WO2023023150A2 - 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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WO2023023150A2
WO2023023150A2 PCT/US2022/040592 US2022040592W WO2023023150A2 WO 2023023150 A2 WO2023023150 A2 WO 2023023150A2 US 2022040592 W US2022040592 W US 2022040592W WO 2023023150 A2 WO2023023150 A2 WO 2023023150A2
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seq
amino acid
sequence
lcvr
hcvr
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PCT/US2022/040592
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French (fr)
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WO2023023150A3 (en
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Liang SCHWEIZER
Francisco Adrian
Matthieu DELINCE
Roshan Moti KUMAR
Qian Zhang
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Hifibio (Hk) Limited
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/732—Antibody-dependent cellular cytotoxicity [ADCC]
    • 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
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C07K2317/734—Complement-dependent cytotoxicity [CDC]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance

Definitions

  • said isolated monoclonal antibody is not naturally occurring; and/or, optionally further comprising a signal peptide sequence of MGWSCIILFLVATATGAHS (SEQ ID NO: 225) at the N-terminus of said HCVR and/or LCVR.
  • SEQ ID NO: 225 signal peptide sequence of MGWSCIILFLVATATGAHS
  • E3. The isolated monoclonal antibody or antigen-binding fragment thereof according to E1 or E2, wherein said monoclonal antibody has: (1) (1a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 7; and/or, (1b) a light chain sequence comprising the amino acid of SEQ ID NO: 8, or, (2) (2a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 17; and/or, (2b) a light chain sequence comprising the amino acid of SEQ ID NO: 18, or, (3) (3a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 27; and/or, (3b) a light chain sequence comprising the amino acid of SEQ ID NO: 28, or, (4) (4a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 37; and/or, (4b) a light chain sequence comprising the amino acid of SEQ ID NO: 38, or, (5) (5a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 57;
  • E4 The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of E1-E3, wherein: (a) the isolated monoclonal antibody is a human antibody, a CDR-grafted antibody, or a resurfaced antibody; (b) the isolated monoclonal antibody is a bi-specific antibody, optionally the bi- specific antibody comprises the HCVR CDR1, CDR2, and CDR3 and LCVR CDR1, CDR2, and CDR3 of any one of a first antibody of claim 1(1) – 1(13), and the HCVR CDR1, CDR2, and CDR3 and LCVR CDR1, CDR2, and CDR3 of any one of a second antibody of claim 1(1) – 1(13), wherein the first and the second antibodies are different or bind to different (preferably non-overlapping) epitopes; optionally, (i) said first antibody is specific for S1 (such as Ab-1, Ab-6, or Ab-7), and said second antibody is specific for S2 (such as Ab-3, Ab-5),
  • E5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of E1- E4, wherein said monoclonal antibody or antigen-binding fragment thereof: (i) binds to the S1 or S2 glycoprotein of SARS-CoV-2; (ii) binds the SARS-CoV-2 antigen with a Kd of less than about 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or 0.05 nM; (iii) binds to SARS-CoV-2 wild-type S protein and/or RBD/S1 variants selected from the group consisting of S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, A222V/D614G, the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (
  • E6 The isolated monoclonal antibody or antigen-binding fragment thereof of any one of E1- E5, which: (i) 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; (ii) exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 50 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 6 nM, less than 3 nM, less than 1 nM, less than 0.6 nM or less than 0.5 nM; (iii) inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 50 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 n
  • E7 The monoclonal antibody or antigen-binding fragment thereof of any one of E1-E6, comprising a heavy chain constant region, wherein the heavy chain constant region is human IgG4, human IgG3 or human IgG2; optionally, the heavy chain constant region is human IgG4 which optionally comprises a YTE (M252Y/S254T/T256E) mutation and/or an LS (M428L/N434S) mutation.
  • YTE M252Y/S254T/T256E
  • LS M428L/N434S
  • E10 The monoclonal antibody or antigen-binding fragment thereof of claim 8 or claim 9, comprising an IgG4 heavy chain (HC) sequence comprising SEQ ID NO: 67 or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
  • HC heavy chain
  • an expression vector e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector
  • a pharmaceutical composition comprising the isolated monoclonal antibody or antigen- binding fragment thereof of any one of E1-E11, or the mixture of E12, optionally the pharmaceutical composition is formulated for intravenous administration, or for inhalational or oral administration; and/or, optionally, the pharmaceutical composition is for treating a subject infected by SARS- CoV-2, and further comprises a pharmaceutically acceptable excipient or diluent.
  • a combination comprising the pharmaceutical composition of E14, and a second therapeutic agent effective to treat infection by SARS-CoV-2, optionally, 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, or combination thereof.
  • 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
  • a method of treating or preventing a disease or condition arising from SARS-CoV-2 infection 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 E1-E11, the mixture of E12; the polynucleotide of E13, or the pharmaceutical composition of E14, optionally, the method is for treating COVID-19 or a subject infected by SARS-CoV-2, wherein the method further comprises administering a second therapeutic agent; and/or, optionally, said 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, CM46
  • a method of identifying an antibody specific for an antigen from a virus or a bacterium, from a B-cell population obtained from a subject having been infected by and recovering from infection by the virus or the bacterium comprises: (i) obtaining a library of paired VH and VL antibody sequences from a B cell population obtained from the subject; and, (ii) obtaining amino acid sequences of fragments of antibodies specific for said antigen, wherein said antibodies are obtained (e.g., affinity purified) from a sample comprising said B cell population; thereby identifying the antibody specific for said antigen when said amino acid sequences obtained in (ii) match the paired VH and VL antibody sequences obtained in (i).
  • E17 The method of E17, wherein the virus is SARS-CoV-2, and the antigen is SARS-CoV-2 S (Spike) protein or N (Nucleocapsid) protein.
  • E19 The method of E17 or E18, wherein the sample is a blood sample or plasma sample.
  • E20 The method of any one of E17-E19, wherein the B cell population is PBMCs isolated from a peripheral blood sample.
  • E21. The method of any one of E17-E20, wherein the B cell population comprises plasmablasts and memory B cells. E22.
  • step (i) comprises: (1) generating a plurality of nanoliter scale droplets, each comprising: (a) one B cell from said B-cell population; (b) multiple (e.g., 1,000-1,500) co-encapsulated paramagnetic beads (e.g., colloidal nanoparticles) coated by a first non-specific antibody-binding molecule; and, (c) the antigen labeled by a first detectable label; (2) allowing, under a pre-determined condition, antibodies secreted by said one B-cell to bind said first non-specific antibody-binding molecule on said paramagnetic beads, and to bind said antigen when / if said antibodies are specific for said antigen; (3) passing the droplets through a magnetic field to aggregate said multiple paramagnetic beads, in order to concentrate the accumulative signal emitted by the first detectable label on the antigen bound by said antibodies over a background signal emitted by the first detectable label on the antigen unbound by
  • E23 The method of E22, wherein said plurality of nanoliter scale droplets are generated by a microfluidic device.
  • E24 The method of E22 or E23, wherein the average size of said plurality of nanoliter scale droplets is about 30-1500 pL, about 40 pL, about 80 pL, about 125 pL, or about 1 nL.
  • E25 The method of any one of E17-E24, wherein the B-cell population is from a human or a non-human mammal (e.g., mouse, rat, rabbit).
  • E26 The method of any one of E17-E25, wherein the multiple co-encapsulated paramagnetic beads are in an amount sufficient to bind substantially all antibodies secreted by said one B cell.
  • E27 The method of any one of E17-E26, wherein said first non-specific antibody-binding molecule is biotin-labeled anti-human IgG-Fc (which biotin binds to streptavidin-coated paramagnetic beads), Protein G (which binds to immunoglobulin Fab and Fc regions, optionally, the Protein G lacks albumin-binding region), Protein A (which binds heavy chain Fc region and within the Fab region of human VH3 family), Protein A/G (which binds all subclasses of human IgG as well as IgA, IgE, IgM and to a lesser extent IgD, and all subclasses of mouse IgG but not mouse IgA, IgM or serum albumin), Protein L (which binds kappa light chain of all antibody classes including IgG, IgM, IgA, IgE, IgD as well as scFv and Fab fragments), or a species-specific antibody or anti
  • E28 The method of any one of E17-E27, wherein the first detectable label is a fluorescent label (such as Alexa Fluor 488 and DayLight550).
  • the nanoliter scale droplets further comprise: (d) a second non-specific antibody-binding molecule labeled by a second detectable label, wherein said second non-specific antibody-binding molecule does not compete or interfere with binding by said first non- specific antibody-binding molecule; wherein in step (3), the accumulative signal emitted by the second detectable label (e.g., DayLight650) on said second non-specific antibody-binding molecule bound by said antibodies is reflective of the relative amount of said antibodies on aggregated paramagnetic beads.
  • the second detectable label e.g., DayLight650
  • E30 The method of E29, wherein said second non-specific antibody-binding molecule is Protein G, Protein A, Protein A/G, Protein L, or a species-specific antibody or antigen- binding fragment thereof (e.g., Goat-anti-human IgG Fc).
  • E31 The method of any one of E17-E30, wherein the pre-determined condition is 15-60 min. at 37°C (under 5% CO 2 ).
  • E32. The method of any one of E17-E31, wherein said multiple paramagnetic beads aggregate to form a geometric shape (such as a straight line) under the magnetic field.
  • E33 The method of any one of E17-E31, wherein said multiple paramagnetic beads aggregate to form a geometric shape (such as a straight line) under the magnetic field.
  • E17-E32 wherein said accumulative signal emitted by the first detectable label is a fluorescent signal emitted after laser excitation.
  • step (4) is carried out by an acoustic sorter device that generates a surface acoustic wave (SAW), or by fluorescence-activated dielectrophoretic sorting.
  • SAW surface acoustic wave
  • E35 The method of any one of E17-E34, further comprising (5) determining the sequences of the paired heavy and light chains (VH and VL) of each identified antibody.
  • VH and VL paired heavy and light chains
  • step (5) comprises compartmentalizing each B cell collected from the droplets in step (4) with a bead comprising a bead-specific nucleotide-based barcode and a reverse-transcription primer for initiating cDNA synthesis from mRNA encoding antibody heavy chain or antibody light chain.
  • the primer is complementary to a heavy chain constant region coding sequence or a light chain constant region coding sequence, and the cDNA synthesized from the primer comprises heavy chain variable region or light chain variable region, or a CDR (such as CDR3) thereof.
  • step (5) further comprises sequencing cDNA synthesized from the primer.
  • E38 wherein cDNA sequencing is performed by next generation sequencing (NGS) (such as using an Illumina MiSeq sequencer with a 2 ⁇ 300 base pair sequencing flow chip).
  • NGS next generation sequencing
  • E40 The method of E38, further comprising cloning and expressing cDNA sequences in a host cell to produce said antibody specific for said antigen.
  • E41 The method of E40, wherein the host cell is a CHO cell.
  • E42. The method of E40 or E41, further comprising isolating and/or purifying said antibody.
  • step (ii) IgM in said sample is first depleted before the remaining antibodies in said sample are affinity purified with said antigen, and the affinity-purified antibodies are digested with protease for sequencing analysis of the resulting fragments using mass spectrometry.
  • step (ii) An antibody identified as specific for an antigen of SARS-CoV-2 by the method of any one of E18-E43.
