibodies are listed in FIG. 4B, and in the disclosed sequences herein, see Example 3.
  • Another aspect of the invention provides a mixture of two or more isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention.
  • the proportion of each of said two or more isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is substantially the same, or is different.
  • Another aspect of the invention provides a method of treating or preventing a disease or condition arising from SARS-CoV-2 infection, the method comprising administering to a patient in need thereof an effective amount of the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention, or an effective amount of the mixture of the invention.
  • the method is for treating COVID-19, wherein the method further comprises administering a second therapeutic agent, which may be effective to treat infection by SARS-CoV-2.
  • the second therapeutic agent comprises chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab) , CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFN ⁇ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, Calquence (acalabrutinib) , Xeljanz (tofacitinib) , Jakafi (ruxolitinib) , Olumiant (baricitinib) , Ilaris (canakinumab) , Otezla (apremilast) , Methos
  • the second therapeutic agent comprises one or more of: an anti-viral agent, an antibiotic, an anti-inflammatory agent or DMARD (disease-modifying anti-rheumatic drug) .
  • Another aspect of the invention provides a polynucleotide encoding the heavy chain or the light chain or the antigen-binding portion thereof of the invention.
  • the polynucleotide is codon optimized for expression in a human cell.
  • Another aspect of the invention provides a vector comprising the polynucleotide of the invention.
  • the vector is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector) .
  • an expression vector e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector.
  • Another aspect of the invention provides a host cell comprising the vector of the invention, which expresses said isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof.
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention, or the mixture of the invention.
  • the pharmaceutical composition further comprises a pharmaceutically acceptable excipient or diluent.
  • the pharmaceutical composition is formulated for intravenous administration.
  • FIGs. 1A and 1B show comparison of patient titers against S protein from SARS-CoV-2.
  • patient sera were measured for binding activity to the S1-RBD (receptor binding domain) .
  • FIG. 1B shows classification of patient samples into high, medium, and low titer bins.
  • FIG. 2 shows results of evaluating the various S protein constructs.
  • FIG. 3 shows flow cytometry dot plots of cells from two convalescent patients and one healthy donor, with population of interest highlighted in boxes: CD38 versus CD27 for the two patients shows substantially higher percentage of memory B cells and plasmablasts compared to the healthy donor.
  • FIGs. 4A-4E show identified antibodies from patient 013 serum.
  • FIG. 4A is an anti-hIgG western blot analysis indicating that for the immuno-precipitated patient samples, the full-length antibody was purified from serum. The healthy donor did not show any antigen specific antibody band.
  • FIG. 4B shows the CDR3 diversity of identified antibodies.
  • FIG. 4C is the tandem mass spectrum for first CDR3 peptide for heavy chain F22a1-VH0.
  • FIG. 4D is the tandem mass spectrum for second CDR3 peptide for heavy chain F22a1-VH0.
  • FIG. 4E is the tandem mass spectrum for CDR3 peptide of light chain F22a1-VL0.
  • FIG. 5 shows lead antibody binding characteristics in terms of binding to SARS-CoV-2 S antigen.
  • FIG. 6A shows the blocking activities of the subject antibodies, based on FACS analysis of blocked S1 binding to ACE2, using Vero E6 cell line expressing ACE2.
  • FIG. 6B shows potent neutralizing activities of the subject antibodies for neutralization of pseudovirus entry.
  • FIG. 7A shows the abilities of the subject antibodies to neutralize live SARS-CoV-2 cell entry.
  • fluorescent labeled nuclear protein of SAR-CoV-2 infected cells can be observed with a florescence microscope.
  • the top, middle, and bottom panels are for Ab-2, Ab-3, and Ab-1, respectively.
  • FIG. 7B shows dose response curves for live virus assay performed with three of the subject antibodies.
  • the left, middle, and right panels are for Ab-2, Ab-1, and Ab-3, respectively.
  • FIG. 8A shows binding to full-length S protein (left panel) and inhibition of S1 protein binding to hACE2 (right panel) by Ab-2 (C1S5-2A2A) and variants thereof (C1S5-2A2A-1, -2, -3, -4, -9, -10, -11, -14, -15, -18, and -19) .
  • FIG. 8B shows sequence alignments of Ab-2 and variants thereof.
  • FIG. 9 shows the PK profiles of the subject neutralizing antibodies in mice.
  • FIG. 10 shows binding of the subject antibodies to full-length S protein.
  • the top to bottom labels are for Ab-2, -4, -3, -1, -6, -7, -5, -8, and -9, respectively.
  • One aspect of the invention provides an antibody isolated from a convalescent COVID-19 patient using the method of the invention.
  • sera from convalescent COVID-19 i.e., SARS-CoV-2
  • a source of antiviral antibodies capable of conferring protective immunity on recipients were obtained to identify effective antibodies against antigens of COVID-19 for therapeutic purposes.
  • Antibodies identified from patients infected with the Ebola virus have been used as therapeutic antibodies (Bornholdt et al., 2016; Casadevall &Pirofski, 2020) .
  • the invention provides a non-naturally occurring or existing therapeutic antibody based on the antigen-binding sequences of an antibody isolated from the patient using the method of the invention.
  • Such therapeutic antibody may share one or more CDRs, such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences, with the antibody isolated from the patient using the method of the invention.
  • CDRs such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences
  • HC-CDR3 heavy chain CDR3 sequences of certain isolated antibodies are listed in FIG. 4B, or any of the CDR sequences or combinations thereof disclosed herein.
  • Such antibodies may also be multi-specific (e.g., bi-specific) , having antigen binding sequences originating from different antibody light and/or heavy chains.
  • Another aspect of the invention provides a mixture of the antibodies of the invention. Such a mixture may provide better therapeutic efficacy compared to the individual component antibodies of the mixture.
  • a convalescent patient s sera is a natural mixture of different antibodies against the same or different viral antigen or epitope.
  • Applicant has identified multiple antibodies from the serum of convalescent patients, including 10 antibodies capable of binding to the full length SARS-CoV-2 S protein, among which 8 antibodies recognizes S1 only, and 2 antibodies interacts with S2 only but not with S1.
  • the data indicates that diverse epitopes on SARS-CoV-2 virus spike protein can be targeted by different antibodies, suggesting that the antibodies of the invention, either alone or in combination with antibodies that target different epitopes via different mechanisms, may serve as potent therapeutic agents to treat COVID-19 patients.
  • Another aspect of the invention provides a polynucleotide encoding the heavy or light chain of the antibodies of the invention.
  • Such polynucleotide sequences may be codon optimized for expression in a host cell, such as a mammalian cell line (e.g., CHO cell line) for large scale production of antibody.
  • Another aspect of the invention provides a vector comprising the polynucleotide of the invention.
  • Such vector may be used for expression of antibody in a suitable host cell.
  • a further aspect of the invention provides a host cell comprising the vector of the invention, or producing the antibody of the invention.
  • Yet another aspect of the invention provides a method of treating or preventing a disease or condition arising from SARS-CoV-2 infection, such as COVID-19, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody of the invention, or a mixture thereof.
  • antibody in the broadest sense, encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) .
  • the term “antibody” may also broadly refers to a molecule comprising complementarity determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to an antigen.
  • CDR complementarity determining region
  • the term “antibody” also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.
  • antibody refers to the various monoclonal antibodies, including chimeric monoclonal antibodies, humanized monoclonal antibodies, and human monoclonal antibodies, particularly humanized or human monoclonal antibodies of the invention.
  • an antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) .
  • an antibody comprises at least one heavy chain (HC) comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain (LC) comprising a light chain variable region and at least a portion of a light chain constant region.
  • an antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region.
  • a single-chain Fv or any other antibody that comprises, for example, a single polypeptide chain comprising all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain.
  • the heavy chain is the region of the antibody that comprises the three heavy chain CDRs and the light chain in the region of the antibody that comprises the three light chain CDRs.
  • HCVR heavy chain variable region
  • a heavy chain variable region refers to, at a minimum, a region comprising heavy chain CDR1 (CDR-H1) , framework 2 (HFR2) , CDR2 (CDR-H2) , FR3 (HFR3) , and CDR3 (CDR-H3) .
  • a heavy chain variable region also comprises at least a portion (e.g., the whole) of an FR1 (HFR1) , which is N-terminal to CDR-H1 , and/or at least a portion (e.g., the whole) of an FR4 (HFR4) , which is C-terminal to CDR-H3.
  • heavy chain constant region refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3.
  • Non-limiting exemplary heavy chain constant regions include ⁇ , ⁇ , and ⁇ .
  • Non-limiting exemplary heavy chain constant regions also include ⁇ and ⁇ .
  • Each heavy constant region corresponds to an antibody isotype.
  • an antibody comprising a ⁇ constant region is an IgG antibody
  • an antibody comprising a ⁇ constant region is an IgD antibody
  • an antibody comprising an ⁇ constant region is an IgA antibody
  • an antibody comprising an ⁇ constant region is an IgE antibody
  • an antibody comprising an ⁇ constant region is an IgM antibody.
  • IgG antibodies include, but are not limited to, IgGl (comprising a ⁇ 1 constant region) , IgG2 (comprising a ⁇ 2 constant region) , and IgG3 (comprising a ⁇ 3 constant region) antibodies;
  • IgA antibodies include, but are not limited to, IgAl (comprising an ⁇ 1 constant region) and IgA2 (comprising an ⁇ 2 constant region) antibodies;
  • IgM antibodies include, but are not limited to, IgM1 (comprising an ⁇ 1 constant region) and IgM2 (comprising an ⁇ 2 constant region) .
  • heavy chain refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence.
  • a heavy chain comprises at least a portion of a heavy chain constant region.
  • full-length heavy chain refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine.
  • LCVR light chain variable region
  • LCVR light chain variable region
  • a light chain variable region also comprises at least a portion (e.g., the whole) of an FR1 (LFR1) and/or at least a portion (e.g., the whole) of an FR4 (LFR4) .
  • light chain constant region refers to a region comprising a light chain constant domain, C L .
  • Non-limiting exemplary light chain constant regions include ⁇ and ⁇ .
  • light chain refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence.
  • a light chain comprises at least a portion of a light chain constant region.
  • full-length light chain refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
  • antibody fragment or “antigen binding portion” (of antibody) includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single-chain Fv (scFv) , Fab, Fab’, and (Fab’) 2 .
  • an antibody fragment includes Fab, Fab’, F (ab’) 2 , F d , single chain Fv or scFv, disulfide linked F v , V-NAR domain, IgNar, intrabody, IgG ⁇ CH 2 , minibody, F (ab’) 3 , tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2 , (scFv) 2 , or scFv-Fc.
  • Fab refers to an antibody fragment with a molecular mass of approximately 50,000 Daltons, and has an activity of binding to the antigen. It comprises approximately half of the N-terminal side of the heavy chain and the whole of the light chain connected by a disulfide bridge.
  • the Fab can be obtained in particular by treatment of immunoglobulin by a protease, papain.
  • F (ab’) 2 designates a fragment of approximately 100,000 Daltons and an activity of binding to the antigen. This fragment is slightly larger than two Fab fragments connected via a disulfide bridge in the hinge region. These fragments are obtained by treating an immunoglobulin with a protease, pepsin. The Fab fragment can be obtained from the F (ab') 2 fragment by cleaving of the disulfide bridge of the hinge region.
  • a single Fv chain “scFv” corresponds to a VH: VL polypeptide synthesized using the genes coding for the VL and VH domains and a sequence coding for a peptide intended to bind these domains.
  • An scFv according to the invention includes the CDRs maintained in an appropriate conformation, for example using genetic recombination techniques.
  • the dimers of “scFv” correspond to two scFv molecules connected together by a peptide bond.
  • This Fv chain is frequently the result of the expression of a fusion gene including the genes coding for VH and VL connected by a linker sequence coding a peptide.
  • the human scFv fragment may include CDR regions that are maintained in an appropriate conformation, preferably by means of the use of genetic recombination techniques.
  • the “dsFv” fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it may be divalent (dsFV 2 ) . Fragments of divalent Sc (Fv) 2 or multivalent antibodies may form spontaneously by the association of monovalent scFvs or be produced by connecting scFvs fragments by peptide binding sequences.
  • the Fc fragment is the support for the biological properties of the antibody, in particular its ability to be recognized by immunity effectors or to activate the complement. It consists of constant fragments of the heavy chains beyond the hinge region.
  • diabodies signifies small antibody fragments having two antigen fixing sites. These fragments comprise, in the same VH-VL polypeptide chain, a variable heavy chain domain VH connected to a variable light chain domain VL. Using a binding sequence that is too short to allow the matching of two domains of the same chain, the matching with two complementary domains of another chain necessarily occurs and thus two antigen fixing sites are created.
  • an “antibody that binds to the same epitope” as a reference antibody can be determined by an antibody competition assay. It refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50%or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50%or more.
  • the term “compete” when used in the context of an antibody that compete for the same epitope means competition between antibodies is determined by an assay in which an antibody being tested prevents or inhibits specific binding of a reference antibody to a common antigen.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay see, e.g., Stahli et al., 1983, Methods in Enzymology 9: 242-253
  • solid phase direct biotin-avidin EIA see, e.g., Kirkland et al., 1986, J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press) ; solid phase direct label RIA using I 125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15) ; solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552) ; and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. ) .
  • such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding protein and a labeled reference antibody.
  • Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody.
  • the test antibody is present in excess.
  • Antibodies identified by competition assay include antibodies binding to the same epitope as the reference antibodies and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur.
  • a competing antibody when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97%or more.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody or immunologically functional fragment thereof, and additionally capable of being used in a mammal to produce antibodies capable of binding to that antigen.
  • a selective binding agent such as an antibody or immunologically functional fragment thereof
  • An antigen may possess one or more epitopes that are capable of interacting with antibodies.
  • epitope is the portion of an antigen molecule that is bound by a selective binding agent, such as an antibody or a fragment thereof.
  • the term includes any determinant capable of specifically binding to an antibody.
  • An epitope can be contiguous or non-contiguous (e.g., in a polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within in context of the molecule are bound by the antigen binding protein) .
  • epitopes may be mimetic in that they comprise a three dimensional structure that is similar to an epitope used to generate the antibody, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antibody.
  • Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three dimensional structural characteristics, and/or specific charge characteristics.
  • an “epitope” is defined by the method used to determine it.
  • an antibody binds to the same epitope as a reference antibody, if they bind to the same region of the antigen, as determined by hydrogen-deuterium exchange (HDX) .
  • HDX hydrogen-deuterium exchange
  • an antibody binds to the same epitope as a reference antibody if they bind to the same region of the antigen, as determined by X-ray crystallography.
  • human antibody refers to antibodies of human origin or antibodies produced in humans, antibodies produced in non-human animals that comprise human immunoglobulin genes, such as and antibodies selected using in vitro methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequences.
  • a “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide.
  • Host cells may be prokaryotic cells or eukaryotic cells.
  • Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER. cells (Crucell) , and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.
  • isolated refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or has been separated from at least some of the components with which it is typically produced.
  • a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced.
  • a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide.
  • an isolated antibody of the invention may have natural human antibody sequence, but is so purified that it consists essentially of the antibody, such as a monoclonal antibody recombinantly produced and isolated /purified from the cells which produce such antibody.
  • the isolated antibody is at least 90%pure, 95%pure, 97%pure, 99%pure, 99.5%pure, 99.9%pure or purer.
  • a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide.
  • a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated” so long as that polynucleotide is not found in that vector in nature.
  • subject and “patient” are used interchangeably herein to refer to a mammal such as human.
  • methods of treating other non-human mammals including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided.
  • a “subject” or “patient” refers to a (human) subject or patient in need of treatment for a disease or disorder.
  • sample refers to material that is obtained or derived from a subject of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics.
  • disease sample and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.
  • tissue or cell sample is meant a collection of similar cells obtained from a tissue of a subject or patient.
  • the source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as sputum, cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject.
  • the tissue sample may also be primary or cultured cells or cell lines.
  • the tissue or cell sample is obtained from a disease tissue/organ.
  • the tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
  • a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of the same subject or patient in whom a disease or condition is being identified using a composition or method of the invention.
  • a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient in whom a disease or condition is being identified using a composition or method of the invention.
  • a reference sample, reference cell or reference tissue was previously obtained from a patient prior to developing a disease or condition or at an earlier stage of the disease or condition.
  • a “disorder” or “disease” is any condition that would benefit from treatment with one or more antibodies of the invention. This includes COVID-19 or any secondary infection by other bacteria or virus, in which the antibody of the invention is used in a combination therapy.
  • Treatment refers to therapeutic treatment, for example, wherein the object is to slow down (lessen) the targeted pathologic condition or disorder as well as, for example, wherein the object is to inhibit recurrence of the condition or disorder.
  • Treatment covers any administration or application of a therapeutic for a disease (also referred to herein as a “disorder” or a “condition” ) in a mammal, including a human, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, partially or fully relieving the disease, partially or fully relieving one or more symptoms of a disease, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process.
  • treatment also includes reducing the severity of any phenotypic characteristic and/or reducing the incidence, degree, or likelihood of that characteristic.
  • Those in need of treatment include those already with the disorder as well as those at risk of recurrence of the disorder or those in whom a recurrence of the disorder is to be prevented or slowed down.
  • an effective amount refers to an amount of a drug effective to treat a disease or disorder in a subject.
  • an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.
  • a therapeutically effective amount of antibody of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antagonist to elicit a desired response in the individual.
  • a therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of subject antibody are outweighed by the therapeutically beneficial effects.
  • prophylactically effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.
  • a “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject.
  • a pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation.
  • the pharmaceutically acceptable carrier is appropriate for the formulation employed.
  • the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.
  • An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder, or a probe for specifically detecting a biomarker described herein.
  • the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.
  • antibodies of the invention may be administered subcutaneously or intravenously.
  • the subject antibody may be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal, and intrathecal, or otherwise, e.g., by implantation.
  • routes including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal, and intrathecal, or otherwise, e.g., by implantation.
  • the subject antibody or antigen-binding fragment thereof is administered intraveneously (i. v. ) or subcutaneously (s. c. ) .
  • compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.
  • compositions comprising the subject antibody are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003) ; Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004) ; Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000) ) .
  • Various pharmaceutically acceptable carriers which include vehicles, adjuvants, and diluents, are available.
  • Nonlimiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
  • the subject antibody may be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
  • an aqueous or nonaqueous solvent such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol
  • solubilizers isotonic agents
  • suspending agents such as glycerides, esters of higher aliphatic acids, or propylene glycol
  • compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifiuoromethane, propane, nitrogen, and the like.