  • E45. 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 E18-E43.
  • 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 E18-E43, and a pharmaceutically acceptable excipient or diluent.
  • 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 E18-E43, and a pharmaceutically acceptable excipient or diluent.
  • a combination comprising the pharmaceutical composition of E46 or E47, and a second therapeutic agent effective to treat infection by SARS-CoV-2.
  • E49. The combination of E48, 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-IFN ⁇ monoclonal antibody such as emapalumab, an IL-1R antagonist such as Anakinra, Danoprevir+Ritonavir, or combination thereof.
  • the second therapeutic agent comprises chloroquine or hydroxychloroquine, remdesivir, lopinavir and ritonavir, azithromycin, an
  • a method of treating a subject infected by SARS-CoV-2 comprises administering a therapeutically effective amount of the antibody of E44 or E45, the pharmaceutical composition of E46 or E47, or the combination of E48 or E49.
  • the monoclonal antibody or antigen-binding fragment thereof is specific for the Spike protein or S protein of SARS-CoV-2, and wherein said monoclonal antibody specifically binds to and/or has a residue within 4 ⁇ of residues T415, G416, K417, D420, Y421, Y453, L455, F456, R457, K458, N460, Y473, Q474, A475, G476, S477, F486, N487, Y489, Q493, S494, Y495, G496, Q498, T500, N501, G502, and Y505 of the S protein, optionally, said monoclonal antibody does not bind to and/or has no residue within 4 ⁇ of residues T415, G416,
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is a human IgG4 antibody, or an Fc ⁇ R null monoclonal antibody engineered to prevent Fc ⁇ R engagement.
  • 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 isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof binds to SARS-CoV-2 wild-type S protein and/or RBD/S1 variants selected from the group consisting of S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, and A222V/D614G.
  • the monoclonal antibody or antigen-binding fragment thereof binds the SARS-CoV-2 antigen with a Kd 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.
  • 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 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).
  • 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 with an EC50 value of less than 2 nM, 1 nM or 0.1 nM.
  • SARS-CoV-2 antigen e.g., the S1 glycoprotein
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof 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 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
  • a target cell such as Vero E6 cell
  • the isolated monoclonal antibody or antigen- binding fragment thereof inhibits entry of wild-type SARS-CoV-2 and/or SARS-CoV-2 variants, e.g. WuhanD614, BavPat D614G, UK B.1.1.7, South Africa B.1.351 lineage, the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and/or the Lambda (C.37) into a target cell.
  • wild-type SARS-CoV-2 and/or SARS-CoV-2 variants e.g. WuhanD614, BavPat D614G, UK B.1.1.7, South Africa B.1.351 lineage, the
  • the isolated monoclonal antibody or antigen- binding fragment thereof inhibits entry of a SARS-CoV-2 variant sharing one or more S protein mutations with the WuhanD614, BavPat D614G, UK B.1.1.7, South Africa B.1.351 strain(s), the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and/or the Lambda (C.37) into a target cell.
  • the isolated monoclonal antibody or antigen- binding fragment thereof does not cause antibody-dependent enhancement (ADE).
  • the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region is human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the heavy chain constant region is human IgG4.
  • 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.
  • CDRs such as CDR1, CDR2, and/or CDR3 of heavy chain and/or light chain sequences
  • heavy chain CDR3 (HC-CDR3) sequences of certain isolated antibodies Ab-1 to Ab-13 and Ab in FIG.5A are listed below.
  • 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), Methosimumab, or combination thereof.
  • 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).
  • 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.
  • 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.
  • FIG.1A shows that sera from SARS-CoV-2 convalescent patients clearly showed higher antibody (IgG) titers as compared to sera from healthy donors.
  • FIG.1B shows titers of antibodies specific for the RBD domain of S1 or the Spike extracellular domain (ECD) of SARS-CoV-2, from hospitalized SARS-CoV-2 patients, SARS- CoV-2 positive patients, and patients displaying COVID-19 symptoms.
  • FIG.2 is a schematic drawing showing representative fluorescent signal patterns for the various picoliter droplets generated in microfluidic devices, including droplets with no IgG- secreting cell, droplets with cell secreting IgG that does not bind SARS-CoV-2 antigen, droplets with cell secreting IgG that does bind SARS-CoV-2 antigen, droplets with memory B cell having membrane-bound IgG, and droplets with memory B cell having membrane-bound SARS-CoV-2 binding IgG.
  • FIG.3 is a schematic drawing showing the bifurcated approach to identify antigen- specific (e.g., SARS-CoV-2 specific) antibodies.
  • the top part shows the microfluidic-device enabled single cell sorting and bar-coded DNA sequencing approach to generate libraries of paired VH and VL sequences.
  • the bottom part shows the immunoprecipitation based mass spectrometry-based peptide sequencing to identify partial sequences of antibodies specific for a selected antigen (e.g., SARS-CoV-2 S1 and/or S2 protein). Matching sequences from both routes identifies VH/VL pairs specific for the target antigen.
  • FIG.4 shows representative antibodies identified using the procedure of FIG.3, having strong S1 or S2 binding affinity.
  • the middle panel is a schematic drawing showing the ELISA screening format used to measure EC50 values.
  • FIG.5A shows multiple sequence alignments of light chain variants with improved binding affinity
  • FIG.5B shows EC50 values of the various S1-binding antibodies.
  • FIG.6A shows that the identified S protein binders can block binding of viral S protein to the human ACE2 receptor expressed on Vero-E6 cell surface.
  • FIG.6B shows that the identified S protein binders can inhibit the binding of the viral S1 ECD domain binding to the human ACE2 receptor expressed on Vero-E6 cell surface.
  • S2 binders show partial inhibition of Spike protein binding to ACE2.
  • FIG.7 shows the results of S2 binding antibody binning assay.
  • FIG.8 shows exemplary bi-specific antibodies against S1 and S2 proteins. DETAILED DESCRIPTION OF THE INVENTION 1.
  • 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
  • light chain CDR sequences of certain isolated antibodies are listed in FIG.5A, 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.
  • Another aspect of the invention provides a bi-specific antibody of the invention having antigen-binding fragments (e.g., defined by 6 CDR sequences) from two different antibodies binding to different epitopes of S1 and/or S2, respectively.
  • a bi-specific antibody may comprise antigen-binding fragments from a first antibody specific for S1, and antigen-binding fragments from a second antibody specific for S2, wherein the S2 antibody fragments may be in scFv format (either VH-VL or VL-VH) and linked to the VL (or VH) of the S1 antibody.
  • Such bi-specific antibodies may provide better therapeutic efficacy compared to the individual component antibodies of the mixture.
  • 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.
  • 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.
  • 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.
  • these neutralizing antibodies can be formulated for use as therapeutic antibodies for patient treatment.
  • they can also be used prophylactically to prevent virus infection.
  • certain binding antibodies can be used in combination with the vaccine approaches, even if they do not have neutralization activities.
  • different antibodies with either S1 or S2 binding capacity are used to generate multi-valent antibodies, such as bi-specific antibodies, or be used together, for combination therapy.
  • multi-valent antibodies such as bi-specific antibodies
  • 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
  • antibody in the broadest sense, encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, and multispecific antibodies (e.g., bi-specific antibodies).
  • 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. 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.
  • 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.
  • Certain isotypes can be further subdivided into subclasses.
  • IgG antibodies include, but are not limited to, IgGl (comprising a ⁇ 1 constant region), IgG2 (comprising a ⁇ 2 constant region), IgG3 (comprising a ⁇ 3 constant region), and IgG4 (comprising a ⁇ 4 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 refers to a region comprising light chain CDR1 (CDR-L1), framework (FR) 2 (LFR2), CDR2 (CDR-L2), FR3 (LFR3), and CDR3 (CDR-L3).
  • 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).
  • LFR1 framework
  • LFR3 LFR3
  • LFR3 CDR3
  • CDR3 CDR3
  • CDR3 CDR3
  • CDR3 CDR3
  • 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).
  • LFR1 framework
  • LFR4 FR4
  • 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 , Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgG ⁇ CH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (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.
  • 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 or VL:VH 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.
  • Compet 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.
  • 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.
  • 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.
  • 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.
  • 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 XENOMOUSE ® , 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.C6 ® 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.
  • 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,” 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.
  • 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.
  • the term “antibody-dependent enhancement” (ADE) refers to a phenomenon in which binding of a virus to suboptimal antibodies enhances its entry into host cells.
  • ADE has been documented to occur through two distinct mechanisms in viral infections: by enhanced antibody-mediated virus uptake into Fc gamma receptor IIa (Fc ⁇ RIIa)-expressing phagocytic cells leading to increased viral infection and replication, or by excessive antibody Fc-mediated effector functions or immune complex formation causing enhanced inflammation and immunopathology. Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing infection.
  • Fc ⁇ RIIa Fc gamma receptor IIa
  • 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.
  • effective amount or therapeutically 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.
  • 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.
  • 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. 3. Routes of Administration and Carriers
  • 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.
  • 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.
  • 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.
  • the compositions may be formulated for inhalation, for example, using pressurized acceptable propellants such as dichlorodifluoromethane, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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), Methosimumab, or combination thereof.
  • 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. 5.
  • 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.
  • the antibody of the invention inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2.
  • 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. 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.
  • 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
  • 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.
  • multispecific antibodies are provided.
  • bi- specific antibodies are provided.
  • Non-limiting exemplary bi-specific 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 bi-specific antibody comprises the antigen-binding fragment (such as scFv, Fab, or Fab’ fragment) of a first antibody specific for a first epitope of an SARS- CoV-2 (such as S1 or S2), and comprises the full-length antibody or antigen-binding fragment (such as scFv, or F(ab’) 2 fragment) of a second antibody specific for a second epitope of an SARS-CoV-2 (such as S2 or S1), wherein the first and the second epitopes do not overlap.
  • an SARS- CoV-2 such as S1 or S2
  • the full-length antibody or antigen-binding fragment such as scFv, or F(ab’) 2 fragment
  • binding of the first epitope by the antigen-binding fragment of the first antibody does not substantially inhibit (e.g., has no effect or enhances) the binding of the second epitope by the antigen-binding fragment of the second antibody.
  • the antigen-binding fragment of the first antibody is linked to the antigen-binding fragment of the second antibody directly.
  • the antigen-binding fragment of the first antibody is linked to the antigen-binding fragment of the second antibody through a flexible linker, such as G4S type linker with one or more repeats.
  • the antigen-binding fragment of the first antibody is an scFv, and is linked through its C-terminus to the N-terminus of a light chain (or a heavy chain) of the antigen-binding fragment of the second antibody.
  • the bi-specific antibody comprises one or two antigen-binding fragment(s) of the first antibody.
  • the bi-specific antibody comprises one or two antigen-binding fragment(s) of the second antibody.
  • the bi- specific antibody comprises one or two antigen-binding fragment(s) of the first antibody and one or two antigen-binding fragment(s) of the second antibody.
  • the bi-specific antibody comprises two antigen-binding fragments of the first antibody and two antigen- binding fragments of the second antibody.
  • 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.
  • TAP Therapeutic Antibody Profiler
  • TAP as freely available at opig.stats.ox.ac.uk/webapps/sabdab-sabpred/TAP.php.
  • barriers to therapeutic mAb development include intrinsic immunogenicity, chemical and conformational instability, self-association, high viscosity, polyspecificity, and poor expression.
  • 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.
  • LC light chain
  • HC heavy chain
  • FR framework regions
  • Antibody C1S5-2A2A comprises the following sequences:
  • Antibody C1S2-6A6A comprises the following sequences:
  • the framework region sequences 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.
  • the framework region sequences 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.
  • the HC and/or LC further includes a signal peptide sequence, such as MGWSCIILFLVATATGAHS (SEQ ID NO: 225).
  • any one of the antibodies of the invention comprise a light chain constant region (CL) selected form the CL sequences in the table below, and/or a heavy chain constant region (C H ) sequences in the table below.
  • the heavy chain constant region (C H ) sequence is that of human IgG4.
  • any one of the antibodies of the invention comprise a LC leader sequence and/or a HC leader sequence below: LC leader sequence: MAWAPLLLTLLAHCTGSWA (SEQ ID NO: 226) or MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 227). HC leader sequence: MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 228) or MEFGLSWLFLVAILKGVQC (SEQ ID NO: 229).
  • FcRn neonatal Fc receptor
  • Recombinant antibodies therapeutic potential may be enhanced by the introduction of defined mutations in the crystallizable fragment (Fc) domains, such as for example, YTE (M252Y/S254T/T256E) and LS (M428L/N434S), as a consequence of increased half-lives and prolonged duration of protection.
  • Fc crystallizable fragment
  • YTE M252Y/S254T/T256E
  • LS M428L/N434S
  • the prototypical example of an FcRn affinity-enhancing Fc mutant is the YTE mutation which, when incorporated into motavizumab IgG1, is able to extend serum half-life in humans by more than four-fold.
  • any one of the antibodies of the invention comprise an Fc region mutation, such as a YTE (M252Y/S254T/T256E) mutation and/or an LS (M428L/N434S) mutation.
  • the YTE mutation and/or the LS mutation is in IgG4 Fc region.
  • the antibody of the invention comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an optional LC leader sequence, followed by any of the VL sequences described herein (such as the VL sequence of any of Ab-1 to Ab-13, and Ab in FIG.5), followed by a light chain constant region such as one described in the table above; and wherein the HC comprises an optional HC leader sequence, followed by any of the VH sequences described herein (such as the VH sequence of any of Ab-1 to Ab-13, and Ab in FIG.5), followed by a heavy chain constant region such as one described in the table above, such as IgG4, optionally an IgG4 with YTE and/or LS mutation. 6.
  • LC light chain
  • HC heavy chain
  • 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.
  • 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.
  • 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, IgG3, or IgG4.
  • 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.
  • an antibody described herein comprises a human IgG4 heavy chain constant region.
  • an antibody described herein comprises an S241P mutation in the human IgG4 constant region.
  • an antibody described herein comprises a human IgG4 constant region and a human ⁇ light chain.
  • 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. In some embodiments, effector function is not desirable.
  • effector function may not be desirable in treatments of inflammatory conditions and/or autoimmune disorders, such as SARS-CoV-2 induced cytokine storm.
  • a human IgG4 or IgG2 heavy chain constant region is selected or engineered.
  • an IgG4 constant region comprises an S241P mutation.
  • 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, is also used for purifying some polypeptides. 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.
  • 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.
  • 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. 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.
  • 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.
  • expression of the polypeptide or polypeptides is under the control of a promoter or promoters that function in a tissue-specific manner.
  • a promoter or promoters that function in a tissue-specific manner.
  • liver-specific promoters are described, e.g., in PCT Publication No. WO 2006/076288.
  • 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-S and DG44 cells; PER.C6® cells (Crucell); and NSO cells.
  • 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.
  • 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.
  • 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.
  • 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 Identification of SARS-CoV-2 Specific Neutralizing Antibodies
  • the SARS-CoV-2 virus utilizes its surface spike protein to mediate its entry into target cells. Briefly, to enter host cells, SARS-CoV-2 first binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) with its surface-anchored spike protein for viral attachment. This spike protein is present as a trimer on viral surface, with three receptor-binding S1 heads sitting on top of a trimeric membrane fusion S2 stalk.
  • ACE2 cell surface receptor angiotensin-converting enzyme 2
  • Each S1 head contains a receptor-binding domain (RBD) that specifically recognizes ACE2 as its receptor.
  • the RBD constantly switches between a “standing-up” position for receptor binding and a “lying-down” position for immune evasion.
  • the spike needs to be proteolytically activated at the S1/S2 boundary, such that S1 dissociates and S2 undergoes a dramatic structural change.
  • SARS-CoV entry-activating proteases include cell surface protease TMPRSS2 and lysosomal proteases cathepsins.
  • SARS-CoV-2 RBD is more potent and less exposed (hidden RBD), facilitating better immune evasion.
  • SARS-CoV-2 cell entry of SARS-CoV-2 is pre-activated by proprotein convertase furin, reducing its dependence on target cell proteases for entry.
  • proprotein convertase furin reducing its dependence on target cell proteases for entry.
  • the high hACE2 binding affinity of its RBD, furin pre-activation of the spike, and hidden RBD in the spike allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance, thus contributing to the wide spread of this virus.
  • COVID-19 becomes pandemic, it is critical to understand its effects on patients’ immune system as well as identify therapeutic treatments.
  • the generation of protective and long- lasting humoral immunity is one major aspect of the adaptive immune response to infection. Therefore, it is important to analyze humoral responses in patients and use the neutralizing antibodies for potential treatment of the disease.
  • This example demonstrates the profiling of humoral responses in patients exposed to SARS-CoV-2 infection, and the identification of neutralizing antibodies against SARS-CoV-2 from the recovered patients’ samples.
  • B cells secreting antibodies with specificity against the viral Spike protein were sorted and then sequenced, using the proprietary single cell barcoding technology.
  • a number of blood donors, either showing COVID-19 symptoms or confirmed infected patients, were profiled to identify a number of non-redundant neutralizing antibody candidates against SARS-CoV-2.
  • mass spectrometry and parallel single B cell antibody sequencing were used to identify antibodies in patient serum isolated through immunoprecipitation using the viral S protein.
  • Patient blood sample collection and characterization Patients participated in this study ranged in age from 8 to 78 years old, with a median age of 47 years old. The average time between the initial positive RT-PCR test and subsequent negative RT-PCR test was 18.7 days, with a median time of 17 days.
  • antibody titers in patient sera were measured against the SARS-CoV-2 Spike (S) protein. 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, and titer is the highest in hospitalized patients (FIG.1B).
  • 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 of China where the SARS-CoV-2 virus first 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.
  • B cell profiling To characterize the B cell populations of patient samples, PBMCs isolated from peripheral blood collected from 19 patients were analyzed by flow cytometry. Cells were stained with a panel of anti-CD19 / CD27 / CD38 antibodies, and plasmablasts (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 (data not shown).
  • the paired VH-VL sequences were recovered by plating the B cells in individual wells.
  • An alternative approach can be based on the rapid droplet-based single-cell antibody discovery platform CELLIGO® to identify paired antibody heavy chain (VH) and light chain (VL) DNA sequences (see addiction et al., High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics. Nat Biotechnol 38, 715–721, 2020, incorporated herein by reference).
  • two different immunological compartments were interrogated: plasmablasts by direct encapsulation of PBMCs, and memory B cells by in vitro memory B cell activation followed by a B cell enrichment kit.
  • the CELLIGO® platform is a microfluidic-based method that can screen millions of cells for antigen specificity in single day. Specifically, Cells of interest are encapsulated in droplets acting as individual picolitre sized reaction chambers. One hour of incubation is sufficient to detect the presence of IgGs secreted by the B cells using a fluorescence-based assay in droplets.
  • the droplets contain paramagnetic nanoparticles coated with an anti- human Fc nanobody, a fluorescently labeled anti-human Fc detection reagent, and a fluorescently labeled soluble antigen (in this case either the full-length extracellular domain or the S1-RBD domain of the SARS-CoV-2 Spike protein (see FIG.2).
  • the droplets are then sorted using a microfluidic droplet sorter by applying a series of selection criteria including exclusion of droplets containing dead cells and selection of droplets showing positive signals for both IgG secretion and antigen specificity (FIG.2). This strategy enables sorting out antigen-specific, IgG-secreting cells, from the rest of the PBMC population (FIG.2).
  • Non-redundant VH/VL paired sequences were filtered for serious residue liabilities that would disqualify them as potential therapeutics, and clustered into families of presumed ancestral recombination and a single representative of each family chosen for synthesis and functional characterization With this approach, screens for multiple samples were performed, from convalescent patients for antigen specificity. Samples analyzed yielded hundreds of VH/VL pairs representing over one hundred non-redundant pairs. The droplet-based assay performed on patient samples was able to detect ⁇ 1% of the IgG secreting cells specific to the antigen. It was observed that sequences of the light chains exhibit substantially less amino-acids difference with respect to germline, compared to the sequences of the heavy chains.
  • the complementary approach used to identify SARS- CoV2-specific antibodies 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 VH and VL chains.
  • the mass spectra peptide data of the VH or VL of affinity-purified antibodies covering the CDR3 region were used to unambiguously map back to full-length VH-VL paired sequences from the repertoire database. Based on titer information, one sample was selected from convalescent patient to interrogate with this approach.
  • the reference antibody C1S5-2A2A has an EC50 of 0.31 nM, while the newly identified antibody H13S1-8A9A has EC50 of 3.67 nM.
  • the reference antibody C1S2-6A6A has an EC50 of 1.71 nM, while the newly identified antibodies H13S1-9A10A, H13S1-14A14A, and H13S4-8A8A has EC50 of 1.57 nM, 5.32 nM, and 20.58 nM, respectively.
  • S2 binder binning assay was also performed using standard technology, and the results were shown in FIG.8.
  • H13S1-9A10A antibody binds to a distinct epitope on S2, while H13S4-8A8A and H13S9.1-7A7A bind closely related epitopes that somewhat overlap with that of the reference antibody C1S2-6A6A.
  • Knowledge of such binning assays can be used to design bi-specific antibodies, such as those exemplified in FIG.9, that have bi-specific binding affinity towards both S1 and S2 portions of the S protein.
  • the complementary approach demonstrated that it can identify anti-viral antibodies from recovered patients at both protein and DNA level with matching sequences. Further, binding and blocking capabilities were also demonstrated from synthesized antibodies based on their DNA sequences.
  • variable region gene encoding the heavy and light chains were synthesized, then cloned into a vector with a selected constant region to produce IgG1 antibodies.
  • this example shows successful identification of multiple SARS-CoV-2 S protein binders using two approaches, the direct screening as well as the LC-MS and single cell sequencing coupled approach.
  • the antibodies can effectively bind to SARS-CoV-2 with EC50 10 -9 to 10 -10 M, where some bind to S1 glycoprotein and at least one antibody binds to S2 protein, showing the diversity of mAb being identified.
  • the identified binders can also block S1 binding to Vero E6 cell line with IC5010 -9 -10 -10 M (FIG.6).
  • neutralizing antibodies can be used as therapeutic antibodies for patient treatment, some of them can be used prophylactically to prevent virus infection. Moreover, certain binding antibodies can be used in combination with the current vaccine approaches, even though they may not have sufficient neutralization activities.