  • compositions may also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers.
  • a non-limiting exemplary biodegradable formulation includes poly lactic acid-glycolic acid (PLGA) polymer.
  • PLGA poly lactic acid-glycolic acid
  • a non-limiting exemplary non-biodegradable formulation includes a polyglycerin fatty acid ester. Certain methods of making such formulations are described, for example, in EP 1125584 Al.
  • compositions comprising one or more containers, each containing one or more types or doses of the subject antibody, are also provided.
  • a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising the subject antibody, with or without one or more additional agents.
  • such a unit dosage is supplied in single-use prefilled syringe for injection.
  • the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range.
  • the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water.
  • the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine.
  • a composition of the invention comprises heparin and/or a proteoglycan.
  • compositions are administered in an amount effective for treatment or prophylaxis of the specific indication.
  • the therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated.
  • the subject antibody may be administered in an amount in the range of about 50 ⁇ g/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 100 ⁇ g/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 100 ⁇ g/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
  • the subject antibody may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 200 mg per dose.
  • the subject antibody compositions may be administered as needed to subjects.
  • an effective dose of the subject antibody is administered to a subject one or more times.
  • an effective dose of the subject antibody is administered to the subject once a day, less than once a week, such as, for example, every two days, every three days, or every six days.
  • an effective dose of the subject antibody is administered more than once a day, such as, for example, once or multiple times per day.
  • An effective dose of the subject antibody is administered to the subject at least once.
  • the effective dose of the subject antibody may be administered multiple times, including for periods of at least a month, at least six months, or at least a year.
  • the subject antibody is administered to a subject as-needed to alleviate one or more symptoms of a condition.
  • the antibodies and functional fragments thereof of the invention may be administered to a subject in need thereof in combination with other biologically active substances or other treatment procedures for the treatment of diseases, e.g., COVID-19 and associated symptoms and/or complications.
  • the antibodies of the invention may be administered alone, together as a mixture or combination, or with other modes of treatment such as a second therapeutic agent effective to treat COVID-19 or symptoms /complications thereof. They may be provided before, substantially contemporaneous with, or after other modes of treatment.
  • the second therapeutic agent comprises one or more of: chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab) , CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFN ⁇ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, Calquence (acalabrutinib) , Xeljanz (tofacitinib) , Jakafi (ruxolitinib) , Olumiant (baricitinib) , Ilaris (canakinumab) , Otezla (apremilast
  • any two or more agents may start at times that are, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks apart, or administration of the second agent may start, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks after the first agent has been administered.
  • the agents are administered simultaneously, e.g., are infused simultaneously, e.g., over a period of 30 or 60 minutes, to a patient.
  • One aspect of the invention provides human antibodies that block binding of SARS-CoV-2 virus to a human cell receptor to gain viral entry of the human cell, such as inhibiting binding of the S1 glycoprotein to the ACE2 receptor.
  • the antibody of the invention has a dissociation constant (K d ) of ⁇ 1 ⁇ , ⁇ 100 nM, ⁇ 10 nM, ⁇ 5 nM, ⁇ 2 nM, ⁇ 1 nM, ⁇ 0.5 nM, ⁇ 0.2 nM, ⁇ 0.1 nM, ⁇ 0.05 nM, ⁇ 0.01 nM, or ⁇ 0.001 nM (e.g. 10 -8 M or less, e.g. from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M) for the SARS-CoV-2, such as the S1 glycoprotein.
  • K d dissociation constant
  • the antibody of the invention inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2.
  • SARS-CoV-2 antigen e.g., the S1 glycoprotein
  • Such binding can be assessed in vitro using, for example, an ELISA assay using immobilized SARS-CoV-2 antigen on a solid support, or binding to a cell expressing ACE2 receptor on the surface.
  • the antibody of the invention inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 with an EC50 value of less than 1 nM or 0.1 nM.
  • SARS-CoV-2 antigen e.g., the S1 glycoprotein
  • the antibody of the invention exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 10 nM, 6 nM, 3 nM, 2 nM, 1 nM, 0.6 nM or less than 0.5 nM.
  • an antibody having any the characteristics provided herein inhibits at least 25%, 50%, 75%, 80%, 90%or 100%of the entry of SARS-CoV-2 into a host cell, such as according to the in vitro assay conditions used in the examples for entry into Vero E6 cells. Inhibition of live virus entry can be assayed based on the concentration of antibodies needed to protect about 50%SARS-CoV-2 susceptible cells, such as Vero E6 cells growing on monolayer, from exhibiting CPE (cytopathic effect) 3-5 days post infection (dpi) .
  • the antibody of the invention inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, less than 0.02 nM, or less than 0.01 nM.
  • a target cell such as Vero E6 cell
  • the antibody of the invention inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 10 nM, 5 nM, 3 nM, 2 nM, 1 nM, 500 pM, 300 pM, 200 pM, 100 pM, 80 pM, 50 pM, 30 pM, 10 pM, or less than 5 pM.
  • a target cell such as Vero E6 cell
  • multispecific antibodies are provided.
  • bispecific antibodies are provided.
  • Non-limiting exemplary bispecific antibodies include antibodies comprising a first arm comprising a heavy chain/light chain combination that binds a first epitope of SARS-CoV-2 and a second arm comprising a heavy chain/light chain combination that binds a second epitope of SARS-CoV-2.
  • a further non-limiting exemplary multispecific antibody is a dual variable domain antibody.
  • the monoclonal antibodies of the invention or antigen-binding fragments thereof include one or more point mutations of in amino acid sequences that are designed to improve developability of the antibody.
  • TAP Therapeutic Antibody Profiler
  • VH-CDR1 CTVSGGSISSSIYYWGW (SEQ ID NO: 1)
  • VH-CDR2 GSIYYSGNAYYN (SEQ ID NO: 2)
  • VH-CDR3 CATPHTRWGPDYW (SEQ ID NO: 3)
  • VL-CDR1 CTRSSGSIASNYVLW (SEQ ID NO: 4)
  • VL-CDR2 EDDQRPS (SEQ ID NO: 5)
  • VL-CDR3 CQSYDGDNLVF (SEQ ID NO: 6)
  • HFR1 -HFR4 are defined by the VH-CDR sequences.
  • HFR1 is the sequence of HCVR that is N-terminal to VH-CDR1.
  • HFR2 is the sequence of HCVR that is between VH-CDR1 and VH-CDR2.
  • HFR3 is the sequence of HCVR that is between VH-CDR2 and VH-CDR3.
  • HFR4 is the most C-terminal sequence of HCVR.
  • LFR1 -LFR4 are defined by the VL-CDR sequences.
  • LFR1 is the sequence of LCVR that is N-terminal to VL-CDR1.
  • LFR2 is the sequence of LCVR that is between VL-CDR1 and VL-CDR2.
  • LFR3 is the sequence of LCVR that is between VL-CDR2 and VL-CDR3.
  • LFR4 is the most C-terminal sequence of LCVR.
  • VH-CDR1 CAASGFIVSSNYMSW (SEQ ID NO: 11)
  • VH-CDR2 SIIYSGGSTFYA (SEQ ID NO: 12)
  • VH-CDR3 CARDLQELGSLDYW (SEQ ID NO: 13)
  • VL-CDR1 CRASQGISSWLAW (SEQ ID NO: 14)
  • VL-CDR2 AASSLQS (SEQ ID NO: 15)
  • VL-CDR3 CQEANSFPYTF (SEQ ID NO: 16)
  • VH-CDR1 CTVSGGSISSTIYYWGW (SEQ ID NO: 21)
  • VH-CDR2 GSIYYSGNAYYN (SEQ ID NO: 22)
  • VH-CDR3 CATPHTRWGPDYW (SEQ ID NO: 23)
  • VL-CDR1 CTRSSGSIASNYVLW (SEQ ID NO: 24)
  • VL-CDR2 EDDQRPS (SEQ ID NO: 25)
  • VL-CDR3 CQSYDGDNLVF (SEQ ID NO: 26)
  • VH-CDR1 CTFSGFSLSTSGVGVGW (SEQ ID NO: 31)
  • VH-CDR2 ALIYWDDDKRYS (SEQ ID NO: 32)
  • VH-CDR3 CAHRLSNFWSGYYTGW (SEQ ID NO: 33)
  • VL-CDR1 CRSSQSLLHSNGYNYLDW (SEQ ID NO: 34)
  • VL-CDR2 LGSNRAS (SEQ ID NO: 35)
  • VL-CDR3 CMQALQTPNTF (SEQ ID NO: 36)
  • VH-CDR1 CGVSGGSISSYYWSW (SEQ ID NO: 51)
  • VH-CDR2 GHIYDSGSTNYN (SEQ ID NO: 52)
  • VH-CDR3 CARQLWLRGAFDIW (SEQ ID NO: 53)
  • VL-CDR1 CTGSSGSIASNYVQW (SEQ ID NO: 54)
  • VL-CDR2 EDQQRPS (SEQ ID NO: 55)
  • VL-CDR3 CQSYDSTNQVF (SEQ ID NO: 56)
  • VH-CDR1 CKVSGYTLTELSMHW (SEQ ID NO: 61)
  • VH-CDR2 GGFDPEDGETIYA (SEQ ID NO: 62)
  • VH-CDR3 CATGHQLLFYNWFDPW (SEQ ID NO: 63)
  • VL-CDR1 CTGTSSDVGGYNYVSW (SEQ ID NO: 64)
  • VL-CDR2 EVSKRPS (SEQ ID NO: 65)
  • VL-CDR3 CSSYAGSNNLVF (SEQ ID NO: 66)
  • VH-CDR1 CAASGFAFSSYTMNW (SEQ ID NO: 71)
  • VH-CDR2 SSISSSSDYIFYA (SEQ ID NO: 72)
  • VH-CDR3 CARGSNTAWGGVPDAFDFW (SEQ ID NO: 73)
  • VL-CDR1 CTGTSSDVGRYNYVSW (SEQ ID NO: 74)
  • VL-CDR2 EVSKRPS (SEQ ID NO: 75)
  • VL-CDR3 CSSYAGSNNLVF (SEQ ID NO: 76)
  • VH-CDR1 CAAPGFIVSSNYMSW (SEQ ID NO: 81)
  • VH-CDR2 SIIYSGGSTFYA (SEQ ID NO: 82)
  • VH-CDR3 CARDLQELGSLDYW (SEQ ID NO: 83)
  • VL-CDR1 CRASQGISSWLAW (SEQ ID NO: 84)
  • VL-CDR2 AASSLQS (SEQ ID NO: 85)
  • VL-CDR3 CQEANSFPYTF (SEQ ID NO: 86)
  • VH-CDR1 CAASGFIVSSNYMSW (SEQ ID NO: 91)
  • VH-CDR2 SIIYSGGSTFYA (SEQ ID NO: 92)
  • VH-CDR3 CARDLQELGSLDCW (SEQ ID NO: 93)
  • VL-CDR1 CRASQGISSWLAW (SEQ ID NO: 94)
  • VL-CDR2 AASSLQS (SEQ ID NO: 95)
  • VL-CDR3 CQEANSFPYTF (SEQ ID NO: 96)
  • the HC and/or LC further includes a signal peptide sequence: MGWSCIILFLVATATGAHS (SEQ ID NO: 41) .
  • the invention described herein provides human antibodies or functional fragment thereof specific for an antigen of SARS-CoV-2, such as the S1 glycoprotein.
  • the human antibodies are isolated /purified from convalescent patients recovering from SARS-CoV-2 infection.
  • the human antibodies share one or more CDR sequences with the patient-isolated antibodies described herein, such as antibodies having the same HCVR and/or LCVR CDR1-3 sequences, or antibodies having the same HCVR and/or LCVR sequences but different constant region sequences, such as modified Fc region sequence, or mutations in the constant region that enhances antibody stability and/or confers additional therapeutic benefits.
  • Human antibodies can be made by any suitable method.
  • Non-limiting exemplary methods include making human antibodies in transgenic mice that comprise human immunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993) ; Jakobovits et al, Nature 362: 255-8 (1993) ; onberg et al, Nature 368: 856-9 (1994) ; and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.
  • Non-limiting exemplary methods also include making human antibodies using phage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992) ; Marks et al, J. Mol. Biol. 222: 581-97 (1991) ; and PCT Publication No. WO 99/10494.
  • a human antibody described herein comprises human constant region sequences.
  • the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD.
  • the human light chain constant region is of an isotype selected from K and ⁇ .
  • an antibody described herein comprises a human IgG constant region, for example, human IgG1, IgG2, or IgG3.
  • an antibody or Fc fusion partner comprises a C237S mutation, for example, in an IgG1 constant region.
  • an antibody described herein comprises a human IgG2 heavy chain constant region.
  • the IgG2 constant region comprises a P331S mutation, as described in U.S. Patent No. 6,900,292.
  • the choice of heavy chain constant region can determine whether or not an antibody will have effector function in vivo.
  • effector function includes antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) , and can result in killing of the cell to which the antibody is bound.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement-dependent cytotoxicity
  • antibodies comprising human IgG1 or IgG3 heavy chains have effector function.
  • any of the antibodies described herein may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography.
  • Suitable affinity ligands include the antigen and/or epitope to which the antibody binds, and ligands that bind antibody constant regions.
  • a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody.
  • hydrophobic interactive chromatography for example, a butyl or phenyl column
  • HIC hydrophobic interactive chromatography
  • Many methods of purifying polypeptides are known in the art.
  • an antibody described herein is produced in a cell-free system.
  • Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009) ; Spirin, Trends Biotechnol. 22: 538-45 (2004) ; Endo et al, Biotechnol. Adv. 21 : 695-713 (2003) .
  • nucleic acid molecules comprising polynucleotides that encode one or more chains of an antibody described herein.
  • a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody described herein.
  • a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an antibody described herein.
  • a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
  • the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides.
  • a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
  • a polynucleotide encoding a heavy chain or light chain of an antibody described herein comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N-terminus of the heavy chain or light chain.
  • the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
  • Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art.
  • a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell, such as a mammalian cell.
  • Vectors comprising polynucleotides that encode heavy chains and/or light chains of the antibodies described herein are provided.
  • Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc.
  • a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain.
  • the heavy chain and light chain are expressed from the vector as two separate polypeptides.
  • the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.
  • a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain.
  • the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts) .
  • a mole-or mass-ratio of between 5: 1 and 1: 5 of the first vector and the second vector is transfected into host cells.
  • a mass ratio of between 1: 1 and 1: 5 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a mass ratio of 1: 2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
  • a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004) .
  • a vector is chosen for in vivo expression of the subject antibody in animals, including humans.
  • expression of the polypeptide or polypeptides is under the control of a promoter or promoters that function in a tissue-specific manner. For example, liver-specific promoters are described, e.g., in PCT Publication No. WO 2006/076288.
  • heavy chains and/or light chains of the antibodies described herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast) , plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art.
  • exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-Sand DG44 cells; PER. cells (Crucell) ; and NSO cells.
  • heavy chains and/or light chains of the antibodies described herein may be expressed in yeast.
  • a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains of the subject antibody.
  • CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
  • nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
  • one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.
  • MS Mass spectrometry
  • Blood samples were collected from 26 patients at Xinhua Hospital. These patients were previously diagnosed with COVID-19, and had since recovered from the disease after treatment. Patients ranged in age from 8 to 78 years old, with a median age of 47 years old. Diagnosis of infection with SARS-CoV-2 was made on the basis of positive RT-PCR test results, and blood samples were collected after patients had recovered and tested negative on a subsequent RT-PCR test. The average time between the initial positive RT-PCR test and the subsequent negative RT-PCR test was about 18.7 days, with a median time of 17 days. Blood samples were collected, and peripheral blood mononuclear cells (PBMCs) and plasma were isolated and frozen for subsequent analysis between 2-4 days after patients tested negative for SARS-CoV-2.
  • PBMCs peripheral blood mononuclear cells
  • Sera from a selected set of patients was further evaluated for binding to the Nucleocapsid (N) protein of SARS-CoV-2.
  • N Nucleocapsid
  • the relative reactivity of patient sera against the S and N proteins were similar, with patients showing higher titers against the S protein also showing higher titers to the N protein. Binding to the N protein could be detected at lower dilutions of sera, but this may be reflective of the ELISA format used to measure antibody titers.
  • the S protein is presented as a trimer on the viral envelope, and it consists of two subunits: a receptor-binding S1 subunit on top of an S2 stalk that mediates membrane fusion and viral entry (Li, Annual Review of Virology, 3 (1) , 237–261. https: //doi. org/10.1146/annurev-virology-110615-042301, 2016) .
  • serum from a high-titer patient was titrated against full-length S1+S2-His protein, an S1-mFc fusion protein, S1-His protein, and the S1 receptor binding domain (S1-RBD) .
  • Binding activity was stronger against the full-length S1+S2-His protein and the S1-mFc fusion protein as compared to the monomeric S1-His and S1-RBD domains (FIG. 2) , suggesting that an immune response had been also generated against the S2 domain and that the multimerization state of the S protein influenced antibody binding.
  • PBMCs isolated from peripheral blood collected from two patients were thawed, and flow cytometry analysis was performed on the cells.
  • Cells were stained with a panel of anti-CD19 /CD27 /CD38 antibodies, and plasmablast (characterized by CD19 + /CD27 + /CD38 + ) and memory B cells (characterized by CD19 + /CD27 + /CD38 - ) were identified.
  • plasmablast characterized by CD19 + /CD27 + /CD38 +
  • memory B cells characterized by CD19 + /CD27 + /CD38 -
  • CD19 positive populations from two patients are somewhat lower than the control healthy volunteer with overall reduced lymphocytes as reported previously (Tian et al., Emerging Microbes and Infections, 9 (1) , 382–385. https: //doi. org/10. 1080/22221751. 2020. 1729069, 2020)
  • the percentages of the two patients for memory B cell are significantly higher than the healthy donor.
  • the percentages plasmablasts are about 20-fold higher compared with healthy donor.
  • immunoprecipitation and mass spectrometry analysis were used to identify antibodies isolated from patient serum specifically against viral spike protein.
  • SARS-CoV-2 specific antibodies were identified using a method consisted of affinity-purification of antigen specific antibodies from patient serum, followed by liquid chromatography-mass spectrometry (LC-MS) analysis.
  • LC-MS liquid chromatography-mass spectrometry
  • the B cells from the same patients were sequenced to generate an IgG repertoire database with paired V H and V L chains.
  • the mass spectra data together with the paired repertoire database were then used to map out the antigen specific antibody sequences (Lavinder, Horton, Georgiou, &Ippolito, Current Opinion in Chemical Biology, 24, 112–120. https: //doi. org/10. 1016/j. cbpa. 2014. 11. 007, 2015) .
  • the immunoprecipitation allows affinity-based enrichment of virus antigen-specific antibodies from patient serum.
  • the high- resolution and high mass accuracy tandem mass spectra allow unambiguous identification of the peptides covering all CDRs from the specific heavy chains and light chains.
  • the identified heavy chains and light chains were then analyzed to form paired antibody sequences.
  • the antibodies purified from serum showed a ⁇ 150 kDa band for patient samples but not for the healthy donor control.
  • Patient 011’s sample was shown in lane 5 in the Western blot for purified antibodies.
  • the mass spectrometry data for Patient 011 was searched against the repertoire database. After manual verification of CDR3 peptide spectra, a total of 52 V H and 25 V L sequences with different CDR3 were identified. Selected CDR3 sequences for V H chains were shown in FIG. 4B, with significant degree of diversity.
  • the broad diversity of the HC CDR3 is not only required for obtaining different epitopes, but also required for screening for an antibody with a specific property, as for the current case, neutralizing virus or blocking virus’ entry to the cell by blocking the S protein-ACE2 interaction.
  • FIG. 4C and FIG. 4D showed 2 consecutive peptides (LNSVTAADTAVYYCATPHTR (SEQ ID NO: 105) and WGPDYWGQGTLVTVTVSSPSTK (SEQ ID NO: 106) ) covering heavy chain F22a1-VH0’s full CDR3 with almost all fragment y ions matched.
  • the paired LC F22a1-VL0 was also identified with high confidence mass spectra with CDR3 peptide TEDEADYYCQSYDGDNLVFGGGTK (SEQ ID NO: 107, FIG. 4E) . Identification of both paired HC and LC further improved the confidence for the identity of the antibody.
  • Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, and Ab-9 antibodies specific for SARS-CoV-2 antigens were identified and further characterized herein, including antibodies Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, and Ab-9.
  • Ab-5 to Ab-7 are weaker binders compared to Ab-1 to Ab-4.
  • Ab-8 and Ab-9 shares the same VL sequence with that of Ab-2, but with 1 amino acid difference in the VH region.
  • the other Ab-2 alternatives, all with the same CDR but different framework region (FR) sequences are not listed.
  • HC and LC signal peptide sequence MGWSCIILFLVATATGAHS (SEQ ID NO: 41) .
  • the antibodies of the invention have been demonstrated to be able to bind to the spike protein of the SARS-CoV-2 virus, based on binding assays for S1 and S2 domains, as well as the full length of the S protein.
  • ELISA results showed that at least 20 antibodies bound to the viral S protein with different affinity, among which Ab-2 was the strongest binder, and Ab-4 is another top candidate which also binds to S full length protein (see FIG. 10) .
  • Ab-1 and Ab-3 are two other binding candidates with reduced B max and EC50 values comparing with Ab-2 and Ab-4, however, with nM level binding affinity, nevertheless. These two antibodies share the same heavy chain, with difference in their light chains.
  • ELISA results showed that the antibodies can effectively bind to full length S protein of SARS-CoV-2 with EC50 ranging from 10 -9 -10 -10 M (FIG. 5, left panel) .
  • Ab-1 to Ab-3 were shown to be binders to the S1 fragment, while Ab-4 to be a binder to the S2 fragment (middle and right panels of FIG. 5) , respectively, showing the diversity of mAb being identified.
  • Antibodies Ab-2 exhibited potent neutralizing activity, with IC50 of 0.53 nM on average.
  • Ab-3 and Ab-1 showed IC50s at 5.80 nM and 4.07 nM respectively, whereas Ab-4 displayed no neutralizing activities (FIG. 6B) .
  • These antibody activities from S protein binding to blockage of S1 binding to Vero E6 cells and further to pesudovirus neutralization suggested that S1 binding is a critical step for virus to bind to host cells, and blocking of S1 binding to host cells could potentially be translated into anti-viral activities. The results are summarized in the table below.
  • Sequence analysis showed that each cluster of antibodies shared at least 93%sequence identity. The difference within the same sequence cluster may result from somatic hypermutation in which point mutations accumulated in the antibody. Because the somatic hypermutation does not distinguish between favorable and unfavorable mutations, 12 closely similar sequences in the cluster comprising Ab-2 were chosen to compare their activity experimentally.
  • 11 antibodies (labeled as “2A2A-x” wherein x is 1-4, 9-11, 14, 15, 18 and 19) from the same family of Ab-2 (also labeled as “2A2A” ) demonstrated various levels of binding abilities to the full-length S protein as well as the abilities to block the S1 protein binding to human ACE2 receptor.
  • Ab-2 demonstrated the best potency in both assays, with a few close family members showing similar activities while 2A2A-4 with 1 amino acid difference in CDR1, and 2A2A-19 with 1 amino acid difference in CDR3, others with differences in the FR regions.
  • the neutralizing activities of the lead antibodies Ab-1 to Ab-3 were further confirmed in live SARS-CoV-2 virus entry assay.
  • Vero E6 cells were infected with SARS-CoV-2 virus at 100 TCID50 in the presence of the lead antibodies at different concentrations.
  • fluorescent labeled nuclear protein of SAR-CoV-2 infected cells can be observed by florescence microscopy.
  • Antibody Ab-2 demonstrated strong anti-virus activities -more than 50%inhibition at 6.5 nM, while Ab-3 demonstrated 74%inhibition at 62.7 nM, and Ab-1 showed 93.8%inhibition at 50.2 nM (FIG. 7A) .
  • the ND50s and ND90s have been further calculated through dose responding curve.
  • Ab-2 has a ND50 of 0.751 nM, a ND90 of 1.682 nM (FIG. 7B, left panel) ;
  • Ab-1 has a ND50 of 4.153 nM, a ND90 of 6.1 nM (FIG. 7B, middle panel) ;
  • Ab-3 has a ND50 of 5.512 nM, a ND90 of 24.08 nM (FIG. 7B, right panel) .
  • the data provided herein provides a deeper understanding of the immune response mounted by patients recovering from COVID-19, and may give insights into disease epidemiology and inform the development of novel therapies.
  • males seem to demonstrate higher antiviral titers as a group. This may be related to higher viral loads during the period of peak infection.
  • Several mechanisms have been proposed to explain the apparently greater susceptibility of males to COVID-19, including 1) A high prevalence of smoking among males in regions where the SARS-CoV-2 virus has spread to humans, and 2) the ACE2 gene that acts as a receptor for the virus on human epithelial cells being located on the X chromosome, which may lead to sex-specific differences in ACE2 expression and susceptibility to infection or viral burden.
  • Children have been observed to have less severe disease symptoms as compared to adults (Dong et al., 2020) .
  • an 8-year-old patient profiled as part of the cohort studied here showed relatively high titers to the SARS-CoV-2 S protein, suggesting that despite their less mature immune systems, children are capable of mounting a robust antiviral immune response.
  • COVID-19 patients tend to display low lymphocyte counts, and reduced levels and functional exhaustion of T cells has been described in patients with severe disease (Ni et al., 2020) . It is therefore notable to find increased levels of circulating plasma cells as well as memory B cells in convalescent patients weeks after initial infection and days after recovery, suggesting that the humoral immune response is critical in limiting viral activity.
  • convalescent patient sera as an immediate way to transfer protective immunity to newly diagnosed patients and at-risk populations is a therapeutic strategy being applied to treat COVID-19, and neutralizing antiviral monoclonal antibodies identified from these sera have the utility to be scalable treatments for the disease. Further mining of immune repertoires from patients recovering from COVID-19, using their methods of the invention described herein can be key to controlling the future spread of this and other similar viruses.
  • these neutralizing antibodies can be formulated for use as therapeutic antibodies for patient treatment. In another embodiment, they can also be used prophylactically to prevent virus infection. In further embodiments, certain binding antibodies can be used in combination with the vaccine approaches, even if they do not have neutralization activities. In yet another embodiments, different antibodies with either S1 or S2 binding capacity are used to generate multi-valent antibodies, or be used together, for combination therapy.
  • the criteria of recovery were normal temperature for at least 3 days, obvious improvement in clinical symptoms, significant absorption of pulmonary inflammation on computer tomography scan and negative tests for SARS-CoV-2 two times in a row with a test interval for at least one day.
  • PBMCs peripheral blood mononuclear cells
  • Plasma samples were collected 3-4 days after donors were discharged from the hospital and separated into plasma and peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque gradient centrifugation. Plasma and PBMCs which were maintained in freezing media and stored at -80°C until ready to use. The plasma were heat-inactivated at 56°C for 1h before use.
  • PBMCs peripheral blood mononuclear cells
  • the ELISA protocol was adapted from previously established protocols. Briefly, 384-well plates were coated overnight at 4°C with PBS containing 1 ⁇ g/mL of the respective protein. The next day the plate was washed 4 times with washing buffer (PBS and 0.05%Tween) and then incubated 1 hour at 37 °C in blocking buffer (PBS with 2%BSA) . After two washes, the plate was incubated for 1 hour at 37 °C with the serum or the positive control ACE2 protein. The human serum samples were diluted to 1: 100 in 1X PBS + 2%BSA followed by 5-fold serial dilutions.
  • the plates were then washed 4 times and incubated for 1 hour in detection reagent (Mouse anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 ⁇ g/mL in 1X PBS with 0.05%Tween and 1%BSA) for 1 hour at room temperature. Following this, the plate was washed again 4 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution.
  • detection reagent Mae anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 ⁇ g/mL in 1X PBS with 0.05%Tween and 1%BSA
  • PBMC peripheral blood mononuclear cells
  • PBMC Patient 2 7.1*10 5 /ml, 200 ⁇ L 90
  • each serum was diluted with PBS to a final volume of 1 mL and then passed through SARS-CoV-2 Spike Protein (RBD, mFc Tag, Sino biological Cat: 40592-V05H) conjugated NHS-sepharose column (200 ⁇ L resin) .
  • RBD SARS-CoV-2 Spike Protein
  • the column was then washed 3 times with 1 mL of HBS-EP buffer and once with 1 mL of ddH 2 O.
  • the bounded antibodies were eluted twice with 300 ⁇ L of 300 mM acetic acid.
  • the eluant was subjected to SDS-gel/Western blot and then dried by a speed vac evaporator.
  • the dried sample was dissolved in 15 ⁇ L of 8 M urea and 20 mM TCEP in 20 mM Tris-HCl (pH8.0) at 37°C for 1hr.
  • the reaction was quenched by adding 5 ⁇ L 20%FA.
  • the tryptic peptides were analyzed by a nano-LC1200 HPLC coupled with a Q Exactive mass spectrometer.
  • Peptides (20 ⁇ L) were first trapped onto a 75 ⁇ m ⁇ 2 cm C18 trap column at 4 ⁇ L/min and were then separated at 250 nL/min using a 75 ⁇ m ⁇ 25 cm C18 column at 40°C with the following gradients: 5%-30%ACN in 157 min; 30%-40%ACN in 15 min; 40%-90%ACN in 2 min; 90%ACN for 15 min.
  • the mass spec spectra were acquired under positive mode using following parameters: MS1 resolution: 70,000; MS1 target: 1E6; maximum injection time: 100 ms; scan range: 350 to 1,800 m/z; MS/MS resolution: 17,500; MS/MS target: 2e5; Top N: 10; isolation window: 2 Th;charge exclusion: 1, >5; dynamic exclusion: 30 sec.
  • the acquired LC-MS data from each patient was searched against the corresponding IgG repertoire database generated using proteome discoverer software.
  • the searching parameters were as follows: Cleavage site: lysine or Arginine; Cleavage site: C-terminal; Digestion specificity: fully specific; Missed cleavages: 2; Precursor mass tolerance: 10 ppm; Fragmentation type: HCD; Fragment mass tolerance: 20 ppm; Fixed modification: carbamidomethyl at cysteine.
  • the binding affinity of antibodies to spike protein was analyzed by ELISA.
  • 384 well plate (Corning#3700) , was coated overnight at 4°C with PBS containing 30 ⁇ L 20 nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein. The next day the plate was washed 5 times with washing buffer (PBS and 0.05%Tween) and then incubated 1 hour at room temperature in blocking buffer (PBS with 2%BSA) . After 5 washes the plate was incubated with serial dilution of purified mAbs for 1 hour at room temperature.
  • the plates were then washed 5 times and incubated for 1 hour in detection reagent (Mouse anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 ⁇ g/ml in 1X PBS with 0.05%Tween and 1%BSA) for 1 hour at room temperature. Following this the plate was washed again 5 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution. The OD values were determined using Thermo MultiSkan or MD SpectraMax i3X at 450 nm wavelength.
  • the blocking with receptor ACE2 was performed using cell surface expressed ACE2.
  • 10 nM SARS-CoV-2 Spike S1, mFc tag spike protein was incubated with serial dilution of purified mAbs at room temperature for 1h and then added to Vero E6 cells (approximately 10 5 per well) in duplicate. Then detection reagent rabbit anti mouse IgG Fc-AF647 was used.
  • Half-maximal inhibitory concentration (IC50) of the evaluated mAbs were determined with Beckman Cytoflex and FlowJo software analysis.
  • Murine leukemia virus-based SARS-CoV-2 S pseudotyped virus were prepared by GenScript as previous described. Neutralization assay were performed by incubating pseudo virus with serial dilution of purified antibodies at room temperature for 1h.
  • ACE2 overexpression Hela cells (approximately 8x10 4 per well) were cultured in DMEM containing 10%FBS, 1 ⁇ g/mL puromycin were added in triplicate into virus-antibody mixture. Following infection at 37°C, 5%CO 2 for 48h, half-maximal inhibitory concentration (IC 50 ) were determined by luciferase activity using Promega Bio-Glo luciferase assay system with GraphPad Prism.
  • Vero E6 cells infected by SARS-CoV-2 at 100 TCID50 or incubated with mixture with viruses and diluted antibodies and incubate for 1 hr, and then fixed with 4%paraformaldehyde diluted in PBS (pH 7.2) for 15 minutes at room temperature and then penetrated with 0.25%triton-X 100 for 10-15 minutes. After three washes, cells were blocked at 37°C for 1 hour using PBS containing 5%BSA, then incubated with in-house prepared anti-SARS-CoV-2 NP rabbit serum as primary antibody and FITC-conjugated goat anti-mouse IgG antibody as the secondary antibody. Cell nuclei were strained using Hoechst 33258 at room temperature for 10 minutes. Images were taken under an inverted fluorescence microscope (Nikon) .

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Abstract

The invention described herein provides neutralizing antibodies against SARS-CoV-2 antigens (such as the S1 subunit of the S antigen), for use in treating human patients with COVID-19.

Description

METHODS AND COMPOSITIONS RELATED TO NEUTRALIZING ANTIBODIES AGAINST HUMAN CORONAVIRUS BACKGROUND OF THE INVENTION
The Coronavirus Disease 2019 (COVID-19) , caused by the novel SARS-CoV-2 coronavirus, has quickly grown into a global pandemic and a major public health crisis. Little is known about the immune response generated against this human coronavirus by infected patients. Thus, a better understanding of the pathological mechanisms caused by the virus, and development of new therapeutic agents against it are urgently needed.
The immune system generates a vast repertoire of antibodies in response to infection or immunization that can potentially be explored for diagnostic, therapeutic or research applications. As the most important class of antibodies in blood, IgGs are secreted by plasmablasts and plasma cells. The target-specific IgGs are matured during ongoing immune responses facilitating a rapid recall of previously-elicited protective immunity in response to repeated pathogen or immunogen exposure. The phenotypic diversity of the target-specific IgG repertoires underlies protection following vaccination or infection, and results from the diversity of IgG sequences in the repertoires. Analyzing the activity and sequence of IgGs secreted by plasmablasts and plasma cells is hence of great interest both to investigate their pivotal role in the humoral immune response and to obtain antibodies for therapeutic or other applications.
Until recently, however, target-specific IgG discovery and repertoire analysis was based on B cell immortalization using hybridoma technology or Epstein-Barr Virus (EBV) transformation, which are relatively inefficient, target dividing plasmablasts but not fully matured plasma cells, and are limited to specific species. Direct PCR cloning of V genes from plasma cells has been reported, but without prior knowledge of antigen specificity. Recently, next generation sequencing (NGS) approaches have been developed to probe the IgG repertoire more deeply, some even recovering the original pairing of heavy-chain and light-chain variable region (V H and V L) . Antigen-labeled IgG + memory B cells and plasmablasts, but not antigen-specific plasma cells that lack surface IgG expression, have been isolated using fluorescence-activated cell sorting (FACS) and sequenced. Phenotypic analyses of antibody-secreting cells has evolved from ELISpot to high-throughput assays based on micro/nanowells or pico/nanolitre droplets in microfluidic systems, but remains non-adapted or reliant on low-throughput manual micromanipulation for further V H-V L  sequencing. Even if in vitro display technologies, notably phage and yeast display, have proved to be powerful tools to discover therapeutic antibodies, screening is based on binding and rarely on function, and was, until recently, unable to preserve the cognate V H-V L pairing, limiting its utility for the analysis of immune responses. However, although maintaining cognate V H-V L pairing yields antibodies with higher sensitivity and specificity than random pairing, yeast display with cognate V H-V L pairing involves multiple steps for library construction, antibody fragment display and multiple rounds of screening, and is restricted to soluble antigens.
Therefore, a system that couples high-throughput single-cell phenotypic screening with paired V H-V L sequencing of IgG secreting primary cells for both soluble and membrane-bound antigens in a flexible format enables direct screening for desired functional activities.