  • ELISA Materials and Methods Titer measurements by ELISA: For ELISA, 384-well plates were coated overnight at 4°C degrees 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 (ThermoFisher 05-4220) at 0.2 ⁇ g/mL in 1X PBS with 0.05% Tween and 1% BSA) for 1 hour at room temperature.
  • detection reagent Mae anti-Human IgG Fc HRP labeled (ThermoFisher 05-4220) at 0.2 ⁇ g/mL in 1X PBS with 0.05% Tween and 1% BSA
  • the plate was then washed again 4 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution.
  • Flow cytometry PBMC was thawed at 37°C, and centrifuged at 450 g for 8 min. The supernatant was discarded, and the cells resuspended in 200 ⁇ L of DMEM.
  • the cells were suspended in 200 ⁇ L PBS.3 ⁇ L of CD19 (FITC labeled, eBioscience 11-0199-42), CD27 (APC labeled, eBioscience 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.
  • CD19 FITC labeled, eBioscience 11-0199-42
  • CD27 APC labeled, eBioscience 17-0279-42
  • anti CD38 PE labeled mouse IgG1 isotype control, eBioscience 12-0388-42
  • Aqueous phase I the PBMC or B cell enriched sample was spun down and resuspended in DMEM supplemented with 0.2% Pluronic F68 (ThermoFisher 24040032), 50 mM HEPES pH 7.4 (LifeTechnologies), 10% HyClone Super Low IgG Defined Fetal Bovine serum (GE Healthcare), the NucGreen dead viability marker (ThermoFisher R37109), and 2% Pen/Strep (ThermoFisher), so as to achieve a ⁇ (mean number of cells per droplet) of ⁇ 0.3 cell per drop.
  • Pluronic F68 ThermoFisher 24040032
  • 50 mM HEPES pH 7.4 LifeTechnologies
  • 10% HyClone Super Low IgG Defined Fetal Bovine serum GE Healthcare
  • the NucGreen dead viability marker ThermoFisher R37109
  • Pen/Strep ThermoFisher
  • Aqueous phase II Paramagnetic nanoparticles (Strep Plus, 300 nm, Ademtech) were coated with biotinylated VHH anti-human IgG-Fc (ThermoFisher 7103322500) as described (Eyer et al., 2017), and resuspended in working buffer containing goat anti human IgG-Fc (DyLight-650-labeled, Abcam ab98622) diluted to 50 nM and Spike-RBD (Sino biologicals# 40592-V05H) or full length spike protein (Sino biologicals#40589-V08B1), labeled with Dyligth550 using the Biorad Lynx labeling kit LNK231D550) diluted to 50 nM.
  • VHH anti-human IgG-Fc ThermoFisher 7103322500
  • aqueous phase I and II concentrations are half of the initial aqueous phase I and II concentrations.
  • a positive control emulsion containing 10 nM ACE2-hFc (kactus cat# ACE-HM501) or 10 nM anti-SARS CoV-2 mAb (Sanyou # AHA001) and a negative control emulsion with 10 nM human IgG isotype (Thermo #31154) were used to establish the assay and set antigen specificity gates for sorting.
  • Droplet were sorted using a droplet surface acoustic wave based sorter (Franke, Braunmüller, Schmid, Wixforth, & Weitz, 2010) with the following gating strategy was the following: live cells were selected (absence of peaks in the Nucgreen channel), then the presence of IgG secreting cells was selected for, by selecting droplets that emit a signal in the detection reagent channel, then droplets emitting a signal in the antigen channel were selected, and finally, droplets were select if the peak emitted in the detection reagent and in the antigen channels are colocalized, confirming the accumulation of those two reagents on the beadline.
  • live cells were selected (absence of peaks in the Nucgreen channel), then the presence of IgG secreting cells was selected for, by selecting droplets that emit a signal in the detection reagent channel, then droplets emitting a signal in the antigen channel were selected, and finally, droplets were select if the peak emitted in the detection reagent
  • the sorted droplets were broken by incubation for 10 min in 100 ⁇ L of DMEM supplemented with 5% serum Low IgG and followed by 100 ⁇ L of 1H,1H,2H,2H-Perfluoro-1- octanol (370533, Sigma) and finally centrifuged at 450 g for 10 min at 4°C to favor complete phase separation.
  • Library preparation for antibody sequencing by single-cell barcoding for VH-VL Single-cell barcoding and library preparation for VH-VL sequencing was performed according to the method described in perpetrat et al., 2020 (supra).
  • VH and VL mRNAs from individual cells in droplets was primed using barcoded primers carrying the T7-SBS12 sequences followed by a cell-specific barcode and the gene- specific primer (GSP) sequences complementary to heavy chain and light chain sequences.
  • GSP gene-specific primer
  • the resulting cDNAs from each single cell have a specific barcode, allowing cognate VH and VL pairs to be identified after NGS.
  • the barcoded VH and VL genes were amplified by nested PCR, with reverse primers priming sequentially on the T7 and on the Illumina SBS12 sequence of the barcode and forward primers priming sequentially on the leader and framework 1 sequences of the V gene.
  • the reverse primer appends the first part of the Illumina P7 and Illumina index sequences by priming on the SBS12 sequence and the forward primer appends the Illumina SBS3 sequence.
  • a third PCR adds the P5 sequence by priming on the SBS3 sequences.
  • Illumina MiSeq 2x300 were used for sequencing the amplified variable genes encoding heavy and light chains obtained. Plate based paired VH-VL sequencing was performed according to the method described in (Gérard et al., 2020, supra).
  • cells were distributed into wells of a 384 well-plate using an acoustic liquid dispenser (Echo 550, Labcyte) at an average of one IgG-secreting cell per well.
  • Each well of the plate contained lysis buffer, barcoded cDNA primers (with a unique barcode per row), reverse transcription buffer, dNTPs and reverse- transcriptase enzyme.
  • the barcoded cDNAs were pooled by columns, purified, and the VH and VL genes amplified separately using a column barcode. After the first PCR all columns were pooled into one reaction for the VH and VL. Two more rounds of PCR were performed priming to the adaptor sequence.
  • reads are filtered and trimmed for quality using the program trim_galore bioinformatics.babraham.ac.uk/projects/trim_galore, with a read quality threshold of 20 and a minimum length of 150.
  • the full-length variable region is then recapitulated from each set of paired-end reads by merging the two reads using the program FLASH.
  • Proprietary 3x11mer combinatorial barcode sequences were assigned by extracting each of the indices and comparing them to a white list of known indices allowing up to 2 mismatches per index. Sequences with no identifiable barcode were filtered out. Only barcodes containing a minimum of 4 reads were retained.
  • Reads assigned to the same barcode are considered to come from the same cell. Consensus sequences for all distinct types of sequence assigned to the same barcode were created by first clustering them using a rapid DNA clustering algorithm MeShClust using a percent identity threshold of 93%. Non-redundant sequences within each cluster were then identified and if the top ranked sequence had more reads associated than all other sequences combined, then this sequence became the consensus. Otherwise, all non-redundant sequences were aligned using ClustalO and the most prevalent base at each position in the alignment is assigned to the consensus. Only consensus sequences made up from at least 4 reads were retained.
  • IgG repertoire database preparation The IgG repertoire was evaluated by directly encapsulating single B cells and performing barcoding RT as described in the previous paragraph. 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 ⁇ L resin).
  • RBD SARS-CoV-2 Spike Protein
  • mFc Tag Sino biological Cat: 40592-V05H
  • 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 1 hr.
  • 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 2min; 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.
  • 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°C with PBS containing 30 ⁇ L 20 nM of the SARS- CoV-2 Spike S1+S2 ECD, his Tag protein.
  • 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 hr 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 hr at room temperature.
  • 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
  • the plate was then 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 1 hr 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.
  • FIG.4 middle panel illustrates a typical ELISA assay format useful for determining binding affinity of the subject antibodies against S1 or S2 domains.
  • FIG.5A shows that two antibodies having the same VH sequence and closely related VL sequences - Ab-6 (H13S1-8A9A) and Ab-7 (H13S1-8A4A) - shared high sequence similarity to two other antibodies in the light chain CDR regions – H13S1-8A097A and H13S1-8A839A, and they all bind the S1 domain with nanomolar to subnanomalor affinity (FIG.5B).
  • Ab-7 differs from Ab-6 only by 1 residue each in light chain CDR2 and CDR3 (see FIG.5A), yet its binding affinity increased by 4 fold (EC50 dropped from 3.7 nM of Ab-6 to 0.9 nM of Ab-7).
  • 384-well plates were coated with 2 ⁇ g/mL of tested antibodies in 30 ⁇ L 1xPBS at 4°C overnight. After blocking with 2% BSA at room temperature for 1 hour, 40 ⁇ L of the competitor antibody-S2 protein mixture was added into a well and incubated at room temperature for 1 hour. The mixture was composed of 20 ⁇ L of 90 ⁇ g/mL competitor anti-S2 antibody or isotype control and 20 ⁇ L of S2 spike protein with HIS tag at a determined EC80 concentration binding to the coated antibody. After washing, the remaining S2 spike protein binding to the coated antibody was detected by anti-His-HRP at 1/15000 dilution.
  • Example 3 Pharmacological profile of the neutralizing antibodies The subject antibodies’ binding to S1/S2 domains is further profiled pharmacologically.
  • Example 4 IgG4 Monoclonal Antibodies Have Comparable Activity This example demonstrates that certain subject antibodies with hIgG4 constant region (which does not engage or minimally engages Fc ⁇ R), compared to their counterpart with the hIgG1 constant region, have comparable binding affinity for the SARS-CoV-2 S1 or S2 antigen, pseudovirus neutralization activity, and live virus neutralization activity. Such hIgG4 antibodies also have comparable favorable developability characteristics.
  • CM5 chip For binding affinity assay, SPR (surface plasmon resonance) with anti-hIgG Fc immobilized on CM5 chip was used to capture several subject monoclonal antibodies with hIgG1 vs. hIgG4 constant region, respectively, in order to compare their respective binding affinity for the soluble S1 (for Ab-7) and S2 (for Ab-5) domain.
  • D614 refers to binding to the full length ectodomain of the S protein.
  • D614G refers to binding to the full length ectodomain of the S protein carrying the D614G mutation.
  • RBD refers to binding to the RBD domain.
  • the pseudovirus neutralization functional assay was performed, in brief, by expressing SARS-CoV-2 S protein (either from the reference strain or from the D614G mutant strain) with GFP as a marker on VSV ⁇ G (Delta-G VSV) to create a pseudovirus.
  • This S protein-expressing VSV ⁇ G pseudovirus could enter the Vero target cell, and the GFP fluorescent intensity inside the Vero cell is an indication for the extent of viral entry.
  • Neutralization of pseudovirus entry by the subject antibodies can be determined based on the extent of GFP signal reduction inside the Vero cells. The concentrations of the antibodies required to achieve 50%, 80%, or 90% pseudovirus neutralization were determined based on GFP fluorescence reduction.
  • Delta-G-VSV vesicular stomatitis virus
  • SARS-CoV-2 S and GFP were inhibited with the respective test antibodies and then used to infect Vero cells.
  • Expression of GFP from the Vero cells were measured to calculate the concentration of antibody needed to have 50%, 80%, or 90% inhibition or neutralization of the Delta-G-VSV expressing SARS-CoV S.