SUMMARY OF THE INVENTION
One aspect of the invention provides an isolated or recombinantly produced monoclonal antibody, or an antigen-binding fragment thereof, wherein said monoclonal antibody or antigen-binding fragment thereof is specific for an antigen of SARS-CoV-2, and wherein said monoclonal antibody comprises: (1a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 1, a HCVR CDR2 sequence of SEQ ID NO: 2, and a HCVR CDR3 sequence of SEQ ID NO: 3; and, (1b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 4, a LCVR CDR2 sequence of SEQ ID NO: 5, and a LCVR CDR3 sequence of SEQ ID NO: 6; or (2a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 11, a HCVR CDR2 sequence of SEQ ID NO: 12, and a HCVR CDR3 sequence of SEQ ID NO: 13; and, (2b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 14, a LCVR CDR2 sequence of SEQ ID NO: 15, and a LCVR CDR3 sequence of SEQ ID NO: 16; or (3a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 21, a HCVR CDR2 sequence of SEQ ID NO: 22, and a HCVR CDR3 sequence of SEQ ID NO: 23; and, (3b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 24, a LCVR CDR2 sequence of SEQ ID NO: 25, and a LCVR CDR3 sequence of SEQ ID NO: 26; or (4a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 31, a HCVR CDR2 sequence of SEQ ID NO: 32, and a HCVR CDR3 sequence of SEQ ID NO: 33; and, (4b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 34, a LCVR CDR2 sequence of SEQ ID NO: 35, and a LCVR CDR3 sequence of SEQ ID NO:  36; (5a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 51, a HCVR CDR2 sequence of SEQ ID NO: 52, and a HCVR CDR3 sequence of SEQ ID NO: 53; and, (5b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 54, a LCVR CDR2 sequence of SEQ ID NO: 55, and a LCVR CDR3 sequence of SEQ ID NO: 56; or (6a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 61, a HCVR CDR2 sequence of SEQ ID NO: 62, and a HCVR CDR3 sequence of SEQ ID NO: 63; and, (6b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 64, a LCVR CDR2 sequence of SEQ ID NO: 65, and a LCVR CDR3 sequence of SEQ ID NO: 66; or (7a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 71, a HCVR CDR2 sequence of SEQ ID NO: 72, and a HCVR CDR3 sequence of SEQ ID NO: 73; and, (7b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 74, a LCVR CDR2 sequence of SEQ ID NO: 75, and a LCVR CDR3 sequence of SEQ ID NO: 76; or (8a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 81, a HCVR CDR2 sequence of SEQ ID NO: 82, and a HCVR CDR3 sequence of SEQ ID NO: 83; and, (8b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 84, a LCVR CDR2 sequence of SEQ ID NO: 85, and a LCVR CDR3 sequence of SEQ ID NO: 86; or (9a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 91, a HCVR CDR2 sequence of SEQ ID NO: 92, and a HCVR CDR3 sequence of SEQ ID NO: 93; and, (9b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 94, a LCVR CDR2 sequence of SEQ ID NO: 95, and a LCVR CDR3 sequence of SEQ ID NO: 96; optionally, said isolated monoclonal antibody is not naturally occurring; and/or, optionally further comprising a signal peptide sequence of SEQ ID NO: 41 at the N-terminus of said HCVR and/or LCVR.
In certain embodiments, in the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof: (1A) the HCVR sequence is SEQ ID NO: 7; and/or, (1B) the LCVR sequence is SEQ ID NO: 8, or, (2A) the HCVR sequence is SEQ ID NO: 17; and/or, (2B) the LCVR sequence is SEQ ID NO: 18, or, (3A) the HCVR sequence is SEQ ID NO: 27; and/or, (3B) the LCVR sequence is SEQ ID NO: 28, or, (4A) the HCVR sequence is SEQ ID NO: 37; and/or, (4B) the LCVR sequence is SEQ ID NO: 38; (5A) the HCVR sequence is SEQ ID NO: 57; and/or, (5B) the LCVR sequence is SEQ ID NO: 58, or, (6A) the HCVR sequence is SEQ ID NO: 67; and/or, (6B) the LCVR sequence is SEQ ID  NO: 68, or, (7A) the HCVR sequence is SEQ ID NO: 77; and/or, (7B) the LCVR sequence is SEQ ID NO: 78, or, (8A) the HCVR sequence is SEQ ID NO: 87; and/or, (8B) the LCVR sequence is SEQ ID NO: 88, or, (9A) the HCVR sequence is SEQ ID NO: 97; and/or, (9B) the LCVR sequence is SEQ ID NO: 98.
In certain embodiments, the monoclonal antibody has: (1a) a heavy chain sequence of SEQ ID NO: 9; and/or, (1b) a light chain sequence of SEQ ID NO: 10, or, (2a) a heavy chain sequence of SEQ ID NO: 19; and/or, (2b) a light chain sequence of SEQ ID NO: 20, or, (3a) a heavy chain sequence of SEQ ID NO: 29; and/or, (3b) a light chain sequence of SEQ ID NO: 30, or, (4a) a heavy chain sequence of SEQ ID NO: 39; and/or, (4b) a light chain sequence of SEQ ID NO: 40; (5a) a heavy chain sequence of SEQ ID NO: 59; and/or, (5b) a light chain sequence of SEQ ID NO: 60, or, (6a) a heavy chain sequence of SEQ ID NO: 69; and/or, (6b) a light chain sequence of SEQ ID NO: 70, or, (7a) a heavy chain sequence of SEQ ID NO: 79; and/or, (7b) a light chain sequence of SEQ ID NO: 80, or, (8a) a heavy chain sequence of SEQ ID NO: 89; and/or, (8b) a light chain sequence of SEQ ID NO: 90, or, (9a) a heavy chain sequence of SEQ ID NO: 99; and/or, (9b) a light chain sequence of SEQ ID NO: 100.
In certain embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is a human antibody, a CDR-grafted antibody, or a resurfaced antibody.
In certain embodiments, the antigen-binding fragment thereof is an Fab, Fab’, F (ab’)  2, F d, single chain Fv or scFv, disulfide linked F v, V-NAR domain, IgNar, intrabody, IgGΔCH 2, minibody, F (ab’)  3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2, (scFv)  2, or scFv-Fc.
In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to the S1 glycoprotein of SARS-CoV-2.
In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to the S2 glycoprotein of SARS-CoV-2.
In certain embodiments, the monoclonal antibody or antigen-binding fragment thereof binds the SARS-CoV-2 antigen with a K d of less than about 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or 0.05 nM.
In certain embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2.
In certain embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 immobilized on a solid support (such as in ELISA assay) , or inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 expressed on the surface of a cell (such as Vero E6 cell) .
In certain embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 with an EC50 value of less than 2 nM, 1 nM or 0.1 nM.
In certain embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 10 nM, 8 nM, 6 nM, 5 nM, 3 nM, 2 nM, 1 nM, 0.6 nM or less than 0.5 nM.
In certain embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, less than 0.02 nM, or less than 0.01 nM.
In certain embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 500 pM, less than 300 pM, less than 200 pM, less than 100 pM, less than 80 pM, less than 50 pM, less than 30 pM, less than 10 pM, or less than 5 pM.
Another aspect of the invention provides an isolated or recombinantly produced monoclonal antibody or an antigen-binding fragment thereof, which competes with the isolated monoclonal antibody or antigen-binding fragment thereof of for binding to the same epitope.
In a related aspect, the invention provides a non-naturally occurring or existing therapeutic antibody based on the antigen-binding sequences of an antibody isolated from the patient using the method of the invention. Such therapeutic antibody may share one or more CDRs, such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences, with the antibody isolated from the patient using the method of the invention.
For example, heavy chain CDR3 (HC-CDR3) sequences of certain isolated antibodies are listed in FIG. 4B, and in the disclosed sequences herein, see Example 3.
Another aspect of the invention provides a mixture of two or more isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention.
In certain embodiments, the proportion of each of said two or more isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is substantially the same, or is different.
Another aspect of the invention provides a method of treating or preventing a disease or condition arising from SARS-CoV-2 infection, the method comprising administering to a patient in need thereof an effective amount of the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention, or an effective amount of the mixture of the invention.
In certain embodiments, the method is for treating COVID-19, wherein the method further comprises administering a second therapeutic agent, which may be effective to treat infection by SARS-CoV-2.
In certain embodiments, the second therapeutic agent comprises chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab) , CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFNγ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, Calquence (acalabrutinib) , Xeljanz (tofacitinib) , Jakafi (ruxolitinib) , Olumiant (baricitinib) , Ilaris (canakinumab) , Otezla (apremilast) , Mavrilimumab, or combination thereof.
In certain embodiments, the second therapeutic agent comprises one or more of: an anti-viral agent, an antibiotic, an anti-inflammatory agent or DMARD (disease-modifying anti-rheumatic drug) .
Another aspect of the invention provides a polynucleotide encoding the heavy chain or the light chain or the antigen-binding portion thereof of the invention.
In certain embodiments, the polynucleotide is codon optimized for expression in a human cell.
Another aspect of the invention provides a vector comprising the polynucleotide of the invention.
In certain embodiments, the vector is an expression vector (e.g., a mammalian  expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector) .
Another aspect of the invention provides a host cell comprising the vector of the invention, which expresses said isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof.
Another aspect of the invention provides a pharmaceutical composition comprising the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof of the invention, or the mixture of the invention. The pharmaceutical composition further comprises a pharmaceutically acceptable excipient or diluent.
In certain embodiments, the pharmaceutical composition is formulated for intravenous administration.
It should be understood that any one embodiment of the invention, including those only described under one aspect or section of the invention, and those only described in the examples or claims, can be combined with any other embodiment (s) of the invention unless improper or expressly disclaimed.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGs. 1A and 1B show comparison of patient titers against S protein from SARS-CoV-2. In FIG. 1A, patient sera were measured for binding activity to the S1-RBD (receptor binding domain) . FIG. 1B shows classification of patient samples into high, medium, and low titer bins.
FIG. 2 shows results of evaluating the various S protein constructs. For a given donor (Patient 016 in this study) , titer shows S1+S2-his =S1-mFc>S1 RBD-mFC> S1-his; Trimer (S1+S2 full length) /dimer with mFc tag seem to be better than monomer S1-his.
FIG. 3 shows flow cytometry dot plots of cells from two convalescent patients and one healthy donor, with population of interest highlighted in boxes: CD38 versus CD27 for the two patients shows substantially higher percentage of memory B cells and plasmablasts compared to the healthy donor.
FIGs. 4A-4E show identified antibodies from patient 013 serum. FIG. 4A is an anti-hIgG western blot analysis indicating that for the immuno-precipitated patient samples, the full-length antibody was purified from serum. The healthy donor did not show any antigen specific antibody band. FIG. 4B shows the CDR3 diversity of identified antibodies. FIG. 4C is the tandem mass spectrum for first CDR3 peptide for heavy chain F22a1-VH0. FIG. 4D is  the tandem mass spectrum for second CDR3 peptide for heavy chain F22a1-VH0. FIG. 4E is the tandem mass spectrum for CDR3 peptide of light chain F22a1-VL0.
FIG. 5 shows lead antibody binding characteristics in terms of binding to SARS-CoV-2 S antigen.
FIG. 6A shows the blocking activities of the subject antibodies, based on FACS analysis of blocked S1 binding to ACE2, using Vero E6 cell line expressing ACE2.
FIG. 6B shows potent neutralizing activities of the subject antibodies for neutralization of pseudovirus entry.
FIG. 7A shows the abilities of the subject antibodies to neutralize live SARS-CoV-2 cell entry. Using fluorescent labeled nuclear protein of SAR-CoV-2, infected cells can be observed with a florescence microscope. The top, middle, and bottom panels are for Ab-2, Ab-3, and Ab-1, respectively.
FIG. 7B shows dose response curves for live virus assay performed with three of the subject antibodies. The left, middle, and right panels are for Ab-2, Ab-1, and Ab-3, respectively.
FIG. 8A shows binding to full-length S protein (left panel) and inhibition of S1 protein binding to hACE2 (right panel) by Ab-2 (C1S5-2A2A) and variants thereof (C1S5-2A2A-1, -2, -3, -4, -9, -10, -11, -14, -15, -18, and -19) .
FIG. 8B shows sequence alignments of Ab-2 and variants thereof.
FIG. 9 shows the PK profiles of the subject neutralizing antibodies in mice.
FIG. 10 shows binding of the subject antibodies to full-length S protein. The top to bottom labels are for Ab-2, -4, -3, -1, -6, -7, -5, -8, and -9, respectively.
DETAILED DESCRIPTION OF THE INVENTION
1. Overview
One aspect of the invention provides an antibody isolated from a convalescent COVID-19 patient using the method of the invention. Specifically, sera from convalescent COVID-19 (i.e., SARS-CoV-2) patients, a source of antiviral antibodies capable of conferring protective immunity on recipients, were obtained to identify effective antibodies against antigens of COVID-19 for therapeutic purposes. Antibodies identified from patients infected with the Ebola virus have been used as therapeutic antibodies (Bornholdt et al., 2016; Casadevall &Pirofski, 2020) .
In a related aspect, the invention provides a non-naturally occurring or existing therapeutic antibody based on the antigen-binding sequences of an antibody isolated from the patient using the method of the invention. Such therapeutic antibody may share one or more CDRs, such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences, with the antibody isolated from the patient using the method of the invention. For example, heavy chain CDR3 (HC-CDR3) sequences of certain isolated antibodies are listed in FIG. 4B, or any of the CDR sequences or combinations thereof disclosed herein. Such antibodies may also be multi-specific (e.g., bi-specific) , having antigen binding sequences originating from different antibody light and/or heavy chains.
Another aspect of the invention provides a mixture of the antibodies of the invention. Such a mixture may provide better therapeutic efficacy compared to the individual component antibodies of the mixture.
Indeed, a convalescent patient’s sera is a natural mixture of different antibodies against the same or different viral antigen or epitope. Further, Applicant has identified multiple antibodies from the serum of convalescent patients, including 10 antibodies capable of binding to the full length SARS-CoV-2 S protein, among which 8 antibodies recognizes S1 only, and 2 antibodies interacts with S2 only but not with S1. The data indicates that diverse epitopes on SARS-CoV-2 virus spike protein can be targeted by different antibodies, suggesting that the antibodies of the invention, either alone or in combination with antibodies that target different epitopes via different mechanisms, may serve as potent therapeutic agents to treat COVID-19 patients.
Another aspect of the invention provides a polynucleotide encoding the heavy or light chain of the antibodies of the invention. Such polynucleotide sequences may be codon optimized for expression in a host cell, such as a mammalian cell line (e.g., CHO cell line) for large scale production of antibody.
Another aspect of the invention provides a vector comprising the polynucleotide of the invention. Such vector may be used for expression of antibody in a suitable host cell.
A further aspect of the invention provides a host cell comprising the vector of the invention, or producing the antibody of the invention.
Yet another aspect of the invention provides a method of treating or preventing a disease or condition arising from SARS-CoV-2 infection, such as COVID-19, the method comprising administering to a patient in need thereof a therapeutically effective amount of the antibody of the invention, or a mixture thereof.
With the general aspects of the inventions described, the following sections provide more detailed aspects of the invention. It should be understood that any one embodiment of the invention, including those only described in one section or one example, can be combined with any one or more additional embodiment of the invention whenever proper.
2. Definitions
The term “antibody, ” in the broadest sense, encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies) . The term “antibody” may also broadly refers to a molecule comprising complementarity determining region (CDR) 1, CDR2, and CDR3 of a heavy chain and CDR1, CDR2, and CDR3 of a light chain, wherein the molecule is capable of binding to an antigen. The term “antibody” also includes, but is not limited to, chimeric antibodies, humanized antibodies, human antibodies, and antibodies of various species such as mouse, human, cynomolgus monkey, etc.
In a narrower sense, however, “antibody” refers to the various monoclonal antibodies, including chimeric monoclonal antibodies, humanized monoclonal antibodies, and human monoclonal antibodies, particularly humanized or human monoclonal antibodies of the invention.
In some embodiments, an antibody comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) . In some embodiments, an antibody comprises at least one heavy chain (HC) comprising a heavy chain variable region and at least a portion of a heavy chain constant region, and at least one light chain (LC) comprising a light chain variable region and at least a portion of a light chain constant region. In some embodiments, an antibody comprises two heavy chains, wherein each heavy chain comprises a heavy chain variable region and at least a portion of a heavy chain constant region, and two light chains, wherein each light chain comprises a light chain variable region and at least a portion of a light chain constant region.
As used herein, a single-chain Fv (scFv) , or any other antibody that comprises, for example, a single polypeptide chain comprising all six CDRs (three heavy chain CDRs and three light chain CDRs) is considered to have a heavy chain and a light chain. In some such embodiments, the heavy chain is the region of the antibody that comprises the three heavy chain CDRs and the light chain in the region of the antibody that comprises the three light chain CDRs.
The term “heavy chain variable region (HCVR) ” as used herein refers to, at a  minimum, a region comprising heavy chain CDR1 (CDR-H1) , framework 2 (HFR2) , CDR2 (CDR-H2) , FR3 (HFR3) , and CDR3 (CDR-H3) . In some embodiments, a heavy chain variable region also comprises at least a portion (e.g., the whole) of an FR1 (HFR1) , which is N-terminal to CDR-H1 , and/or at least a portion (e.g., the whole) of an FR4 (HFR4) , which is C-terminal to CDR-H3.
The term “heavy chain constant region” as used herein refers to a region comprising at least three heavy chain constant domains, CH1, CH2, and CH3. Non-limiting exemplary heavy chain constant regions include γ, δ, and α. Non-limiting exemplary heavy chain constant regions also include ε and μ. Each heavy constant region corresponds to an antibody isotype. For example, an antibody comprising a γ constant region is an IgG antibody, an antibody comprising a δ constant region is an IgD antibody, an antibody comprising an α constant region is an IgA antibody, an antibody comprising an ε constant region is an IgE antibody, and an antibody comprising an μ constant region is an IgM antibody.
Certain isotypes can be further subdivided into subclasses. For example, IgG antibodies include, but are not limited to, IgGl (comprising a γ1 constant region) , IgG2 (comprising a γ2 constant region) , and IgG3 (comprising a γ3 constant region) antibodies; IgA antibodies include, but are not limited to, IgAl (comprising an α1 constant region) and IgA2 (comprising an α2 constant region) antibodies; and IgM antibodies include, but are not limited to, IgM1 (comprising an μ1 constant region) and IgM2 (comprising an μ2 constant region) .
The term “heavy chain” as used herein refers to a polypeptide comprising at least a heavy chain variable region, with or without a leader sequence. In some embodiments, a heavy chain comprises at least a portion of a heavy chain constant region. The term “full-length heavy chain” as used herein refers to a polypeptide comprising a heavy chain variable region and a heavy chain constant region, with or without a leader sequence, and with or without a C-terminal lysine.
The term “light chain variable region (LCVR) ” as used herein refers to a region comprising light chain CDR1 (CDR-L1) , framework (FR) 2 (LFR2) , CDR2 (CDR-L2) , FR3 (LFR3) , and CDR3 (CDR-L3) . In some embodiments, a light chain variable region also comprises at least a portion (e.g., the whole) of an FR1 (LFR1) and/or at least a portion (e.g., the whole) of an FR4 (LFR4) .
The term “light chain constant region” as used herein refers to a region comprising a light chain constant domain, C L. Non-limiting exemplary light chain constant regions include  λ and κ.
The term “light chain” as used herein refers to a polypeptide comprising at least a light chain variable region, with or without a leader sequence. In some embodiments, a light chain comprises at least a portion of a light chain constant region. The term “full-length light chain” as used herein refers to a polypeptide comprising a light chain variable region and a light chain constant region, with or without a leader sequence.
The term “antibody fragment” or “antigen binding portion” (of antibody) includes, but is not limited to, fragments that are capable of binding antigen, such as Fv, single-chain Fv (scFv) , Fab, Fab’, and (Fab’)  2. In certain embodiments, an antibody fragment includes Fab, Fab’, F (ab’)  2, F d, single chain Fv or scFv, disulfide linked F v, V-NAR domain, IgNar, intrabody, IgGΔCH 2, minibody, F (ab’)  3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2, (scFv)  2, or scFv-Fc.
The term “Fab” refers to an antibody fragment with a molecular mass of approximately 50,000 Daltons, and has an activity of binding to the antigen. It comprises approximately half of the N-terminal side of the heavy chain and the whole of the light chain connected by a disulfide bridge. The Fab can be obtained in particular by treatment of immunoglobulin by a protease, papain.
The term “F (ab’)  2” designates a fragment of approximately 100,000 Daltons and an activity of binding to the antigen. This fragment is slightly larger than two Fab fragments connected via a disulfide bridge in the hinge region. These fragments are obtained by treating an immunoglobulin with a protease, pepsin. The Fab fragment can be obtained from the F (ab') 2 fragment by cleaving of the disulfide bridge of the hinge region.
A single Fv chain “scFv” corresponds to a VH: VL polypeptide synthesized using the genes coding for the VL and VH domains and a sequence coding for a peptide intended to bind these domains. An scFv according to the invention includes the CDRs maintained in an appropriate conformation, for example using genetic recombination techniques.
The dimers of “scFv” correspond to two scFv molecules connected together by a peptide bond. This Fv chain is frequently the result of the expression of a fusion gene including the genes coding for VH and VL connected by a linker sequence coding a peptide. The human scFv fragment may include CDR regions that are maintained in an appropriate conformation, preferably by means of the use of genetic recombination techniques.
The “dsFv” fragment is a VH-VL heterodimer stabilized by a disulfide bridge; it may be divalent (dsFV 2) . Fragments of divalent Sc (Fv)  2 or multivalent antibodies may form  spontaneously by the association of monovalent scFvs or be produced by connecting scFvs fragments by peptide binding sequences.
The Fc fragment is the support for the biological properties of the antibody, in particular its ability to be recognized by immunity effectors or to activate the complement. It consists of constant fragments of the heavy chains beyond the hinge region.
The term “diabodies” signifies small antibody fragments having two antigen fixing sites. These fragments comprise, in the same VH-VL polypeptide chain, a variable heavy chain domain VH connected to a variable light chain domain VL. Using a binding sequence that is too short to allow the matching of two domains of the same chain, the matching with two complementary domains of another chain necessarily occurs and thus two antigen fixing sites are created.
An “antibody that binds to the same epitope” as a reference antibody can be determined by an antibody competition assay. It refers to an antibody that blocks binding of the reference antibody to its antigen in a competition assay by 50%or more, and conversely, the reference antibody blocks binding of the antibody to its antigen in a competition assay by 50%or more. The term “compete” when used in the context of an antibody that compete for the same epitope means competition between antibodies is determined by an assay in which an antibody being tested prevents or inhibits specific binding of a reference antibody to a common antigen.
Numerous types of competitive binding assays can be used, for example: solid phase direct or indirect radioimmunoassay (RIA) , solid phase direct or indirect enzyme immunoassay (EIA) , sandwich competition assay (see, e.g., Stahli et al., 1983, Methods in Enzymology 9: 242-253) ; solid phase direct biotin-avidin EIA (see, e.g., Kirkland et al., 1986, J. Immunol. 137: 3614-3619) ; solid phase direct labeled assay; solid phase direct labeled sandwich assay (see, e.g., Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press) ; solid phase direct label RIA using I 125 label (see, e.g., Morel et al., 1988, Molec. Immunol. 25: 7-15) ; solid phase direct biotin-avidin EIA (see, e.g., Cheung, et al., 1990, Virology 176: 546-552) ; and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. ) .
Typically, such an assay involves the use of purified antigen bound to a solid surface or cells bearing either of these, an unlabeled test antigen binding protein and a labeled reference antibody. Competitive inhibition is measured by determining the amount of label bound to the solid surface or cells in the presence of the test antibody. Usually the test  antibody is present in excess. Antibodies identified by competition assay (competing antibodies) include antibodies binding to the same epitope as the reference antibodies and antibodies binding to an adjacent epitope sufficiently proximal to the epitope bound by the reference antibody for steric hindrance to occur. In some embodiments, when a competing antibody is present in excess, it will inhibit specific binding of a reference antibody to a common antigen by at least 40%, 45%, 50%, 55%, 60%, 65%, 70%or 75%. In some instance, binding is inhibited by at least 80%, 85%, 90%, 95%, or 97%or more.
The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody or immunologically functional fragment thereof, and additionally capable of being used in a mammal to produce antibodies capable of binding to that antigen. An antigen may possess one or more epitopes that are capable of interacting with antibodies.
The term “epitope” is the portion of an antigen molecule that is bound by a selective binding agent, such as an antibody or a fragment thereof. The term includes any determinant capable of specifically binding to an antibody. An epitope can be contiguous or non-contiguous (e.g., in a polypeptide, amino acid residues that are not contiguous to one another in the polypeptide sequence but that within in context of the molecule are bound by the antigen binding protein) . In some embodiments, epitopes may be mimetic in that they comprise a three dimensional structure that is similar to an epitope used to generate the antibody, yet comprise none or only some of the amino acid residues found in that epitope used to generate the antibody. Epitope determinants may include chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl or sulfonyl groups, and may have specific three dimensional structural characteristics, and/or specific charge characteristics.
In some embodiments, an “epitope” is defined by the method used to determine it. For example, in some embodiments, an antibody binds to the same epitope as a reference antibody, if they bind to the same region of the antigen, as determined by hydrogen-deuterium exchange (HDX) .
In certain embodiments, an antibody binds to the same epitope as a reference antibody if they bind to the same region of the antigen, as determined by X-ray crystallography.
A “human antibody” as used herein refers to antibodies of human origin or antibodies produced in humans, antibodies produced in non-human animals that comprise human immunoglobulin genes, such as
Figure PCTCN2021100414-appb-000001
and antibodies selected using in vitro  methods, such as phage display, wherein the antibody repertoire is based on a human immunoglobulin sequences.
A “host cell” refers to a cell that may be or has been a recipient of a vector or isolated polynucleotide. Host cells may be prokaryotic cells or eukaryotic cells. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate animal cells; fungal cells, such as yeast; plant cells; and insect cells. Non-limiting exemplary mammalian cells include, but are not limited to, NSO cells, PER. 
Figure PCTCN2021100414-appb-000002
cells (Crucell) , and 293 and CHO cells, and their derivatives, such as 293-6E and DG44 cells, respectively.
The term “isolated” as used herein refers to a molecule that has been separated from at least some of the components with which it is typically found in nature or has been separated from at least some of the components with which it is typically produced. For example, a polypeptide is referred to as “isolated” when it is separated from at least some of the components of the cell in which it was produced. Where a polypeptide is secreted by a cell after expression, physically separating the supernatant containing the polypeptide from the cell that produced it is considered to be “isolating” the polypeptide.
In certain embodiments, an isolated antibody of the invention may have natural human antibody sequence, but is so purified that it consists essentially of the antibody, such as a monoclonal antibody recombinantly produced and isolated /purified from the cells which produce such antibody.
In certain embodiments, the isolated antibody is at least 90%pure, 95%pure, 97%pure, 99%pure, 99.5%pure, 99.9%pure or purer.
Similarly, a polynucleotide is referred to as “isolated” when it is not part of the larger polynucleotide (such as, for example, genomic DNA or mitochondrial DNA, in the case of a DNA polynucleotide) in which it is typically found in nature, or is separated from at least some of the components of the cell in which it was produced, e.g., in the case of an RNA polynucleotide. Thus, a DNA polynucleotide that is contained in a vector inside a host cell may be referred to as “isolated” so long as that polynucleotide is not found in that vector in nature.
The terms “subject” and “patient” are used interchangeably herein to refer to a mammal such as human. In some embodiments, methods of treating other non-human mammals, including, but not limited to, rodents, simians, felines, canines, equines, bovines, porcines, ovines, caprines, mammalian laboratory animals, mammalian farm animals, mammalian sport animals, and mammalian pets, are also provided. In some instances, a  “subject” or “patient” refers to a (human) subject or patient in need of treatment for a disease or disorder.
The term “sample” or “patient sample” as used herein, refers to material that is obtained or derived from a subject of interest that contains a cellular and/or other molecular entity that is to be characterized and/or identified, for example based on physical, biochemical, chemical and/or physiological characteristics. For example, the phrase “disease sample” and variations thereof refers to any sample obtained from a subject of interest that would be expected or is known to contain the cellular and/or molecular entity that is to be characterized.
By “tissue or cell sample” is meant a collection of similar cells obtained from a tissue of a subject or patient. The source of the tissue or cell sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as sputum, cerebral spinal fluid, amniotic fluid, peritoneal fluid, or interstitial fluid; cells from any time in gestation or development of the subject. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue or cell sample is obtained from a disease tissue/organ. The tissue sample may contain compounds which are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like.
A “reference sample, ” “reference cell, ” or “reference tissue, ” as used herein, refers to a sample, cell or tissue obtained from a source known, or believed, not to be afflicted with the disease or condition for which a method or composition of the invention is being used to identify. In one embodiment, a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of the same subject or patient in whom a disease or condition is being identified using a composition or method of the invention. In one embodiment, a reference sample, reference cell or reference tissue is obtained from a healthy part of the body of at least one individual who is not the subject or patient in whom a disease or condition is being identified using a composition or method of the invention. In some embodiments, a reference sample, reference cell or reference tissue was previously obtained from a patient prior to developing a disease or condition or at an earlier stage of the disease or condition.
A “disorder” or “disease” is any condition that would benefit from treatment with one or more antibodies of the invention. This includes COVID-19 or any secondary infection by other bacteria or virus, in which the antibody of the invention is used in a combination  therapy.
“Treatment” refers to therapeutic treatment, for example, wherein the object is to slow down (lessen) the targeted pathologic condition or disorder as well as, for example, wherein the object is to inhibit recurrence of the condition or disorder. “Treatment” covers any administration or application of a therapeutic for a disease (also referred to herein as a “disorder” or a “condition” ) in a mammal, including a human, and includes inhibiting the disease or progression of the disease, inhibiting or slowing the disease or its progression, arresting its development, partially or fully relieving the disease, partially or fully relieving one or more symptoms of a disease, or restoring or repairing a lost, missing, or defective function; or stimulating an inefficient process. The term “treatment” also includes reducing the severity of any phenotypic characteristic and/or reducing the incidence, degree, or likelihood of that characteristic. Those in need of treatment include those already with the disorder as well as those at risk of recurrence of the disorder or those in whom a recurrence of the disorder is to be prevented or slowed down.
The term “effective amount” or “therapeutically effective amount” refers to an amount of a drug effective to treat a disease or disorder in a subject. In some embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of antibody of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antagonist to elicit a desired response in the individual. A therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of subject antibody are outweighed by the therapeutically beneficial effects.
A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.
A “pharmaceutically acceptable carrier” refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a “pharmaceutical composition” for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other  ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.
An “article of manufacture” is any manufacture (e.g., a package or container) or kit comprising at least one reagent, e.g., a medicament for treatment of a disease or disorder, or a probe for specifically detecting a biomarker described herein. In some embodiments, the manufacture or kit is promoted, distributed, or sold as a unit for performing the methods described herein.
3. Routes of Administration and Carriers
In various embodiments, antibodies of the invention may be administered subcutaneously or intravenously.
In some embodiments, the subject antibody may be administered in vivo by various routes, including, but not limited to, oral, intra-arterial, parenteral, intranasal, intramuscular, intracardiac, intraventricular, intratracheal, buccal, rectal, intraperitoneal, by inhalation, intradermal, topical, transdermal, and intrathecal, or otherwise, e.g., by implantation.
In some embodiments, the subject antibody or antigen-binding fragment thereof is administered intraveneously (i. v. ) or subcutaneously (s. c. ) .
The subject compositions may be formulated into preparations in solid, semi-solid, liquid, or gaseous forms; including, but not limited to, tablets, capsules, powders, granules, ointments, solutions, suppositories, enemas, injections, inhalants, and aerosols.
In various embodiments, compositions comprising the subject antibody are provided in formulations with a wide variety of pharmaceutically acceptable carriers (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003) ; Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004) ; Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000) ) . Various pharmaceutically acceptable carriers, which include vehicles, adjuvants, and diluents, are available. Moreover, various pharmaceutically acceptable auxiliary substances, such as pH adjusting and buffering agents, tonicity adjusting agents, stabilizers, wetting agents and the like, are also available. Nonlimiting exemplary carriers include saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof.
In various embodiments, the subject antibody may be formulated for injection, including subcutaneous administration, by dissolving, suspending, or emulsifying them in an aqueous or nonaqueous solvent, such as vegetable or other oils, synthetic aliphatic acid glycerides, esters of higher aliphatic acids, or propylene glycol; and if desired, with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifying agents, stabilizers and preservatives.
In various embodiments, the compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifiuoromethane, propane, nitrogen, and the like.
The compositions may also be formulated, in various embodiments, into sustained release microcapsules, such as with biodegradable or non-biodegradable polymers. A non-limiting exemplary biodegradable formulation includes poly lactic acid-glycolic acid (PLGA) polymer. A non-limiting exemplary non-biodegradable formulation includes a polyglycerin fatty acid ester. Certain methods of making such formulations are described, for example, in EP 1125584 Al.
Pharmaceutical dosage packs comprising one or more containers, each containing one or more types or doses of the subject antibody, are also provided. In some embodiments, a unit dosage is provided wherein the unit dosage contains a predetermined amount of a composition comprising the subject antibody, with or without one or more additional agents. In some embodiments, such a unit dosage is supplied in single-use prefilled syringe for injection. In various embodiments, the composition contained in the unit dosage may comprise saline, sucrose, or the like; a buffer, such as phosphate, or the like; and/or be formulated within a stable and effective pH range. Alternatively, in some embodiments, the composition may be provided as a lyophilized powder that may be reconstituted upon addition of an appropriate liquid, for example, sterile water. In some embodiments, the composition comprises one or more substances that inhibit protein aggregation, including, but not limited to, sucrose and arginine. In some embodiments, a composition of the invention comprises heparin and/or a proteoglycan.
Pharmaceutical compositions are administered in an amount effective for treatment or prophylaxis of the specific indication. The therapeutically effective amount is typically dependent on the weight of the subject being treated, his or her physical or health condition, the extensiveness of the condition to be treated, or the age of the subject being treated.
In some embodiments, the subject antibody may be administered in an amount in the  range of about 50 μg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 100 μg/kg body weight to about 50 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 100 μg/kg body weight to about 20 mg/kg body weight per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 0.5 mg/kg body weight to about 20 mg/kg body weight per dose.
In some embodiments, the subject antibody may be administered in an amount in the range of about 10 mg to about 1,000 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 500 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 300 mg per dose. In some embodiments, the subject antibody may be administered in an amount in the range of about 20 mg to about 200 mg per dose.
The subject antibody compositions may be administered as needed to subjects. In some embodiments, an effective dose of the subject antibody is administered to a subject one or more times. In various embodiments, an effective dose of the subject antibody is administered to the subject once a day, less than once a week, such as, for example, every two days, every three days, or every six days. In other embodiments, an effective dose of the subject antibody is administered more than once a day, such as, for example, once or multiple times per day. An effective dose of the subject antibody is administered to the subject at least once. In some embodiments, the effective dose of the subject antibody may be administered multiple times, including for periods of at least a month, at least six months, or at least a year. In some embodiments, the subject antibody is administered to a subject as-needed to alleviate one or more symptoms of a condition.
4. Combination Therapy
The antibodies and functional fragments thereof of the invention may be administered to a subject in need thereof in combination with other biologically active substances or other treatment procedures for the treatment of diseases, e.g., COVID-19 and associated symptoms and/or complications. For example, the antibodies of the invention may be administered alone, together as a mixture or combination, or with other modes of treatment such as a second therapeutic agent effective to treat COVID-19 or symptoms /complications thereof. They may be provided before, substantially contemporaneous with, or after other modes of treatment.
In certain embodiments, the second therapeutic agent comprises one or more of: chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab) , CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFNγ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, Calquence (acalabrutinib) , Xeljanz (tofacitinib) , Jakafi (ruxolitinib) , Olumiant (baricitinib) , Ilaris (canakinumab) , Otezla (apremilast) , Mavrilimumab, or combination thereof.
The administration of any two or more agents may start at times that are, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks apart, or administration of the second agent may start, e.g., 30 minutes, 60 minutes, 90 minutes, 120 minutes, 3 hours, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours, 3 days, 5 days, 7 days, or one or more weeks after the first agent has been administered.
In certain aspects, the agents are administered simultaneously, e.g., are infused simultaneously, e.g., over a period of 30 or 60 minutes, to a patient.
5. Exemplary Antibodies
One aspect of the invention provides human antibodies that block binding of SARS-CoV-2 virus to a human cell receptor to gain viral entry of the human cell, such as inhibiting binding of the S1 glycoprotein to the ACE2 receptor.
In some embodiments, the antibody of the invention has a dissociation constant (K d) of ≤ 1 μΜ, ≤ 100 nM, ≤ 10 nM, ≤ 5 nM, ≤ 2 nM, ≤ 1 nM, ≤ 0.5 nM, ≤ 0.2 nM, ≤ 0.1 nM, ≤0.05 nM, ≤ 0.01 nM, or ≤ 0.001 nM (e.g. 10 -8 M or less, e.g. from 10 -8 M to 10 -13 M, e.g., from 10 -9 M to 10 -13 M) for the SARS-CoV-2, such as the S1 glycoprotein.
In some embodiments, the antibody of the invention inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2. Such binding can be assessed in vitro using, for example, an ELISA assay using immobilized SARS-CoV-2 antigen on a solid support, or binding to a cell expressing ACE2 receptor on the surface.
In some embodiments, the antibody of the invention inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 with an EC50 value of less than 1 nM or 0.1 nM.
In some embodiments, the antibody of the invention exhibits neutralizing activity  against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 10 nM, 6 nM, 3 nM, 2 nM, 1 nM, 0.6 nM or less than 0.5 nM.