  • IC501, IC801, and IC901 refer to concentration of antibody to result in 50%, 80%, and 90% inhibition, respectively.
  • the results of the affinity and pseudovirus neutralization assays are summarized in the table below.
  • authentic SARS-CoV-2 virus infection and blockage thereof can be assessed in a BSL-3 or BSL-4 laboratory with adequate safety measure, and the results can be compared with that of pseudovirus neutralization assay.
  • Dual targeting bi-specific antibodies e.g., the Ab-7/Ab-5 bi-specific antibody 8A4A/9A10A-scFv
  • Bi-specific antibodies with an S1 binder/blocker (such as Ab-7 or 8A4A) together and an S2 binder (such as Ab-5 or 9A10A) can show potential synergetic effect in neutralization.
  • Example 5 Antibody Dependent Phagocytosis, Complement Deposition, and Natural Killer Activation Antibodies were incubated with various cells to determine activity for dependent phagocytosis, complement deposition, and natural killer activation. The results are summarized in the table below.
  • the antibodies were assayed to determine binding affinity to Fc ⁇ R and FcR molecules. The results are summarized in the table below.
  • Example 6 Developability Assay A series of developability assays are performed for the IgG4 version of lead antibodies, such as Ab-6 and Ab-7, including accelerated stability (2-3 mg/mL of Ab at 25 and 40°C in D- PBS, pH7.4, for up to 14 days); forced degradation (2-3 mg/mL of Ab at 25°C in 100 mM acetic acid at pH3.5, for up to 6 hours); and up to 5 freeze-thaw cycles (2-3 mg/mL of Ab). The results show that all samples are stable in the accelerated stability study; all antibodies show aggregation formation under the low pH stress condition; and all samples remain stable after 5 cycles of freeze-thaw.
  • Example 7 Efficacy of antibodies against SARS-CoV-2 variants Live virus variant study Vero E6 cells are infected by the WuhanD614, BavPat D614G and other variants such as the UK B.1.1.7 (also named 20I/501Y.V1) SARS-CoV-2 variants and incubated with the subject antibodies with a series of two-fold dilutions in the 0.97-1000 ng/ml range in triplicate. Viral RNA in the supernatant is determined, and % of inhibition is calculated based on infected but untreated control. The results are shown in the table in Example 4.
  • SARS-CoV-2 Various variants of SARS-CoV-2 are tested in this experiment: SARS-CoV-2/Wild-type (WT), the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and the Lambda (C.37) variant. Those variants represent commonly isolated SARS-CoV-2 clinical isolates.
  • Example 8 SARS-CoV-2 S protein variant binding by Ab-2 IgG4 The binding of the Ab-6 or Ab-7 IgG4 antibody to SARS-CoV-2 S protein variants (S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, A222V/D614G, the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and the Lambd
  • the 384-well plates are coated with 20 nM of the different SARS-CoV-2 S protein RBD variants.
  • the binding of Ab-2 IgG4 (12 concentrations obtained by 3-fold serial dilutions starting from 300 nM, in triplicate) to the SARS-CoV-2 S protein RBD (receptor binding domain) variants is detected by goat F(ab’)2 anti-human IgG (H+L)-HRP.
  • the blocking activity of Ab-6 or Ab-7 IgG4 is determined by enzyme linked immunosorbent assay (ELISA).
  • the 384-well plates are coated with 20 nM of the hACE2-mFc protein.
  • SARS-CoV-2 S protein RBD/S1 variants (with His tag) at fixed concentration (binding EC90 of corresponding variant to hACE2-mFc) was pre-incubated with Ab-6 or Ab-7 IgG4 or isotype control at different concentrations (12 concentrations obtained by 3-fold serial dilutions starting from final concentration of 300 nM, in duplicates), before incubated with coated hACE2- mFc protein.
  • the binding of SARS-CoV-2 S protein RBD/S1 variants (with His tag) to the hACE2-mFc protein is detected by HRP anti-6X His tag antibody.
  • binding affinities of Ab-6 or Ab-7 IgG4 to WT RBD or the above mutant RBD variants are evaluated using SPR (Biacore T200).
  • SPR Biacore T200.
  • Example 9 Absence of antibody-dependent enhancement (ADE) by Ab-6/7 IgG4 One potential hurdle for antibody-based therapeutics is the risk of exacerbating COVID- 19 severity via antibody-dependent enhancement (ADE).
  • ADE has been documented to occur through two distinct mechanisms in viral infections: by enhanced antibody-mediated virus uptake into Fc gamma receptor IIa (Fc ⁇ RIIa)-expressing phagocytic cells leading to increased viral infection and replication, or by excessive antibody Fc-mediated effector functions or immune complex formation causing enhanced inflammation and immunopathology. Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing infection. Raji cells, originally derived from a Burkitt’s lymphoma patient, have been shown to facilitate SARS-CoV-1 infection in the presence of anti-S-protein immune serum.
  • Fc ⁇ RIIa Fc gamma receptor IIa
  • this Fc ⁇ RII-bearing human B lymphoblast cell line is used to study the antibody-dependent viral entry of SARS-CoV-2 as an indicator of ADE. Briefly, Raji cells are seeded in 96-well plates. Antibodies at different concentrations are pre-incubated with the SARS-CoV-2 pseudo virus encoding wild-type spike protein and luciferase. The mixture of antibody and pseudo virus are then added to plated Raji cells. The plates are incubated and pseudovirus infection of Raji cells was quantified by measuring luciferase activity.
  • Example 10 Pharmacokinetics of Ab-6/7 IgG4 in rhesus monkeys Systemic circulation of antibody A total of 9 Rhesus monkeys are included for efficacy study of Ab-6/7 IgG4. Monkeys are allocated into 3 groups (three per group) receiving a single intravenous infusion of isotype control at 50 mg/kg (group 1), Ab-6/7 IgG4 at 10 mg/kg (group 2) and Ab-6/7 IgG4 at 50 mg/kg (group 3) one day after intratracheal inoculation of SARS-CoV-2 at 1 ⁇ 10 5 TCID 50 . Serum samples are collected once daily from 0-7 days post infection (d.p.i.).
  • the concentration of Ab-2 IgG4 in plasma samples is determined using validated ELISA methods. Briefly, plates are coated at 4°C overnight with anti-human (h) IgG (quantification of total hIgG) or SARS-CoV-2 S protein S1 subunit recombinant protein (quantification of unbound Ab-6/7 IgG4), before incubated with rhesus monkey plasma collected during Ab-6/7 IgG4 Study. The concentration of plasma antibodies is detected by HRP conjugated anti-hIgG antibody.
  • Pharmacokinetics and immunogenicity study A study is conducted to evaluate serum pharmacokinetics (PK) and immunogenicity following a single IV infusion administration of Ab-2 IgG4 in na ⁇ ve male and female cynomolgus monkeys. On Day 1 of the study, 3 male and 3 female cynomolgus monkeys are administered a single 10 mg/kg dose of Ab-2 IgG4 by IV infusion (60 minutes; 4 mL/kg). Blood is collected and processed for PK, anti-drug antibody (ADA), hematology, and clinical chemistry evaluations (data not shown). The concentration of Ab-6/7 IgG4 and ADA, is monitored in serum for up to 56 days (1345 hours) after beginning of infusion and are provided below.
  • PK serum pharmacokinetics
  • ADA anti-drug antibody
  • Example 11 In vivo efficacy of Ab-6/7 IgG4 in rhesus monkeys The efficacy of Ab-6/7 IgG4 to treat SARS-CoV-2 infection is evaluated in Rhesus monkeys. On Day 0, 3 groups of 3 Rhesus monkeys (2 females and 1 male per group) are infected by intratracheal (IT) inoculation with SARS-CoV-2 at 1 ⁇ 10 5 TCID50/animal.
  • IT intratracheal
  • One monkey from each group is euthanized 5, 6 and 7 days post-infection and selected organs (lungs [6 lobes, trachea, left and right bronchia], spleen, pulmonary hilar lymph node, liver, and kidney) are processed, stained with hematoxylin and eosin [H&E] and Masson's trichrome staining, and associated pathological changes were evaluated microscopically. Serum samples are collected prior to viral challenge (pi0d) and on Days 1 (before Ab-2 IgG4 administration), 2, 3, 4, 5, 6, and 7, for evaluation of Ab-6/7 IgG4 levels.
  • 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°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).
  • 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.
  • Antibody neutralization activity against pseudovirus 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.
  • IC 50 half-maximal inhibitory concentration
  • cells are 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 or Alexa Fluor ® 488-conjugated goat anti-mouse IgG antibody as the secondary antibody.
  • Cell nuclei were stained using Hoechst 33258 at room temperature for 10 minutes. Images were taken under an inverted fluorescence microscope (Nikon). In some experiments, numbers of nuclei and cells infected with viruses are counted using an Operetta CLSTM system. % inhibition is calculated by (total nuclei-infected cells)/ (total nuclei) x 100%.
  • Fifty percent neutralization dose (ND50) and ninety percent neutralization dose (ND90) are calculated using 4-parameter non-linear regression with GraphPad Prism 8.0.

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Abstract

The invention described herein provides neutralizing antibodies against SARS-CoV-2 antigens (such as the S 1 subunit of the S antigen) for use in treating human patients having COVID-19 and for methods of screening, in particular, the combined droplet-based single cell screening with mass spectrometry.