In some embodiments, an antibody having any the characteristics provided herein inhibits at least 25%, 50%, 75%, 80%, 90%or 100%of the entry of SARS-CoV-2 into a host cell, such as according to the in vitro assay conditions used in the examples for entry into Vero E6 cells. Inhibition of live virus entry can be assayed based on the concentration of antibodies needed to protect about 50%SARS-CoV-2 susceptible cells, such as Vero E6 cells growing on monolayer, from exhibiting CPE (cytopathic effect) 3-5 days post infection (dpi) .
In some embodiments, the antibody of the invention inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 10 nM, less than 5 nM, less than 2 nM, less than 1 nM, less than 0.5 nM, less than 0.2 nM, less than 0.1 nM, less than 0.08 nM, less than 0.06 nM, less than 0.02 nM, or less than 0.01 nM.
In some embodiments, the antibody of the invention inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 10 nM, 5 nM, 3 nM, 2 nM, 1 nM, 500 pM, 300 pM, 200 pM, 100 pM, 80 pM, 50 pM, 30 pM, 10 pM, or less than 5 pM.
In some embodiments, multispecific antibodies are provided. In some embodiments, bispecific antibodies are provided. Non-limiting exemplary bispecific antibodies include antibodies comprising a first arm comprising a heavy chain/light chain combination that binds a first epitope of SARS-CoV-2 and a second arm comprising a heavy chain/light chain combination that binds a second epitope of SARS-CoV-2. A further non-limiting exemplary multispecific antibody is a dual variable domain antibody.
In certain embodiments, the monoclonal antibodies of the invention or antigen-binding fragments thereof, including human monoclonal antibodies or antigen-binding fragments thereof, include one or more point mutations of in amino acid sequences that are designed to improve developability of the antibody. For example, Raybould et al. (Five computational developability guidelines for therapeutic antibody profiling, PNAS 116 (10) : 4025-4030, 2019) described Therapeutic Antibody Profiler (TAP) , a computational tool that builds downloadable homology models of variable domain sequences, tests them against five developability guidelines, and reports potential sequence liabilities and canonical forms. The authors further provide TAP as freely available at opig. stats. ox. ac. uk/webapps/sabdab-sabpred/TAP. php.
There are many barriers to therapeutic mAb development, besides achieving the desired affinity to the antigen. These include intrinsic immunogenicity, chemical and  conformational instability, self-association, high viscosity, polyspecificity, and poor expression. For example, high levels of hydrophobicity, particularly in the highly variable complementarity-determining regions (CDRs) , have repeatedly been implicated in aggregation, viscosity, and polyspecificity. Asymmetry in the net charge of the heavy-and light-chain variable domains is also correlated with self-association and viscosity at high concentrations. Patches of positive and negative charge in the CDRs are linked to high rates of clearance and poor expression levels. Product heterogeneity (e.g., through oxidation, isomerization, or glycosylation) often results from specific sequence motifs liable to post-or co-translational modification. Computational tools are available to facilitate the identification of sequence liabilities. Warszawski et al. (Optimizing antibody affinity and stability by the automated design of the variable light-heavy chain interfaces. PLoS Comput Biol 15 (8) : e1007207. https: //doi. org/10.1371/journal. pcbi. 1007207) also described methods of optimizing antibody affinity and stability by an automated design of the variable light-heave chain interfaces. Additional methods are available to identify potential developability issues of a candidate antibody, and in preferred embodiments of this invention, one or more point mutations can be introduced, via conventional methods, to the candidate antibody to address such issues to lead to an optimized therapeutic antibody of the invention.
The sequences of certain representative antibodies, including the light chain (LC) and heavy chain (HC) variable regions, the CDR regions, and the framework regions (FR) , are listed below.
Ab-1
VH-CDR1: CTVSGGSISSSIYYWGW (SEQ ID NO: 1)
VH-CDR2: GSIYYSGNAYYN (SEQ ID NO: 2)
VH-CDR3: CATPHTRWGPDYW (SEQ ID NO: 3)
HCVR:
Figure PCTCN2021100414-appb-000003
Heavy chain sequence:
Figure PCTCN2021100414-appb-000004
Figure PCTCN2021100414-appb-000005
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000006
VL-CDR1: CTRSSGSIASNYVLW (SEQ ID NO: 4)
VL-CDR2: EDDQRPS (SEQ ID NO: 5)
VL-CDR3: CQSYDGDNLVF (SEQ ID NO: 6)
LCVR:
Figure PCTCN2021100414-appb-000007
Light chain sequence:
Figure PCTCN2021100414-appb-000008
For all the antibody heavy chain sequences, the framework region sequences HFR1 -HFR4 are defined by the VH-CDR sequences. For example, HFR1 is the sequence of HCVR that is N-terminal to VH-CDR1. HFR2 is the sequence of HCVR that is between VH-CDR1 and VH-CDR2. HFR3 is the sequence of HCVR that is between VH-CDR2 and VH-CDR3. HFR4 is the most C-terminal sequence of HCVR.
Likewise, for all the antibody light chain sequences, the framework region sequences LFR1 -LFR4 are defined by the VL-CDR sequences. For example, LFR1 is the sequence of LCVR that is N-terminal to VL-CDR1. LFR2 is the sequence of LCVR that is between VL-CDR1 and VL-CDR2. LFR3 is the sequence of LCVR that is between VL-CDR2 and VL-CDR3. LFR4 is the most C-terminal sequence of LCVR.
Ab-2
VH-CDR1: CAASGFIVSSNYMSW (SEQ ID NO: 11)
VH-CDR2: SIIYSGGSTFYA (SEQ ID NO: 12)
VH-CDR3: CARDLQELGSLDYW (SEQ ID NO: 13)
HCVR:
Figure PCTCN2021100414-appb-000009
HC:
Figure PCTCN2021100414-appb-000010
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000011
VL-CDR1: CRASQGISSWLAW (SEQ ID NO: 14)
VL-CDR2: AASSLQS (SEQ ID NO: 15)
VL-CDR3: CQEANSFPYTF (SEQ ID NO: 16)
LCVR:
Figure PCTCN2021100414-appb-000012
LC:
Figure PCTCN2021100414-appb-000013
Figure PCTCN2021100414-appb-000014
Ab-3
VH-CDR1: CTVSGGSISSTIYYWGW (SEQ ID NO: 21)
VH-CDR2: GSIYYSGNAYYN (SEQ ID NO: 22)
VH-CDR3: CATPHTRWGPDYW (SEQ ID NO: 23)
HCVR:
Figure PCTCN2021100414-appb-000015
HC:
Figure PCTCN2021100414-appb-000016
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000017
VL-CDR1: CTRSSGSIASNYVLW (SEQ ID NO: 24)
VL-CDR2: EDDQRPS (SEQ ID NO: 25)
VL-CDR3: CQSYDGDNLVF (SEQ ID NO: 26)
LCVR:
Figure PCTCN2021100414-appb-000018
Figure PCTCN2021100414-appb-000019
LC:
Figure PCTCN2021100414-appb-000020
Ab-4
VH-CDR1: CTFSGFSLSTSGVGVGW (SEQ ID NO: 31)
VH-CDR2: ALIYWDDDKRYS (SEQ ID NO: 32)
VH-CDR3: CAHRLSNFWSGYYTGW (SEQ ID NO: 33)
HCVR:
Figure PCTCN2021100414-appb-000021
HC:
Figure PCTCN2021100414-appb-000022
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000023
VL-CDR1: CRSSQSLLHSNGYNYLDW (SEQ ID NO: 34)
VL-CDR2: LGSNRAS (SEQ ID NO: 35)
VL-CDR3: CMQALQTPNTF (SEQ ID NO: 36)
LCVR:
Figure PCTCN2021100414-appb-000024
LC:
Figure PCTCN2021100414-appb-000025
Ab-5
VH-CDR1: CGVSGGSISSYYWSW (SEQ ID NO: 51)
VH-CDR2: GHIYDSGSTNYN (SEQ ID NO: 52)
VH-CDR3: CARQLWLRGAFDIW (SEQ ID NO: 53)
HCVR:
Figure PCTCN2021100414-appb-000026
HC:
Figure PCTCN2021100414-appb-000027
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000028
Figure PCTCN2021100414-appb-000029
VL-CDR1: CTGSSGSIASNYVQW (SEQ ID NO: 54)
VL-CDR2: EDQQRPS (SEQ ID NO: 55)
VL-CDR3: CQSYDSTNQVF (SEQ ID NO: 56)
LCVR:
Figure PCTCN2021100414-appb-000030
LC:
Figure PCTCN2021100414-appb-000031
Ab-6
VH-CDR1: CKVSGYTLTELSMHW (SEQ ID NO: 61)
VH-CDR2: GGFDPEDGETIYA (SEQ ID NO: 62)
VH-CDR3: CATGHQLLFYNWFDPW (SEQ ID NO: 63)
HCVR:
Figure PCTCN2021100414-appb-000032
HC:
Figure PCTCN2021100414-appb-000033
Figure PCTCN2021100414-appb-000034
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000035
VL-CDR1: CTGTSSDVGGYNYVSW (SEQ ID NO: 64)
VL-CDR2: EVSKRPS (SEQ ID NO: 65)
VL-CDR3: CSSYAGSNNLVF (SEQ ID NO: 66)
LCVR:
Figure PCTCN2021100414-appb-000036
LC:
Figure PCTCN2021100414-appb-000037
Ab-7
VH-CDR1: CAASGFAFSSYTMNW (SEQ ID NO: 71)
VH-CDR2: SSISSSSDYIFYA (SEQ ID NO: 72)
VH-CDR3: CARGSNTAWGGVPDAFDFW (SEQ ID NO: 73)
HCVR:
Figure PCTCN2021100414-appb-000038
HC:
Figure PCTCN2021100414-appb-000039
Figure PCTCN2021100414-appb-000040
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000041
VL-CDR1: CTGTSSDVGRYNYVSW (SEQ ID NO: 74)
VL-CDR2: EVSKRPS (SEQ ID NO: 75)
VL-CDR3: CSSYAGSNNLVF (SEQ ID NO: 76)
LCVR:
Figure PCTCN2021100414-appb-000042
LC:
Figure PCTCN2021100414-appb-000043
Ab-8
VH-CDR1: CAAPGFIVSSNYMSW (SEQ ID NO: 81)
VH-CDR2: SIIYSGGSTFYA (SEQ ID NO: 82)
VH-CDR3: CARDLQELGSLDYW (SEQ ID NO: 83)
HCVR:
Figure PCTCN2021100414-appb-000044
Figure PCTCN2021100414-appb-000045
HC:
Figure PCTCN2021100414-appb-000046
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000047
VL-CDR1: CRASQGISSWLAW (SEQ ID NO: 84)
VL-CDR2: AASSLQS (SEQ ID NO: 85)
VL-CDR3: CQEANSFPYTF (SEQ ID NO: 86)
LCVR:
Figure PCTCN2021100414-appb-000048
LC:
Figure PCTCN2021100414-appb-000049
Ab-9
VH-CDR1: CAASGFIVSSNYMSW (SEQ ID NO: 91)
VH-CDR2: SIIYSGGSTFYA (SEQ ID NO: 92)
VH-CDR3: CARDLQELGSLDCW (SEQ ID NO: 93)
HCVR:
Figure PCTCN2021100414-appb-000050
HC:
Figure PCTCN2021100414-appb-000051
HCVR-encoding polynucleotide sequence:
Figure PCTCN2021100414-appb-000052
VL-CDR1: CRASQGISSWLAW (SEQ ID NO: 94)
VL-CDR2: AASSLQS (SEQ ID NO: 95)
VL-CDR3: CQEANSFPYTF (SEQ ID NO: 96)
LCVR:
Figure PCTCN2021100414-appb-000053
LC:
Figure PCTCN2021100414-appb-000054
In certain embodiments, the HC and/or LC further includes a signal peptide sequence: MGWSCIILFLVATATGAHS (SEQ ID NO: 41) .
6. Human Antibodies
The invention described herein provides human antibodies or functional fragment thereof specific for an antigen of SARS-CoV-2, such as the S1 glycoprotein.
In certain embodiments, the human antibodies are isolated /purified from convalescent patients recovering from SARS-CoV-2 infection.
In certain embodiments, the human antibodies share one or more CDR sequences with the patient-isolated antibodies described herein, such as antibodies having the same HCVR and/or LCVR CDR1-3 sequences, or antibodies having the same HCVR and/or LCVR sequences but different constant region sequences, such as modified Fc region sequence, or mutations in the constant region that enhances antibody stability and/or confers additional therapeutic benefits.
Human antibodies can be made by any suitable method. Non-limiting exemplary methods include making human antibodies in transgenic mice that comprise human immunoglobulin loci. See, e.g., Jakobovits et al., Proc. Natl. Acad. Sci. USA 90: 2551-55 (1993) ; Jakobovits et al, Nature 362: 255-8 (1993) ; onberg et al, Nature 368: 856-9 (1994) ; and U.S. Patent Nos. 5,545,807; 6,713,610; 6,673,986; 6,162,963; 5,545,807; 6,300,129; 6,255,458; 5,877,397; 5,874,299; and 5,545,806.
Non-limiting exemplary methods also include making human antibodies using phage display libraries. See, e.g., Hoogenboom et al., J. Mol. Biol. 227: 381-8 (1992) ; Marks et al, J. Mol. Biol. 222: 581-97 (1991) ; and PCT Publication No. WO 99/10494.
Human Antibody Constant Regions
In some embodiments, a human antibody described herein comprises human constant region sequences. In some embodiments, the human heavy chain constant region is of an isotype selected from IgA, IgG, and IgD. In some embodiments, the human light chain constant region is of an isotype selected from K and λ. In some embodiments, an antibody described herein comprises a human IgG constant region, for example, human IgG1, IgG2, or IgG3. In some embodiments, an antibody or Fc fusion partner comprises a C237S mutation, for example, in an IgG1 constant region. In some embodiments, an antibody described herein comprises a human IgG2 heavy chain constant region. In some such embodiments, the IgG2 constant region comprises a P331S mutation, as described in U.S. Patent No. 6,900,292.
The choice of heavy chain constant region can determine whether or not an antibody will have effector function in vivo. Such effector function, in some embodiments, includes  antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) , and can result in killing of the cell to which the antibody is bound. Typically, antibodies comprising human IgG1 or IgG3 heavy chains have effector function.
Any of the antibodies described herein may be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include the antigen and/or epitope to which the antibody binds, and ligands that bind antibody constant regions. For example, a Protein A, Protein G, Protein A/G, or an antibody affinity column may be used to bind the constant region and to purify an antibody.
In some embodiments, hydrophobic interactive chromatography (HIC) , for example, a butyl or phenyl column, is also used for purifying some polypeptides. Many methods of purifying polypeptides are known in the art.
Alternatively, in some embodiments, an antibody described herein is produced in a cell-free system. Nonlimiting exemplary cell-free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009) ; Spirin, Trends Biotechnol. 22: 538-45 (2004) ; Endo et al, Biotechnol. Adv. 21 : 695-713 (2003) .
7. Nucleic Acid Molecules Encoding Antibody
The invention also provides nucleic acid molecules comprising polynucleotides that encode one or more chains of an antibody described herein. In some embodiments, a nucleic acid molecule comprises a polynucleotide that encodes a heavy chain or a light chain of an antibody described herein. In some embodiments, a nucleic acid molecule comprises both a polynucleotide that encodes a heavy chain and a polynucleotide that encodes a light chain, of an antibody described herein. In some embodiments, a first nucleic acid molecule comprises a first polynucleotide that encodes a heavy chain and a second nucleic acid molecule comprises a second polynucleotide that encodes a light chain.
In some such embodiments, the heavy chain and the light chain are expressed from one nucleic acid molecule, or from two separate nucleic acid molecules, as two separate polypeptides. In some embodiments, such as when an antibody is an scFv, a single polynucleotide encodes a single polypeptide comprising both a heavy chain and a light chain linked together.
In some embodiments, a polynucleotide encoding a heavy chain or light chain of an antibody described herein comprises a nucleotide sequence that encodes a leader sequence, which, when translated, is located at the N-terminus of the heavy chain or light chain. As  discussed above, the leader sequence may be the native heavy or light chain leader sequence, or may be another heterologous leader sequence.
Nucleic acid molecules may be constructed using recombinant DNA techniques conventional in the art. In some embodiments, a nucleic acid molecule is an expression vector that is suitable for expression in a selected host cell, such as a mammalian cell.
8. Vectors
Vectors comprising polynucleotides that encode heavy chains and/or light chains of the antibodies described herein are provided. Such vectors include, but are not limited to, DNA vectors, phage vectors, viral vectors, retroviral vectors, etc. In some embodiments, a vector comprises a first polynucleotide sequence encoding a heavy chain and a second polynucleotide sequence encoding a light chain. In some embodiments, the heavy chain and light chain are expressed from the vector as two separate polypeptides. In some embodiments, the heavy chain and light chain are expressed as part of a single polypeptide, such as, for example, when the antibody is an scFv.
In some embodiments, a first vector comprises a polynucleotide that encodes a heavy chain and a second vector comprises a polynucleotide that encodes a light chain. In some embodiments, the first vector and second vector are transfected into host cells in similar amounts (such as similar molar amounts or similar mass amounts) . In some embodiments, a mole-or mass-ratio of between 5: 1 and 1: 5 of the first vector and the second vector is transfected into host cells. In some embodiments, a mass ratio of between 1: 1 and 1: 5 for the vector encoding the heavy chain and the vector encoding the light chain is used. In some embodiments, a mass ratio of 1: 2 for the vector encoding the heavy chain and the vector encoding the light chain is used.
In some embodiments, a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells. Exemplary such vectors are described, e.g., in Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004) . In some embodiments, a vector is chosen for in vivo expression of the subject antibody in animals, including humans. In some such embodiments, expression of the polypeptide or polypeptides is under the control of a promoter or promoters that function in a tissue-specific manner. For example, liver-specific promoters are described, e.g., in PCT Publication No. WO 2006/076288.
9. Host Cells
In various embodiments, heavy chains and/or light chains of the antibodies described  herein may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast) , plant cells, insect cells, and mammalian cells. Such expression may be carried out, for example, according to procedures known in the art. Exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-Sand DG44 cells; PER. 
Figure PCTCN2021100414-appb-000055
cells (Crucell) ; and NSO cells. In some embodiments, heavy chains and/or light chains of the antibodies described herein may be expressed in yeast. See, e.g., U.S. Publication No. US 2006/0270045 Al. In some embodiments, a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the heavy chains and/or light chains of the subject antibody. For example, in some embodiments, CHO cells produce polypeptides that have a higher level of sialylation than the same polypeptide produced in 293 cells.
Introduction of one or more nucleic acids into a desired host cell may be accomplished by any method, including but not limited to, calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, etc., Nonlimiting exemplary methods are described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd ed. Cold Spring Harbor Laboratory Press (2001) . Nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
In some embodiments, one or more polypeptides may be produced in vivo in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptides, according to any suitable method.
EXAMPLES
Example 1 Profiling B cell Immune Responses to SARS-CoV-2 in Convalescent Patients Recovering from Coronavirus Disease 2019 (COVID-19)
The generation of a protective and long-lasting humoral immunity is one major aspect of the adaptive immune response to infection. Using the methods and systems described herein, this example profiled humoral responses in infected and recovering COVID-19 patients and identified antibodies targeting SARS-CoV-2 from recovered patients’s amples.
From the 26 patients profiled, antibodies in patient serum were isolated through immunoprecipitation using the viral S protein. Mass spectrometry (MS) was then applied to analyse these isolated antibodies. More than 80 antibodies were identified from mass  spectrometry analysis.
Patient blood sample collection and characterization
Blood samples were collected from 26 patients at Xinhua Hospital. These patients were previously diagnosed with COVID-19, and had since recovered from the disease after treatment. Patients ranged in age from 8 to 78 years old, with a median age of 47 years old. Diagnosis of infection with SARS-CoV-2 was made on the basis of positive RT-PCR test results, and blood samples were collected after patients had recovered and tested negative on a subsequent RT-PCR test. The average time between the initial positive RT-PCR test and the subsequent negative RT-PCR test was about 18.7 days, with a median time of 17 days. Blood samples were collected, and peripheral blood mononuclear cells (PBMCs) and plasma were isolated and frozen for subsequent analysis between 2-4 days after patients tested negative for SARS-CoV-2.
To characterize antiviral immune responses, antibody titers in patient sera were measured against the SARS-CoV-2 Spike (S) protein (Wrapp et al., Science, 367 (6483) : 1260 LP –1263. https: //doi. org/10. 1126/science. abb25072020) . Sera from convalescent patients clearly showed higher titers as compared to sera from healthy donors (FIG. 1A) . In general, sera from male patients showed higher titers relative to female patients (FIG. 1B) .
Sera from a selected set of patients was further evaluated for binding to the Nucleocapsid (N) protein of SARS-CoV-2. The relative reactivity of patient sera against the S and N proteins were similar, with patients showing higher titers against the S protein also showing higher titers to the N protein. Binding to the N protein could be detected at lower dilutions of sera, but this may be reflective of the ELISA format used to measure antibody titers.
The S protein is presented as a trimer on the viral envelope, and it consists of two subunits: a receptor-binding S1 subunit on top of an S2 stalk that mediates membrane fusion and viral entry (Li, Annual Review of Virology, 3 (1) , 237–261. https: //doi. org/10.1146/annurev-virology-110615-042301, 2016) . To assess the immune response to different domains and conformations of the SARS-CoV-2 S protein, serum from a high-titer patient was titrated against full-length S1+S2-His protein, an S1-mFc fusion protein, S1-His protein, and the S1 receptor binding domain (S1-RBD) . Binding activity was stronger against the full-length S1+S2-His protein and the S1-mFc fusion protein as compared to the monomeric S1-His and S1-RBD domains (FIG. 2) , suggesting that an immune response had been also generated against the S2 domain and that the multimerization  state of the S protein influenced antibody binding.
B cell profiling
To characterize the B cell populations of patient samples, PBMCs isolated from peripheral blood collected from two patients were thawed, and flow cytometry analysis was performed on the cells. Cells were stained with a panel of anti-CD19 /CD27 /CD38 antibodies, and plasmablast (characterized by CD19 +/CD27 +/CD38 +) and memory B cells (characterized by CD19 +/CD27 +/CD38 -) were identified. Compared to cells isolated from a healthy donor, patient samples showed higher proportions of both plasmablasts and memory B cells within the CD19 + B cell compartment (FIG. 3) . While CD19 positive populations from two patients are somewhat lower than the control healthy volunteer with overall reduced lymphocytes as reported previously (Tian et al., Emerging Microbes and Infections, 9 (1) , 382–385. https: //doi. org/10. 1080/22221751. 2020. 1729069, 2020) , the percentages of the two patients for memory B cell (CD27 high CD38 low) are significantly higher than the healthy donor. In addition, the percentages plasmablasts (CD27 high CD38 high) are about 20-fold higher compared with healthy donor.
Example 2 Identification of SARS-CoV-2 specific antibodies
Since humoral responses against virus can be applied further for potential diagnosis and treatment of infected patients (Casadevall &Pirofski, The Journal of Clinical Investigation, 2. https: //doi. org/10. 1172/JCI138003, 2020) , together with the observation of increased fraction of memory B cells as well as plasmablasts, antibodies from convalescent patients were identified.
Specifically, to identify SARS-CoV-2 specific antibodies, immunoprecipitation and mass spectrometry analysis were used to identify antibodies isolated from patient serum specifically against viral spike protein.
SARS-CoV-2 specific antibodies were identified using a method consisted of affinity-purification of antigen specific antibodies from patient serum, followed by liquid chromatography-mass spectrometry (LC-MS) analysis. In parallel the B cells from the same patients were sequenced to generate an IgG repertoire database with paired V H and V L chains. The mass spectra data together with the paired repertoire database were then used to map out the antigen specific antibody sequences (Lavinder, Horton, Georgiou, &Ippolito, Current Opinion in Chemical Biology, 24, 112–120. https: //doi. org/10. 1016/j. cbpa. 2014. 11. 007, 2015) . The immunoprecipitation allows affinity-based enrichment of virus antigen-specific antibodies from patient serum. After analyzing those antibodies by LC-MS, the high- resolution and high mass accuracy tandem mass spectra allow unambiguous identification of the peptides covering all CDRs from the specific heavy chains and light chains. The identified heavy chains and light chains were then analyzed to form paired antibody sequences.
For convalescent Patient 011, as shown in FIG. 4A, the antibodies purified from serum showed a ～150 kDa band for patient samples but not for the healthy donor control. Patient 011’s sample was shown in lane 5 in the Western blot for purified antibodies. The mass spectrometry data for Patient 011 was searched against the repertoire database. After manual verification of CDR3 peptide spectra, a total of 52 V H and 25 V L sequences with different CDR3 were identified. Selected CDR3 sequences for V H chains were shown in FIG. 4B, with significant degree of diversity. The broad diversity of the HC CDR3 is not only required for obtaining different epitopes, but also required for screening for an antibody with a specific property, as for the current case, neutralizing virus or blocking virus’ entry to the cell by blocking the S protein-ACE2 interaction.
In summary, by analyzing those sequences for their pairing information, a total of 80 antibodies belonging to 31 different CDR3 groups were retrieved and synthesized. Example mass spectra for an identified antibody with HC F22a1-VH0 and LC F22a1-VL0 were shown as in FIGs. 4C-4E. FIG. 4C and FIG. 4D showed 2 consecutive peptides (LNSVTAADTAVYYCATPHTR (SEQ ID NO: 105) and WGPDYWGQGTLVTVTVSSPSTK (SEQ ID NO: 106) ) covering heavy chain F22a1-VH0’s full CDR3 with almost all fragment y ions matched. The paired LC F22a1-VL0 was also identified with high confidence mass spectra with CDR3 peptide TEDEADYYCQSYDGDNLVFGGGTK (SEQ ID NO: 107, FIG. 4E) . Identification of both paired HC and LC further improved the confidence for the identity of the antibody.
Example 3 Characterization of SARS-CoV-2 specific antibodies
Many antibodies specific for SARS-CoV-2 antigens were identified and further characterized herein, including antibodies Ab-1, Ab-2, Ab-3, Ab-4, Ab-5, Ab-6, Ab-7, Ab-8, and Ab-9. Among them, Ab-5 to Ab-7 are weaker binders compared to Ab-1 to Ab-4. Ab-8 and Ab-9 shares the same VL sequence with that of Ab-2, but with 1 amino acid difference in the VH region. The other Ab-2 alternatives, all with the same CDR but different framework region (FR) sequences are not listed.
The relevant sequences of these representative antibodies are listed below.
Figure PCTCN2021100414-appb-000056
Figure PCTCN2021100414-appb-000057
Figure PCTCN2021100414-appb-000058
Figure PCTCN2021100414-appb-000059
Figure PCTCN2021100414-appb-000060
Figure PCTCN2021100414-appb-000061
Figure PCTCN2021100414-appb-000062
Figure PCTCN2021100414-appb-000063
Figure PCTCN2021100414-appb-000064
Figure PCTCN2021100414-appb-000065
o Signal peptide sequences are not included; the variable regions are in bold font; and the constant regions are in normal font
HC and LC signal peptide sequence: MGWSCIILFLVATATGAHS (SEQ ID NO: 41) .
The antibodies of the invention have been demonstrated to be able to bind to the spike protein of the SARS-CoV-2 virus, based on binding assays for S1 and S2 domains, as well as  the full length of the S protein. ELISA results showed that at least 20 antibodies bound to the viral S protein with different affinity, among which Ab-2 was the strongest binder, and Ab-4 is another top candidate which also binds to S full length protein (see FIG. 10) . Ab-1 and Ab-3 are two other binding candidates with reduced B max and EC50 values comparing with Ab-2 and Ab-4, however, with nM level binding affinity, nevertheless. These two antibodies share the same heavy chain, with difference in their light chains. ELISA results showed that the antibodies can effectively bind to full length S protein of SARS-CoV-2 with EC50 ranging from 10 -9 -10 -10 M (FIG. 5, left panel) . Interestingly, when the binding of the specific domain of S protein was evaluated, Ab-1 to Ab-3 were shown to be binders to the S1 fragment, while Ab-4 to be a binder to the S2 fragment (middle and right panels of FIG. 5) , respectively, showing the diversity of mAb being identified.
Next, the blocking effects of the top four antibodies on the binding of S1 to Vero E6 cell line was analyzed by FACS analysis (FIG. 6A) . As expected, Ab-1 to Ab-3 blocked S1 binding to E6 at the nM range, while S2 binder Ab-4 did not affect S1 binding to the receptor on Vero E6 cells as expected.
The blocking activities of these antibodies were also tested for blocking S1 binding to ACE2 using ELISA assay (data not shown) . Again, Ab-1, Ab-2, &Ab-3 showed potent blocking activities with potency at 10 -9 -10 -10 M, and Ab-4 did not show any blocking activity on S1 binding to ACE2.
Next, the neutralizing activities of these antibodies against HIV-vectored pseudovirus were examined. Antibodies Ab-2 exhibited potent neutralizing activity, with IC50 of 0.53 nM on average. Ab-3 and Ab-1 showed IC50s at 5.80 nM and 4.07 nM respectively, whereas Ab-4 displayed no neutralizing activities (FIG. 6B) . These antibody activities from S protein binding to blockage of S1 binding to Vero E6 cells and further to pesudovirus neutralization suggested that S1 binding is a critical step for virus to bind to host cells, and blocking of S1 binding to host cells could potentially be translated into anti-viral activities. The results are summarized in the table below.
Figure PCTCN2021100414-appb-000066
Figure PCTCN2021100414-appb-000067
Sequence analysis showed that each cluster of antibodies shared at least 93%sequence identity. The difference within the same sequence cluster may result from somatic hypermutation in which point mutations accumulated in the antibody. Because the somatic hypermutation does not distinguish between favorable and unfavorable mutations, 12 closely similar sequences in the cluster comprising Ab-2 were chosen to compare their activity experimentally.
As shown in FIGs. 8A and 8B, 11 antibodies (labeled as “2A2A-x” wherein x is 1-4, 9-11, 14, 15, 18 and 19) from the same family of Ab-2 (also labeled as “2A2A” ) demonstrated various levels of binding abilities to the full-length S protein as well as the abilities to block the S1 protein binding to human ACE2 receptor. However, Ab-2 demonstrated the best potency in both assays, with a few close family members showing similar activities while 2A2A-4 with 1 amino acid difference in CDR1, and 2A2A-19 with 1 amino acid difference in CDR3, others with differences in the FR regions.
  2A2A 2A2A-4 2A2A-3 2A2A-1 2A2A-2 2A2A-10 2A2A-9 2A2A-11 2A2A-15 2A2A-18 2A2A-19 2A2A-14
EC50 0.55 0.57 0.66 0.68 0.81 1.05 1.27 4.35 4.65 14.12 56.5 ～124.2
IC50 (nM) 1.42 1.6 1.62 1.7 2.31 3.42 3.46 4.14 13.55 35.54 127.72 No inh
Difference   1AA 1AA 1AA 1AA 2AA 1AA 1AA 1AA 1AA 1AA 1AA
    CDR1 FR4 FR4 FR4 FR4 FR4 FR1 FR3 FR1 CDR3 FR3
The neutralizing activities of the lead antibodies Ab-1 to Ab-3 were further confirmed in live SARS-CoV-2 virus entry assay. Vero E6 cells were infected with SARS-CoV-2 virus at 100 TCID50 in the presence of the lead antibodies at different concentrations. Using fluorescent labeled nuclear protein of SAR-CoV-2, infected cells can be observed by florescence microscopy. Antibody Ab-2 demonstrated strong anti-virus activities -more than 50%inhibition at 6.5 nM, while Ab-3 demonstrated 74%inhibition at 62.7 nM, and Ab-1 showed 93.8%inhibition at 50.2 nM (FIG. 7A) . The ND50s and ND90s have been further calculated through dose responding curve. Ab-2 has a ND50 of 0.751 nM, a ND90 of 1.682 nM (FIG. 7B, left panel) ; Ab-1 has a ND50 of 4.153 nM, a ND90 of 6.1 nM (FIG. 7B, middle panel) ; and Ab-3 has a ND50 of 5.512 nM, a ND90 of 24.08 nM (FIG. 7B, right panel) . 
  Ab-1 Ab-2 Ab-3
ND50 (μg/mL) 4.153 0.751 5.512
ND90 (μg/mL) 6.100 1.682 14.076
As the pandemics progressed, the mutations located in S protein accumulated and may confer selective advantages in transmission and resistance to the antibody intervention. Thus, the affinity of Ab-2 for 4 dominant spike RBD mutations, and its ability to competing with ACE2 for RBD binding were examined. SPR results showed that Ab-2 bound to mutations with similar affinity as to the wild-type S protein. Ab-2 also blocked the interaction of ACE2 with all RBD mutations (data not shown) .
Example 4 Pharmacological profile of the neutralizing antibodies
The three most potent antibodies binding to S1 protein has been further profiled pharmacologically. A snapshot PK study at 1 h, 24 h and 72 h separately in wild-type (WT) mice showed good PK profile. While no significant difference was observed among the 3 Abs, Ab-2 showed slightly better exposure comparing with Ab-1 and Ab-3 (FIG. 9) . This results provide further confidence that these antibodies can be utilized for further development as a drug candidate (s) to treat COVID-19 patients.
The data provided herein provides a deeper understanding of the immune response mounted by patients recovering from COVID-19, and may give insights into disease epidemiology and inform the development of novel therapies.
For example, despite the documented greater severity of the disease in males as compared to females, males seem to demonstrate higher antiviral titers as a group. This may be related to higher viral loads during the period of peak infection. Several mechanisms have been proposed to explain the apparently greater susceptibility of males to COVID-19, including 1) A high prevalence of smoking among males in regions where the SARS-CoV-2 virus has spread to humans, and 2) the ACE2 gene that acts as a receptor for the virus on human epithelial cells being located on the X chromosome, which may lead to sex-specific differences in ACE2 expression and susceptibility to infection or viral burden. Children have been observed to have less severe disease symptoms as compared to adults (Dong et al., 2020) . Interestingly, an 8-year-old patient profiled as part of the cohort studied here showed relatively high titers to the SARS-CoV-2 S protein, suggesting that despite their less mature immune systems, children are capable of mounting a robust antiviral immune response.
COVID-19 patients tend to display low lymphocyte counts, and reduced levels and functional exhaustion of T cells has been described in patients with severe disease (Ni et al., 2020) . It is therefore notable to find increased levels of circulating plasma cells as well as  memory B cells in convalescent patients weeks after initial infection and days after recovery, suggesting that the humoral immune response is critical in limiting viral activity.
Use of convalescent patient sera as an immediate way to transfer protective immunity to newly diagnosed patients and at-risk populations is a therapeutic strategy being applied to treat COVID-19, and neutralizing antiviral monoclonal antibodies identified from these sera have the utility to be scalable treatments for the disease. Further mining of immune repertoires from patients recovering from COVID-19, using their methods of the invention described herein can be key to controlling the future spread of this and other similar viruses.
The methods described herein identified antibodies binding to SARS-CoV-2 coronavirus, thus permitting further characterization of the neutralizing activities of these antibodies, as well as mapping out binding epitopes of these antibodies. In one embodiment, these neutralizing antibodies can be formulated for use as therapeutic antibodies for patient treatment. In another embodiment, they can also be used prophylactically to prevent virus infection. In further embodiments, certain binding antibodies can be used in combination with the vaccine approaches, even if they do not have neutralization activities. In yet another embodiments, different antibodies with either S1 or S2 binding capacity are used to generate multi-valent antibodies, or be used together, for combination therapy.
Materials and Methods
Study design and participants
COVID-19 patients who subsequently recovered were clinically diagnosed and laboratory-confirmed. A laboratory-confirmed case of COVID-19 was defined as a positive result on high-throughput sequencing or real-time reverse-transcriptase polymerase-chain-reaction (RT-PCR) assay of nasal and pharyngeal swab specimens on the basis of the WHO interim guidance. The criteria of recovery were normal temperature for at least 3 days, obvious improvement in clinical symptoms, significant absorption of pulmonary inflammation on computer tomography scan and negative tests for SARS-CoV-2 two times in a row with a test interval for at least one day. Patients tested positive for HIV, HBV, HAV, HCV or syphilis were excluded. Finally, 26 patients and two healthy volunteers were included in the samples. Blood samples were collected and peripheral blood mononuclear cells (PBMCs) and plasma were isolated and frozen for subsequent analysis between 2-4 days after patients recovered from COVID-19.
PBMC and serum sample preparation
Blood samples were collected 3-4 days after donors were discharged from the hospital  and separated into plasma and peripheral blood mononuclear cells (PBMCs) by Ficoll-Hypaque gradient centrifugation. Plasma and PBMCs which were maintained in freezing media and stored at -80℃ until ready to use. The plasma were heat-inactivated at 56℃ for 1h before use.