Description

METHODS AND COMPOSITIONS RELATED TO NEUTRALIZING ANTIBODIES AGAINST HUMAN CORONAVIRUS REFERENCE TO RELATED APPLICATION This application claims priority to and the benefit of the filing date of U.S. Provisional Patent Application No.63/234,296, filed on August 18, 2021, the entire contents of which are incorporated herein by reference. 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. Since the virus has only recently emerged from bats and crossed over into humans, little is known about the immune response generated against it 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. SUMMARY OF THE INVENTION The invention described herein is summarized in the following numbered embodiments: E1. 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 (e.g., the Spike or S protein responsible for ACE2 binding) of SARS-CoV-2, and wherein said monoclonal antibody comprises: (1) (1a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 1, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 2, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 3; and, (1b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 4, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 5, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 6; and/or (2) (2a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 11, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 12, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 13; and, (2b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 14, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 15, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 16; and/or (3) (3a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 21, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 22, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 23; and, (3b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 24, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 25, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 26; and/or (4) (4a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 31, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 12, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 33; and, (4b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 34 or SEQ ID NO: 115, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 35, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 36; and/or (5) (5a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 51, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 12, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 53; and, (5b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 54, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 55, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 56; and/or (6) (6a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and (6b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 65, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 66; and/or (7) (7a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (7b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 75, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 76; and/or (8) (8a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (8b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 75, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 66; and/or (9) (9a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (9b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 65, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 96; and/or (10) (10a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (10b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 14 a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 15, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 106; and/or (11) (11a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (11b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 114, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 115, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 106; and/or (12) (12a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (12b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 124, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 15, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 106; and/or (13) (13a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (13b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 134, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 115, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 136; and/or (14) (14a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (14b) a light chain variable region (LCVR), comprising the same LCVR CDR1, LCVR CDR2, and LCVR CDR3 of H13S1-8A839A or H13S1-8A097A (in FIG. 5A); optionally, said isolated monoclonal antibody is not naturally occurring; and/or, optionally further comprising a signal peptide sequence of MGWSCIILFLVATATGAHS (SEQ ID NO: 225) at the N-terminus of said HCVR and/or LCVR. E2. The isolated monoclonal antibody or antigen-binding fragment thereof of E1, wherein: (1) (1A) the HCVR sequence comprising the amino acid of SEQ ID NO: 7; and/or, (1B) the LCVR sequence comprising the amino acid of SEQ ID NO: 8, or, (2) (2A) the HCVR sequence comprising the amino acid of SEQ ID NO: 17; and/or, (2B) the LCVR sequence comprising the amino acid of SEQ ID NO: 18, or, (3) (3A) the HCVR sequence comprising the amino acid of SEQ ID NO: 27; and/or, (3B) the LCVR sequence comprising the amino acid of SEQ ID NO: 28, or, (4) (4A) the HCVR sequence comprising the amino acid of SEQ ID NO: 37; and/or, (4B) the LCVR sequence comprising the amino acid of SEQ ID NO: 38, or, (5) (5A) the HCVR sequence comprising the amino acid of SEQ ID NO: 57; and/or, (5B) the LCVR sequence comprising the amino acid of SEQ ID NO: 58, or, (6) (6A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (6B) the LCVR sequence comprising the amino acid of SEQ ID NO: 68, or, (7) (7A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (7B) the LCVR sequence comprising the amino acid of SEQ ID NO: 78, or, (8) (8A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (8B) the LCVR sequence comprising the amino acid of SEQ ID NO: 88, or, (9) (9A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (9B) the LCVR sequence comprising the amino acid of SEQ ID NO: 98, or, (10) (10A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (10B) the LCVR sequence comprising the amino acid of SEQ ID NO: 109, or, (11) (11A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (11B) the LCVR sequence comprising the amino acid of SEQ ID NO: 119, or, (12) (12A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (12B) the LCVR sequence comprising the amino acid of SEQ ID NO: 129, or, (13) (13A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (13B) the LCVR sequence comprising the amino acid of SEQ ID NO: 139, or, (14) (14A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (14B) the same LCVR sequence of H13S1-8A839A or H13S1-8A097A (in FIG. 5A). E3. The isolated monoclonal antibody or antigen-binding fragment thereof according to E1 or E2, wherein said monoclonal antibody has: (1) (1a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 7; and/or, (1b) a light chain sequence comprising the amino acid of SEQ ID NO: 8, or, (2) (2a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 17; and/or, (2b) a light chain sequence comprising the amino acid of SEQ ID NO: 18, or, (3) (3a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 27; and/or, (3b) a light chain sequence comprising the amino acid of SEQ ID NO: 28, or, (4) (4a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 37; and/or, (4b) a light chain sequence comprising the amino acid of SEQ ID NO: 38, or, (5) (5a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 57; and/or, (5b) a light chain sequence comprising the amino acid of SEQ ID NO: 58, or, (6) (6a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (6b) a light chain sequence comprising the amino acid of SEQ ID NO: 68, or, (7) (7a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (7b) a light chain sequence comprising the amino acid of SEQ ID NO: 78, or, (8) (8a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (8b) a light chain sequence comprising the amino acid of SEQ ID NO: 88, or, (9) (9a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (9b) a light chain sequence comprising the amino acid of SEQ ID NO: 98, or, (10) (10a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (10b) a light chain sequence comprising the amino acid of SEQ ID NO: 109, or, (11) (11a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (11b) a light chain sequence comprising the amino acid of SEQ ID NO: 119, or, (12) (12a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (12b) a light chain sequence comprising the amino acid of SEQ ID NO: 129, or, (13) (13a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (13b) a light chain sequence comprising the amino acid of SEQ ID NO: 139, or, (14) (14a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (14B) the same light chain sequence of H13S1-8A839A or H13S1-8A097A (in FIG.5A). E4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of E1-E3, wherein: (a) the isolated monoclonal antibody is a human antibody, a CDR-grafted antibody, or a resurfaced antibody; (b) the isolated monoclonal antibody is a bi-specific antibody, optionally the bi- specific antibody comprises the HCVR CDR1, CDR2, and CDR3 and LCVR CDR1, CDR2, and CDR3 of any one of a first antibody of claim 1(1) – 1(13), and the HCVR CDR1, CDR2, and CDR3 and LCVR CDR1, CDR2, and CDR3 of any one of a second antibody of claim 1(1) – 1(13), wherein the first and the second antibodies are different or bind to different (preferably non-overlapping) epitopes; optionally, (i) said first antibody is specific for S1 (such as Ab-1, Ab-6, or Ab-7), and said second antibody is specific for S2 (such as Ab-3, Ab-5), and/or, (ii) said bi-specific antibody comprises an antigen-binding fragment (such as an scFv, Fab, or Fab’ fragment) of one of said first antibody and said second antibody, fused to the light chain (or heavy chain) of the other of said first antibody and said second antibody), and/or, (c) the antigen-binding fragment thereof is an Fab, Fab’, F(ab’)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc. E5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of E1- E4, wherein said monoclonal antibody or antigen-binding fragment thereof: (i) binds to the S1 or S2 glycoprotein of SARS-CoV-2; (ii) binds the SARS-CoV-2 antigen with a Kd of less than about 5 nM, 2 nM, 1 nM, 0.5 nM, 0.2 nM, 0.1 nM, or 0.05 nM; (iii) binds to SARS-CoV-2 wild-type S protein and/or RBD/S1 variants selected from the group consisting of S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, A222V/D614G, the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and the Lambda (C.37) variant, and/or, (iv) inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2, optionally 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), and/or optionally 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). E6. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of E1- E5, which: (i) 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; (ii) exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 50 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 6 nM, less than 3 nM, less than 1 nM, less than 0.6 nM or less than 0.5 nM; (iii) inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 50 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 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; (iv) inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 50 nM, less than 30 nM, less than 20 nM, 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; (v) inhibits entry of wild-type SARS-CoV-2, and/or SARS-CoV-2 variants (e.g., Wuhan D614, BavPat D614G, UK B.1.1.7, or South Africa B.1.351 strain, or a SARS-CoV-2 variant sharing one or more S1 protein mutations with the WuhanD614, BavPat D614G, UK B.1.1.7, and/or South Africa B.1.351 strain(s)) into a target cell; and/or, (vi) does not cause antibody-dependent enhancement (ADE). E7. The monoclonal antibody or antigen-binding fragment thereof of any one of E1-E6, comprising a heavy chain constant region, wherein the heavy chain constant region is human IgG4, human IgG3 or human IgG2; optionally, the heavy chain constant region is human IgG4 which optionally comprises a YTE (M252Y/S254T/T256E) mutation and/or an LS (M428L/N434S) mutation. E8. 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 (e.g., the S protein responsible for ACE2 binding) of SARS- CoV-2, and wherein said monoclonal antibody comprises a heavy chain variable region (HCVR) comprising a HCVR CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 65, and a LCVR CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 66, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises: (i) an HCVR sequence comprising the amino acid sequence of SEQ ID NO: 67 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 67; and (ii) an LCVR sequence comprising the amino acid sequence of SEQ ID NO: 68 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 68; and/or, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG4, human IgG3, or human IgG2, preferably human IgG4. E9. 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 (e.g., the S protein responsible for ACE2 binding) of SARS- CoV-2, and wherein said monoclonal antibody comprises 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 a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO: 64, a LCVR CDR2 sequence of SEQ ID NO: 75, and a LCVR CDR3 sequence of SEQ ID NO: 76, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises: (i) an HCVR sequence of SEQ ID NO: 67 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 67; and (ii) an LCVR sequence of SEQ ID NO: 78 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 78; and/or, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG4, human IgG3, or human IgG2, preferably human IgG4. E10. The monoclonal antibody or antigen-binding fragment thereof of claim 8 or claim 9, comprising an IgG4 heavy chain (HC) sequence comprising SEQ ID NO: 67 or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto. E11. 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 E1-E10 for binding to the same or substantially the same epitope (e.g., based on binning assay). E12. A mixture of two or more isolated monoclonal antibodies or antigen-binding fragments thereof of any one of E1-E10, optionally, the proportion of each of said two or more isolated monoclonal antibodies or antigen-binding fragments thereof is substantially the same, or is different. E13. A polynucleotide encoding the heavy chain and/or the light chain, or the antigen-binding portion thereof, of any one of E1-E11, optionally, the polynucleotide is codon optimized for expression in a human cell; and/or, optionally, the polynucleotide is in a vector, such as an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector), wherein the vector is optionally in a host cell that expresses said isolated monoclonal antibody or antigen-binding fragment thereof. E14. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen- binding fragment thereof of any one of E1-E11, or the mixture of E12, optionally the pharmaceutical composition is formulated for intravenous administration, or for inhalational or oral administration; and/or, optionally, the pharmaceutical composition is for treating a subject infected by SARS- CoV-2, and further comprises a pharmaceutically acceptable excipient or diluent. E15. A combination comprising the pharmaceutical composition of E14, and a second therapeutic agent effective to treat infection by SARS-CoV-2, optionally, 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. E16. 