Titer measurements by ELISA:
The ELISA protocol was adapted from previously established protocols. Briefly, 384-well plates were coated overnight at 4℃ with PBS containing 1 μg/mL of the respective protein. The next day the plate was washed 4 times with washing buffer (PBS and 0.05%Tween) and then incubated 1 hour at 37 ℃ in blocking buffer (PBS with 2%BSA) . After two washes, the plate was incubated for 1 hour at 37 ℃ with the serum or the positive control ACE2 protein. The human serum samples were diluted to 1: 100 in 1X PBS + 2%BSA followed by 5-fold serial dilutions. The plates were then washed 4 times and incubated for 1 hour in detection reagent (Mouse anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 μg/mL in 1X PBS with 0.05%Tween and 1%BSA) for 1 hour at room temperature. Following this, the plate was washed again 4 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution.
Flow cytometry for B cell immune profiling
PBMC was thawed at 37 ℃ and then centrifuged at 450 g for 8 min. The supernatant was discarded, and the cells resuspended in 200 μL of DMEM. Following the addition of 1 μL of DNase I, cells were incubated for 3 min and spun down again. The pellet was resuspended in 20 μL of FcR Blocking Reagent (vendor) , incubated for 10 mins and centrifuged. The cells were suspended in 200 μL PBS. 3 μL of CD19 (FITC labeled, eBioscience 11-0199-42) , CD27 (APC labeled, eBiosciences 17-0279-42) , anti CD38 (PE labeled mouse IgG1 isotype control, eBioscience 12-0388-42) or its isotype (PE Mouse isotype control, BioLegend 400114 and FITC labeled mouse IgG1 isotype control, ebioscience 11-4714-41 and APC labeled mouse IgG1 isotype control, BD 550854) was then added, and incubated for 30 mins at RT. Following centrifugation, cells were resuspended in a 100 μL of 4%PFA. After 10 min the cells were washed twice by centrifugation and finally resuspended in PBS and ready for flow cytometry analysis using a Cytoflex, Beckman Coulter. The median fluorescence intensity (MFI) was calculated with FlowJo.
Patient sample information for FACS
  labeling Cell Number Viability (%) comments
PBMC Patient  1 2.54*10 5/ml, 200 μL 84 Patient
PBMC Patient  2 7.1*10 5/ml, 200 μL 90 Patient
PBMC Healthy donor 7.0*10 5/ml, 200 μL 92 Healthy Volunteer
Serum antibody IP and MS analysis
In brief, each serum was diluted with PBS to a final volume of 1 mL and then passed through SARS-CoV-2 Spike Protein (RBD, mFc Tag, Sino biological Cat: 40592-V05H) conjugated NHS-sepharose column (200 μL resin) . The column was then washed 3 times with 1 mL of HBS-EP buffer and once with 1 mL of ddH 2O. The bounded antibodies were eluted twice with 300 μL of 300 mM acetic acid. The eluant was subjected to SDS-gel/Western blot and then dried by a speed vac evaporator.
The dried sample was dissolved in 15 μL of 8 M urea and 20 mM TCEP in 20 mM Tris-HCl (pH8.0) at 37℃ for 1hr. The denatured and reduced sample was then alkylated with 5 mM iodoacetamide for 30 min followed by overnight trypsin (w/v=1: 20) digestion at 37℃ with a total final volume of 80 μL in 100 mM Tris-HCl pH 7.5. The reaction was quenched by adding 5 μL 20%FA. The tryptic peptides were analyzed by a nano-LC1200 HPLC coupled with a Q Exactive mass spectrometer. Peptides (20 μL) were first trapped onto a 75 μm × 2 cm C18 trap column at 4 μL/min and were then separated at 250 nL/min using a 75 μm × 25 cm C18 column at 40℃ with the following gradients: 5%-30%ACN in 157 min; 30%-40%ACN in 15 min; 40%-90%ACN in 2 min; 90%ACN for 15 min. The mass spec spectra were acquired under positive mode using following parameters: MS1 resolution: 70,000; MS1 target: 1E6; maximum injection time: 100 ms; scan range: 350 to 1,800 m/z; MS/MS resolution: 17,500; MS/MS target: 2e5; Top N: 10; isolation window: 2 Th;charge exclusion: 1, >5; dynamic exclusion: 30 sec. 
The acquired LC-MS data from each patient was searched against the corresponding IgG repertoire database generated using proteome discoverer software. The searching parameters were as follows: Cleavage site: lysine or Arginine; Cleavage site: C-terminal; Digestion specificity: fully specific; Missed cleavages: 2; Precursor mass tolerance: 10 ppm; Fragmentation type: HCD; Fragment mass tolerance: 20 ppm; Fixed modification: carbamidomethyl at cysteine.
Antibody binding and competition with receptor ACE2:
The binding affinity of antibodies to spike protein was analyzed by ELISA. 384 well plate (Corning#3700) , was coated overnight at 4℃ with PBS containing 30 μL 20 nM of the SARS-CoV-2 Spike S1+S2 ECD, his Tag protein. The next day the plate was washed 5  times with washing buffer (PBS and 0.05%Tween) and then incubated 1 hour at room temperature in blocking buffer (PBS with 2%BSA) . After 5 washes the plate was incubated with serial dilution of purified mAbs for 1 hour at room temperature. The plates were then washed 5 times and incubated for 1 hour in detection reagent (Mouse anti-Human IgG Fc HRP labeled (Thermo Fisher 05-4220) at 0.2 μg/ml in 1X PBS with 0.05%Tween and 1%BSA) for 1 hour at room temperature. Following this the plate was washed again 5 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution. The OD values were determined using Thermo MultiSkan or MD SpectraMax i3X at 450 nm wavelength.
The blocking with receptor ACE2 was performed using cell surface expressed ACE2. 10 nM SARS-CoV-2 Spike S1, mFc tag spike protein was incubated with serial dilution of purified mAbs at room temperature for 1h and then added to Vero E6 cells (approximately 10 5 per well) in duplicate. Then detection reagent rabbit anti mouse IgG Fc-AF647 was used. Half-maximal inhibitory concentration (IC50) of the evaluated mAbs were determined with Beckman Cytoflex and FlowJo software analysis.
Antibody neutralization activity against pseudo virus
Murine leukemia virus-based SARS-CoV-2 S pseudotyped virus were prepared by GenScript as previous described. Neutralization assay were performed by incubating pseudo virus with serial dilution of purified antibodies at room temperature for 1h. ACE2 overexpression Hela cells (approximately 8x10 4 per well) were cultured in DMEM containing 10%FBS, 1 μg/mL puromycin were added in triplicate into virus-antibody mixture. Following infection at 37℃, 5%CO 2 for 48h, half-maximal inhibitory concentration (IC 50) were determined by luciferase activity using Promega Bio-Glo luciferase assay system with GraphPad Prism.
Live virus assay
Vero E6 cells infected by SARS-CoV-2 at 100 TCID50 or incubated with mixture with viruses and diluted antibodies and incubate for 1 hr, and then fixed with 4%paraformaldehyde diluted in PBS (pH=7.2) for 15 minutes at room temperature and then penetrated with 0.25%triton-X 100 for 10-15 minutes. After three washes, cells were blocked at 37℃ for 1 hour using PBS containing 5%BSA, then incubated with in-house prepared anti-SARS-CoV-2 NP rabbit serum as primary antibody and FITC-conjugated goat anti-mouse IgG antibody as the secondary antibody. Cell nuclei were strained using Hoechst 33258 at room temperature for 10 minutes. Images were taken under an inverted fluorescence microscope (Nikon) .
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Claims (34)

  1. An isolated or recombinantly produced monoclonal antibody, or an antigen-binding fragment thereof, wherein said monoclonal antibody or antigen-binding fragment thereof is specific for an antigen of SARS-CoV-2, and wherein said monoclonal antibody comprises:
    (1a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 1, a HCVR CDR2 sequence of SEQ ID NO: 2, and a HCVR CDR3 sequence of SEQ ID NO: 3; and,
    (1b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 4, a LCVR CDR2 sequence of SEQ ID NO: 5, and a LCVR CDR3 sequence of SEQ ID NO: 6; or
    (2a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 11, a HCVR CDR2 sequence of SEQ ID NO: 12, and a HCVR CDR3 sequence of SEQ ID NO: 13; and,
    (2b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 14, a LCVR CDR2 sequence of SEQ ID NO: 15, and a LCVR CDR3 sequence of SEQ ID NO: 16; or
    (3a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 21, a HCVR CDR2 sequence of SEQ ID NO: 22, and a HCVR CDR3 sequence of SEQ ID NO: 23; and,
    (3b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 24, a LCVR CDR2 sequence of SEQ ID NO: 25, and a LCVR CDR3 sequence of SEQ ID NO: 26; or
    (4a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 31, a HCVR CDR2 sequence of SEQ ID NO: 32, and a HCVR CDR3 sequence of SEQ ID NO: 33; and,
    (4b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 34, a LCVR CDR2 sequence of SEQ ID NO: 35, and a LCVR CDR3 sequence of SEQ ID NO: 36; or
    (5a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 51, a HCVR CDR2 sequence of SEQ ID NO: 52, and a HCVR CDR3 sequence of SEQ ID NO: 53; and,
    (5b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of  SEQ ID NO: 54, a LCVR CDR2 sequence of SEQ ID NO: 55, and a LCVR CDR3 sequence of SEQ ID NO: 56; or
    (6a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 61, a HCVR CDR2 sequence of SEQ ID NO: 62, and a HCVR CDR3 sequence of SEQ ID NO: 63; and,
    (6b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 64, a LCVR CDR2 sequence of SEQ ID NO: 65, and a LCVR CDR3 sequence of SEQ ID NO: 66; or
    (7a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 71, a HCVR CDR2 sequence of SEQ ID NO: 72, and a HCVR CDR3 sequence of SEQ ID NO: 73; and,
    (7b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 74, a LCVR CDR2 sequence of SEQ ID NO: 75, and a LCVR CDR3 sequence of SEQ ID NO: 76; or
    (8a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 81, a HCVR CDR2 sequence of SEQ ID NO: 82, and a HCVR CDR3 sequence of SEQ ID NO: 83; and,
    (8b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 84, a LCVR CDR2 sequence of SEQ ID NO: 85, and a LCVR CDR3 sequence of SEQ ID NO: 86; or
    (9a) a heavy chain variable region (HCVR) , comprising a HCVR CDR1 sequence of SEQ ID NO: 91, a HCVR CDR2 sequence of SEQ ID NO: 92, and a HCVR CDR3 sequence of SEQ ID NO: 93; and,
    (9b) a light chain variable region (LCVR) , comprising a LCVR CDR1 sequence of SEQ ID NO: 94, a LCVR CDR2 sequence of SEQ ID NO: 95, and a LCVR CDR3 sequence of SEQ ID NO: 96;
    optionally, said isolated monoclonal antibody is not naturally occurring; and/or,
    optionally further comprising a signal peptide sequence of SEQ ID NO: 41 at the N-terminus of said HCVR and/or LCVR.
  2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein:
    (1A) the HCVR sequence is SEQ ID NO: 7; and/or,
    (1B) the LCVR sequence is SEQ ID NO: 8, or,
    (2A) the HCVR sequence is SEQ ID NO: 17; and/or,
    (2B) the LCVR sequence is SEQ ID NO: 18, or,
    (3A) the HCVR sequence is SEQ ID NO: 27; and/or,
    (3B) the LCVR sequence is SEQ ID NO: 28, or,
    (4A) the HCVR sequence is SEQ ID NO: 37; and/or,
    (4B) the LCVR sequence is SEQ ID NO: 38, or,
    (5A) the HCVR sequence is SEQ ID NO: 57; and/or,
    (5B) the LCVR sequence is SEQ ID NO: 58, or,
    (6A) the HCVR sequence is SEQ ID NO: 67; and/or,
    (6B) the LCVR sequence is SEQ ID NO: 68, or,
    (7A) the HCVR sequence is SEQ ID NO: 77; and/or,
    (7B) the LCVR sequence is SEQ ID NO: 78, or,
    (8A) the HCVR sequence is SEQ ID NO: 87; and/or,
    (8B) the LCVR sequence is SEQ ID NO: 88, or,
    (9A) the HCVR sequence is SEQ ID NO: 97; and/or,
    (9B) the LCVR sequence is SEQ ID NO: 98.
  3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said monoclonal antibody has:
    (1a) a heavy chain sequence of SEQ ID NO: 9; and/or,
    (1b) a light chain sequence of SEQ ID NO: 10, or,
    (2a) a heavy chain sequence of SEQ ID NO: 19; and/or,
    (2b) a light chain sequence of SEQ ID NO: 20, or,
    (3a) a heavy chain sequence of SEQ ID NO: 29; and/or,
    (3b) a light chain sequence of SEQ ID NO: 30, or,
    (4a) a heavy chain sequence of SEQ ID NO: 39; and/or,
    (4b) a light chain sequence of SEQ ID NO: 40, or,
    (5a) a heavy chain sequence of SEQ ID NO: 59; and/or,
    (5b) a light chain sequence of SEQ ID NO: 60, or,
    (6a) a heavy chain sequence of SEQ ID NO: 69; and/or,
    (6b) a light chain sequence of SEQ ID NO: 70, or,
    (7a) a heavy chain sequence of SEQ ID NO: 79; and/or,
    (7b) a light chain sequence of SEQ ID NO: 80, or,
    (8a) a heavy chain sequence of SEQ ID NO: 89; and/or,
    (8b) a light chain sequence of SEQ ID NO: 90, or,
    (9a) a heavy chain sequence of SEQ ID NO: 99; and/or,
    (9b) a light chain sequence of SEQ ID NO: 100.
  4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, which is a human antibody, a CDR-grafted antibody, or a resurfaced antibody.
  5. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-4, wherein said antigen-binding fragment thereof is an Fab, Fab’, F(ab’)  2, F d, single chain Fv or scFv, disulfide linked F v, V-NAR domain, IgNar, intrabody, IgGΔCH 2, minibody, F (ab’)  3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb 2, (scFv)  2, or scFv-Fc.
  6. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein said monoclonal antibody or antigen-binding fragment thereof binds to the S1 glycoprotein of SARS-CoV-2.
  7. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, wherein said monoclonal antibody or antigen-binding fragment thereof binds to the S2 glycoprotein of SARS-CoV-2.
  8. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-7, wherein said monoclonal antibody or antigen-binding fragment thereof binds the SARS-CoV-2 antigen with a K d of less than about 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or 0.05 nM.
  9. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-8, which inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2.
  10. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 9, which inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 immobilized on a solid support (such as in ELISA assay) , or inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2 expressed on the surface of a cell (such as Vero E6 cell) .
  11. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-10, which inhibits binding of the SARS-CoV-2 antigen (e.g., the S1  glycoprotein) to ACE2 with an EC50 value of less than 1 nM or 0.1 nM.
  12. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-11, which exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 10 nM, 6 nM, 3 nM, 1 nM, 0.6 nM or less than 0.5 nM.
  13. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-12, which inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 10 nM, 5 nM, 3 nM, 2 nM, 1 nM, 0.1 nM, 0.08 nM, 0.06 nM, 0.02 nM, or less than 0.01 nM.
  14. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-13, which inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 nM, less than 500 pM, less than 200 pM, less than 100 pM, less than 80 pM, less than 50 pM, less than 30 pM, less than 10 pM, or less than 5 pM.
  15. An isolated monoclonal antibody or an antigen-binding fragment thereof, which competes with the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-14 for binding to the same epitope.
  16. A mixture of two or more isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-15.
  17. The mixture of claim 16, wherein the proportion of each of said two or more isolated monoclonal antibody or antigen-binding fragment thereof is substantially the same, or is different.
  18. A method of treating or preventing a disease or condition arising from SARS-CoV-2 infection, the method comprising administering to a patient in need thereof an effective amount of the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-15, or an effective amount of the mixture of claim 16 or 17.
  19. The method of claim 18, which is for treating COVID-19, wherein the method further comprises administering a second therapeutic agent.
  20. The method of claim 19, wherein the second therapeutic agent comprises chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune  system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab) , CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFNγ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, Calquence (acalabrutinib) , Xeljanz (tofacitinib) , Jakafi (ruxolitinib) , Olumiant (baricitinib) , Ilaris (canakinumab) , Otezla (apremilast) , Mavrilimumab, or combination thereof.
  21. A polynucleotide encoding the heavy chain or the light chain or the antigen-binding portion thereof of any one of claims 1-15.
  22. The polynucleotide of claim 21, which is codon optimized for expression in a human cell.
  23. A vector comprising the polynucleotide of claim 21 or 22.
  24. The vector of claim 23, which is an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector) .
  25. A host cell comprising the vector of claim 23 or 24, which expresses said isolated monoclonal antibody or antigen-binding fragment thereof.
  26. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-15, or the mixture of claim 16 or 17.
  27. The pharmaceutical composition of claim 26, formulated for intravenous administration.
  28. The pharmaceutical composition of claim 26, formulated for inhalational or oral administration.
  29. A recombinant monoclonal antibody comprising HCVR CDR1-CDR3 and/or LCVR CDR1-CDR3 of an antibody identified as specific for an antigen of SARS-CoV-2.
  30. A pharmaceutical composition for treating a subject infected by SARS-CoV-2, comprising a monoclonal antibody identified as specific for an antigen of SARS-CoV-2 using any of the methods of claims 2-27, and a pharmaceutically acceptable excipient or diluent.
  31. A pharmaceutical composition for treating a subject infected by SARS-CoV-2, comprising a recombinant monoclonal antibody comprising HCVR CDR1-CDR3 and/or LCVR CDR1-CDR3 of an antibody identified as specific for an antigen of SARS-CoV-2 using any of the methods of claims 2-27, and a pharmaceutically acceptable excipient or diluent.
  32. A combination comprising the pharmaceutical composition of claim 30 or 31, and a second therapeutic agent effective to treat infection by SARS-CoV-2.
  33. The combination of claim 32, wherein the second therapeutic agent comprises chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an immune system inhibitor to inhibits cytokine storm (such as an anti-IL-6 neutralizing antibody such as tocilizumab or sarilumab) , CD24Fc, IFX-1, an anti-CCR5 antibody such as Leronlimab, DAS181, CM4620, an anti-IFNγ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, or combination thereof.
  34. A method of treating a subject infected by SARS-CoV-2, the method comprises administering a therapeutically effective amount of the antibody of claim 28 or 29, the pharmaceutical composition of claim 30 or 31, or the combination of claim 32 or 33.
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