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 E1-E11, the mixture of E12; the polynucleotide of E13, or the pharmaceutical composition of E14, optionally, the method is for treating COVID-19 or a subject infected by SARS-CoV-2, wherein the method further comprises administering a second therapeutic agent; and/or, optionally, said 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. E17. A method of identifying an antibody specific for an antigen from a virus or a bacterium, from a B-cell population obtained from a subject having been infected by and recovering from infection by the virus or the bacterium, the method comprises: (i) obtaining a library of paired VH and VL antibody sequences from a B cell population obtained from the subject; and, (ii) obtaining amino acid sequences of fragments of antibodies specific for said antigen, wherein said antibodies are obtained (e.g., affinity purified) from a sample comprising said B cell population; thereby identifying the antibody specific for said antigen when said amino acid sequences obtained in (ii) match the paired VH and VL antibody sequences obtained in (i). E18. The method of E17, wherein the virus is SARS-CoV-2, and the antigen is SARS-CoV-2 S (Spike) protein or N (Nucleocapsid) protein. E19. The method of E17 or E18, wherein the sample is a blood sample or plasma sample. E20. The method of any one of E17-E19, wherein the B cell population is PBMCs isolated from a peripheral blood sample. E21. The method of any one of E17-E20, wherein the B cell population comprises plasmablasts and memory B cells. E22. The method of any one of E17-E21, wherein step (i) comprises: (1) generating a plurality of nanoliter scale droplets, each comprising: (a) one B cell from said B-cell population; (b) multiple (e.g., 1,000-1,500) co-encapsulated paramagnetic beads (e.g., colloidal nanoparticles) coated by a first non-specific antibody-binding molecule; and, (c) the antigen labeled by a first detectable label; (2) allowing, under a pre-determined condition, antibodies secreted by said one B-cell to bind said first non-specific antibody-binding molecule on said paramagnetic beads, and to bind said antigen when / if said antibodies are specific for said antigen; (3) passing the droplets through a magnetic field to aggregate said multiple paramagnetic beads, in order to concentrate the accumulative signal emitted by the first detectable label on the antigen bound by said antibodies over a background signal emitted by the first detectable label on the antigen unbound by said antibodies; and, (4) collecting droplets having significantly enhanced accumulative signal emitted by the first detectable label over the background signal; thereby identifying the antibody secreted by said one B-cell as being specific for said antigen. E23. The method of E22, wherein said plurality of nanoliter scale droplets are generated by a microfluidic device. E24. The method of E22 or E23, wherein the average size of said plurality of nanoliter scale droplets is about 30-1500 pL, about 40 pL, about 80 pL, about 125 pL, or about 1 nL. E25. The method of any one of E17-E24, wherein the B-cell population is from a human or a non-human mammal (e.g., mouse, rat, rabbit). E26. The method of any one of E17-E25, wherein the multiple co-encapsulated paramagnetic beads are in an amount sufficient to bind substantially all antibodies secreted by said one B cell. E27. The method of any one of E17-E26, wherein said first non-specific antibody-binding molecule is biotin-labeled anti-human IgG-Fc (which biotin binds to streptavidin-coated paramagnetic beads), Protein G (which binds to immunoglobulin Fab and Fc regions, optionally, the Protein G lacks albumin-binding region), Protein A (which binds heavy chain Fc region and within the Fab region of human VH3 family), Protein A/G (which binds all subclasses of human IgG as well as IgA, IgE, IgM and to a lesser extent IgD, and all subclasses of mouse IgG but not mouse IgA, IgM or serum albumin), Protein L (which binds kappa light chain of all antibody classes including IgG, IgM, IgA, IgE, IgD as well as scFv and Fab fragments), or a species-specific antibody or antigen-binding fragment thereof (such as an anti-mouse κ light chain (Igκ) nanobody VHH). E28. The method of any one of E17-E27, wherein the first detectable label is a fluorescent label (such as Alexa Fluor 488 and DayLight550). E29. The method of any one of E17-E28, wherein the nanoliter scale droplets further comprise: (d) a second non-specific antibody-binding molecule labeled by a second detectable label, wherein said second non-specific antibody-binding molecule does not compete or interfere with binding by said first non- specific antibody-binding molecule; wherein in step (3), the accumulative signal emitted by the second detectable label (e.g., DayLight650) on said second non-specific antibody-binding molecule bound by said antibodies is reflective of the relative amount of said antibodies on aggregated paramagnetic beads. E30. The method of E29, wherein said second non-specific antibody-binding molecule is Protein G, Protein A, Protein A/G, Protein L, or a species-specific antibody or antigen- binding fragment thereof (e.g., Goat-anti-human IgG Fc). E31. The method of any one of E17-E30, wherein the pre-determined condition is 15-60 min. at 37°C (under 5% CO2). E32. The method of any one of E17-E31, wherein said multiple paramagnetic beads aggregate to form a geometric shape (such as a straight line) under the magnetic field. E33. The method of any one of E17-E32, wherein said accumulative signal emitted by the first detectable label is a fluorescent signal emitted after laser excitation. E34. The method of any one of E17-E33, wherein step (4) is carried out by an acoustic sorter device that generates a surface acoustic wave (SAW), or by fluorescence-activated dielectrophoretic sorting. E35. The method of any one of E17-E34, further comprising (5) determining the sequences of the paired heavy and light chains (VH and VL) of each identified antibody. E36. The method of E35, wherein step (5) comprises compartmentalizing each B cell collected from the droplets in step (4) with a bead comprising a bead-specific nucleotide-based barcode and a reverse-transcription primer for initiating cDNA synthesis from mRNA encoding antibody heavy chain or antibody light chain. E37. The method of E36, wherein the primer is complementary to a heavy chain constant region coding sequence or a light chain constant region coding sequence, and the cDNA synthesized from the primer comprises heavy chain variable region or light chain variable region, or a CDR (such as CDR3) thereof. E38. The method of E36, wherein step (5) further comprises sequencing cDNA synthesized from the primer. E39. The method of E38, wherein cDNA sequencing is performed by next generation sequencing (NGS) (such as using an Illumina MiSeq sequencer with a 2 × 300 base pair sequencing flow chip). E40. The method of E38, further comprising cloning and expressing cDNA sequences in a host cell to produce said antibody specific for said antigen. E41. The method of E40, wherein the host cell is a CHO cell. E42. The method of E40 or E41, further comprising isolating and/or purifying said antibody. E43. The method of any one of E17-E42, wherein in step (ii), IgM in said sample is first depleted before the remaining antibodies in said sample are affinity purified with said antigen, and the affinity-purified antibodies are digested with protease for sequencing analysis of the resulting fragments using mass spectrometry. E44. An antibody identified as specific for an antigen of SARS-CoV-2 by the method of any one of E18-E43. E45. 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 E18-E43. E46. 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 E18-E43, and a pharmaceutically acceptable excipient or diluent. E47. 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 E18-E43, and a pharmaceutically acceptable excipient or diluent. E48. A combination comprising the pharmaceutical composition of E46 or E47, and a second therapeutic agent effective to treat infection by SARS-CoV-2. E49. The combination of E48, 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. E50. A method of treating a subject infected by SARS-CoV-2, the method comprises administering a therapeutically effective amount of the antibody of E44 or E45, the pharmaceutical composition of E46 or E47, or the combination of E48 or E49. In any of the preceding embodiment, the monoclonal antibody or antigen-binding fragment thereof is specific for the Spike protein or S protein of SARS-CoV-2, and wherein said monoclonal antibody specifically binds to and/or has a residue within 4 Å of residues T415, G416, K417, D420, Y421, Y453, L455, F456, R457, K458, N460, Y473, Q474, A475, G476, S477, F486, N487, Y489, Q493, S494, Y495, G496, Q498, T500, N501, G502, and Y505 of the S protein, optionally, said monoclonal antibody does not bind to and/or has no residue within 4 Å of residues G446 and Y449 of the S protein. In any of the proceeding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof is a human IgG4 antibody, or an FcγR null monoclonal antibody engineered to prevent FcγR engagement. In any of the proceeding embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to the S1 glycoprotein of SARS-CoV-2. In any of the proceeding embodiments, the monoclonal antibody or antigen-binding fragment thereof binds to the S2 glycoprotein of SARS-CoV-2. In any of the proceeding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof binds to SARS-CoV-2 wild-type S protein and/or RBD/S1 variants selected from the group consisting of S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, and A222V/D614G. In any of the proceeding embodiments, the monoclonal antibody or antigen-binding fragment thereof binds the SARS-CoV-2 antigen with a Kd 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 any of the proceeding 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 any of the proceeding 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 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 any of the proceeding 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 with an EC50 value of less than 2 nM, 1 nM or 0.1 nM. In any of the proceeding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof 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 any of the proceeding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof 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 any of the proceeding 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. In any of the proceeding embodiments, the isolated monoclonal antibody or antigen- binding fragment thereof inhibits entry of wild-type SARS-CoV-2 and/or SARS-CoV-2 variants, e.g. WuhanD614, BavPat D614G, UK B.1.1.7, South Africa B.1.351 lineage, the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and/or the Lambda (C.37) into a target cell. In any of the proceeding embodiments, the isolated monoclonal antibody or antigen- binding fragment thereof inhibits entry of a SARS-CoV-2 variant sharing one or more S protein mutations with the WuhanD614, BavPat D614G, UK B.1.1.7, South Africa B.1.351 strain(s), the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and/or the Lambda (C.37) into a target cell. In any of the proceeding embodiments, the isolated monoclonal antibody or antigen- binding fragment thereof does not cause antibody-dependent enhancement (ADE). In any of the proceeding embodiments, the isolated or recombinantly produced monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region, wherein the heavy chain constant region is human IgG1, human IgG2, human IgG3 or human IgG4. In some embodiments, the heavy chain constant region is human IgG4. 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 Ab-1 to Ab-13 and Ab in FIG.5A are listed below. 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 FIG.1A shows that sera from SARS-CoV-2 convalescent patients clearly showed higher antibody (IgG) titers as compared to sera from healthy donors. FIG.1B shows titers of antibodies specific for the RBD domain of S1 or the Spike extracellular domain (ECD) of SARS-CoV-2, from hospitalized SARS-CoV-2 patients, SARS- CoV-2 positive patients, and patients displaying COVID-19 symptoms. FIG.2 is a schematic drawing showing representative fluorescent signal patterns for the various picoliter droplets generated in microfluidic devices, including droplets with no IgG- secreting cell, droplets with cell secreting IgG that does not bind SARS-CoV-2 antigen, droplets with cell secreting IgG that does bind SARS-CoV-2 antigen, droplets with memory B cell having membrane-bound IgG, and droplets with memory B cell having membrane-bound SARS-CoV-2 binding IgG. FIG.3 is a schematic drawing showing the bifurcated approach to identify antigen- specific (e.g., SARS-CoV-2 specific) antibodies. The top part shows the microfluidic-device enabled single cell sorting and bar-coded DNA sequencing approach to generate libraries of paired VH and VL sequences. The bottom part shows the immunoprecipitation based mass spectrometry-based peptide sequencing to identify partial sequences of antibodies specific for a selected antigen (e.g., SARS-CoV-2 S1 and/or S2 protein). Matching sequences from both routes identifies VH/VL pairs specific for the target antigen. FIG.4 shows representative antibodies identified using the procedure of FIG.3, having strong S1 or S2 binding affinity. The middle panel is a schematic drawing showing the ELISA screening format used to measure EC50 values. FIG.5A shows multiple sequence alignments of light chain variants with improved binding affinity FIG.5B shows EC50 values of the various S1-binding antibodies. FIG.6A shows that the identified S protein binders can block binding of viral S protein to the human ACE2 receptor expressed on Vero-E6 cell surface. FIG.6B shows that the identified S protein binders can inhibit the binding of the viral S1 ECD domain binding to the human ACE2 receptor expressed on Vero-E6 cell surface. S2 binders show partial inhibition of Spike protein binding to ACE2. FIG.7 shows the results of S2 binding antibody binning assay. FIG.8 shows exemplary bi-specific antibodies against S1 and S2 proteins. 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, light chain CDR sequences of certain isolated antibodies are listed in FIG.5A, 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. Another aspect of the invention provides a bi-specific antibody of the invention having antigen-binding fragments (e.g., defined by 6 CDR sequences) from two different antibodies binding to different epitopes of S1 and/or S2, respectively. For example, as shown in FIG.8, a bi-specific antibody may comprise antigen-binding fragments from a first antibody specific for S1, and antigen-binding fragments from a second antibody specific for S2, wherein the S2 antibody fragments may be in scFv format (either VH-VL or VL-VH) and linked to the VL (or VH) of the S1 antibody. Such bi-specific antibodies may provide better therapeutic efficacy compared to the individual component antibodies of the mixture. 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. In summary, 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, such as bi-specific antibodies, or be used together, for combination therapy. 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., bi-specific 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), IgG3 (comprising a γ3 constant region), and IgG4 (comprising a γ4 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, CL. 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, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (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 or VL:VH 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 (dsFV2). 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 I125 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 XENOMOUSE®, 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.C6® 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. The term “antibody-dependent enhancement” (ADE) refers to a phenomenon in which binding of a virus to suboptimal antibodies enhances its entry into host cells. While not wishing to be bound by any particular theory and not implying that any actual mechanisms of ADE may have been operating in connection with any antibodies tested herein, ADE has been documented to occur through two distinct mechanisms in viral infections: by enhanced antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa)-expressing phagocytic cells leading to increased viral infection and replication, or by excessive antibody Fc-mediated effector functions or immune complex formation causing enhanced inflammation and immunopathology. Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing infection. “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 dichlorodifluoromethane, 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 (Kd) 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, bi- specific antibodies are provided. Non-limiting exemplary bi-specific 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 bi-specific antibody comprises the antigen-binding fragment (such as scFv, Fab, or Fab’ fragment) of a first antibody specific for a first epitope of an SARS- CoV-2 (such as S1 or S2), and comprises the full-length antibody or antigen-binding fragment (such as scFv, or F(ab’)2 fragment) of a second antibody specific for a second epitope of an SARS-CoV-2 (such as S2 or S1), wherein the first and the second epitopes do not overlap. In certain embodiments, binding of the first epitope by the antigen-binding fragment of the first antibody does not substantially inhibit (e.g., has no effect or enhances) the binding of the second epitope by the antigen-binding fragment of the second antibody. In certain embodiments, the antigen-binding fragment of the first antibody is linked to the antigen-binding fragment of the second antibody directly. In certain other embodiments, the antigen-binding fragment of the first antibody is linked to the antigen-binding fragment of the second antibody through a flexible linker, such as G4S type linker with one or more repeats. In certain embodiments, the antigen-binding fragment of the first antibody is an scFv, and is linked through its C-terminus to the N-terminus of a light chain (or a heavy chain) of the antigen-binding fragment of the second antibody. In certain embodiments, the bi-specific antibody comprises one or two antigen-binding fragment(s) of the first antibody. In certain embodiments, the bi-specific antibody comprises one or two antigen-binding fragment(s) of the second antibody. In certain embodiments, the bi- specific antibody comprises one or two antigen-binding fragment(s) of the first antibody and one or two antigen-binding fragment(s) of the second antibody. In certain embodiments, the bi- specific antibody comprises two antigen-binding fragments of the first antibody and two antigen- binding fragments of the second 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.
Figure imgf000038_0001
Figure imgf000039_0001
Figure imgf000040_0001
Figure imgf000041_0001
Figure imgf000042_0001
Figure imgf000043_0001
Figure imgf000044_0001
Figure imgf000045_0001
Figure imgf000046_0001
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Figure imgf000050_0001
Exemplary Bi-Specific Antibody (see FIG. 8)
Figure imgf000050_0002
Figure imgf000051_0001
Figure imgf000052_0001
Figure imgf000053_0001
Control SARS-CoV-2 specific antibodies Antibody C1S5-2A2A comprises the following sequences:
Figure imgf000053_0002
Figure imgf000054_0001
Antibody C1S2-6A6A comprises the following sequences:
Figure imgf000054_0002
Figure imgf000055_0001
For all the antibody HCVR 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 LCVR 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. In certain embodiments, the HC and/or LC further includes a signal peptide sequence, such as MGWSCIILFLVATATGAHS (SEQ ID NO: 225). In certain embodiments, any one of the antibodies of the invention comprise a light chain constant region (CL) selected form the CL sequences in the table below, and/or a heavy chain constant region (CH) sequences in the table below. In certain embodiments, the heavy chain constant region (CH) sequence is that of human IgG4.
Figure imgf000055_0002
Figure imgf000056_0001
In certain embodiments, any one of the antibodies of the invention comprise a LC leader sequence and/or a HC leader sequence below: LC leader sequence: MAWAPLLLTLLAHCTGSWA (SEQ ID NO: 226) or MDMRVPAQLLGLLLLWLRGARC (SEQ ID NO: 227). HC leader sequence: MKHLWFFLLLVAAPRWVLS (SEQ ID NO: 228) or MEFGLSWLFLVAILKGVQC (SEQ ID NO: 229). Engineering of antibodies for improved pharmacokinetics through enhanced binding to the neonatal Fc receptor (FcRn) has been demonstrated in transgenic mice, non-human primates and humans. Booth et al., “Extending human IgG half-life using structure-guided design,” MAbs, 2018 Oct.10(7) 1098-1110. Recombinant antibodies’ therapeutic potential may be enhanced by the introduction of defined mutations in the crystallizable fragment (Fc) domains, such as for example, YTE (M252Y/S254T/T256E) and LS (M428L/N434S), as a consequence of increased half-lives and prolonged duration of protection. For example, The prototypical example of an FcRn affinity-enhancing Fc mutant is the YTE mutation which, when incorporated into motavizumab IgG1, is able to extend serum half-life in humans by more than four-fold. Robbie et al., A novel investigational Fc-modified humanized monoclonal antibody, motavizumab-YTE, has an extended half-life in healthy adults. Antimicrob Agents Chemother.2013;57:6147–6153. Booth et al. and Robbie et al. are incorporated herein. Thus, in certain embodiments, any one of the antibodies of the invention comprise an Fc region mutation, such as a YTE (M252Y/S254T/T256E) mutation and/or an LS (M428L/N434S) mutation. In certain embodiments, the YTE mutation and/or the LS mutation is in IgG4 Fc region. In certain embodiments, the antibody of the invention comprises a light chain (LC) and a heavy chain (HC), wherein the LC comprises an optional LC leader sequence, followed by any of the VL sequences described herein (such as the VL sequence of any of Ab-1 to Ab-13, and Ab in FIG.5), followed by a light chain constant region such as one described in the table above; and wherein the HC comprises an optional HC leader sequence, followed by any of the VH sequences described herein (such as the VH sequence of any of Ab-1 to Ab-13, and Ab in FIG.5), followed by a heavy chain constant region such as one described in the table above, such as IgG4, optionally an IgG4 with YTE and/or LS mutation. 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, IgG3, or IgG4. 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. In some embodiments, an antibody described herein comprises a human IgG4 heavy chain constant region. In some such embodiments, an antibody described herein comprises an S241P mutation in the human IgG4 constant region. See, e.g., Angal et al. Mol. Immunol.30(1):105-108 (1993). In some embodiments, an antibody described herein comprises a human IgG4 constant region and a human κ light chain. 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. In some embodiments, effector function is not desirable. For example, in some embodiments, effector function may not be desirable in treatments of inflammatory conditions and/or autoimmune disorders, such as SARS-CoV-2 induced cytokine storm. In some such embodiments, a human IgG4 or IgG2 heavy chain constant region is selected or engineered. In some embodiments, an IgG4 constant region comprises an S241P mutation. 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-S and DG44 cells; PER.C6® 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 Identification of SARS-CoV-2 Specific Neutralizing Antibodies The SARS-CoV-2 virus utilizes its surface spike protein to mediate its entry into target cells. Briefly, to enter host cells, SARS-CoV-2 first binds to the cell surface receptor angiotensin-converting enzyme 2 (ACE2) with its surface-anchored spike protein for viral attachment. This spike protein is present as a trimer on viral surface, with three receptor-binding S1 heads sitting on top of a trimeric membrane fusion S2 stalk. Each S1 head contains a receptor-binding domain (RBD) that specifically recognizes ACE2 as its receptor. The RBD constantly switches between a “standing-up” position for receptor binding and a “lying-down” position for immune evasion. To fuse membranes, the spike needs to be proteolytically activated at the S1/S2 boundary, such that S1 dissociates and S2 undergoes a dramatic structural change. These SARS-CoV entry-activating proteases include cell surface protease TMPRSS2 and lysosomal proteases cathepsins. Compared to the related SARS-CoV RBD, SARS-CoV-2 RBD is more potent and less exposed (hidden RBD), facilitating better immune evasion. Further, unlike SARS-CoV, cell entry of SARS-CoV-2 is pre-activated by proprotein convertase furin, reducing its dependence on target cell proteases for entry. Collectively, the high hACE2 binding affinity of its RBD, furin pre-activation of the spike, and hidden RBD in the spike, allow SARS-CoV-2 to maintain efficient cell entry while evading immune surveillance, thus contributing to the wide spread of this virus. As COVID-19 becomes pandemic, it is critical to understand its effects on patients’ immune system as well as identify therapeutic treatments. The generation of protective and long- lasting humoral immunity is one major aspect of the adaptive immune response to infection. Therefore, it is important to analyze humoral responses in patients and use the neutralizing antibodies for potential treatment of the disease. This example demonstrates the profiling of humoral responses in patients exposed to SARS-CoV-2 infection, and the identification of neutralizing antibodies against SARS-CoV-2 from the recovered patients’ samples. Specifically, B cells secreting antibodies with specificity against the viral Spike protein were sorted and then sequenced, using the proprietary single cell barcoding technology. A number of blood donors, either showing COVID-19 symptoms or confirmed infected patients, were profiled to identify a number of non-redundant neutralizing antibody candidates against SARS-CoV-2. As a complementary approach, mass spectrometry and parallel single B cell antibody sequencing were used to identify antibodies in patient serum isolated through immunoprecipitation using the viral S protein. Patient blood sample collection and characterization Patients participated in this study ranged in age from 8 to 78 years old, with a median age of 47 years old. The average time between the initial positive RT-PCR test and subsequent negative RT-PCR test was 18.7 days, with a median time of 17 days. To characterize antiviral immune responses, antibody titers in patient sera were measured against the SARS-CoV-2 Spike (S) protein. 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, and titer is the highest in hospitalized patients (FIG.1B). Thus, 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 of China where the SARS-CoV-2 virus first 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. 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. 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. 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 (mouse antibody Fc) 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 (data not shown), suggesting that an immune response is also generated against the S2 domain and that the multimerization state of the S protein influences antibody binding. B cell profiling To characterize the B cell populations of patient samples, PBMCs isolated from peripheral blood collected from 19 patients were analyzed by flow cytometry. Cells were stained with a panel of anti-CD19 / CD27 / CD38 antibodies, and plasmablasts (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 (data not shown). While total CD19 positive populations from two patients are somewhat lower than the control healthy donor with overall reduced lymphocytes as reported previously, the fraction of memory B cells (CD27high CD38low) are significantly higher in the two convalescent patient samples than in the healthy donor control, and the percentages of plasmablasts (CD27high CD38high) are about 20-fold higher compared to healthy donor sample. Identification of SARS-CoV-2 specific antibodies To identify SARS-CoV-2 specific antibodies, two complementary approaches were used. First - direct screening of plasmablasts from patient blood using droplet-based assays. Second - affinity-based enrichment of antibodies followed by mass spectrometry and parallel single-cell sequencing of B cells from the same patient. For the first approach, the paired VH-VL sequences were recovered by plating the B cells in individual wells. An alternative approach can be based on the rapid droplet-based single-cell antibody discovery platform CELLIGO® to identify paired antibody heavy chain (VH) and light chain (VL) DNA sequences (see Gérard et al., High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics. Nat Biotechnol 38, 715–721, 2020, incorporated herein by reference). In the primary approach, two different immunological compartments were interrogated: plasmablasts by direct encapsulation of PBMCs, and memory B cells by in vitro memory B cell activation followed by a B cell enrichment kit. Then proprietary droplet-based CELLIGO® platform was used to screen antibody-secreting B cells for antigen specificity. The CELLIGO® platform is a microfluidic-based method that can screen millions of cells for antigen specificity in single day. Specifically, Cells of interest are encapsulated in droplets acting as individual picolitre sized reaction chambers. One hour of incubation is sufficient to detect the presence of IgGs secreted by the B cells using a fluorescence-based assay in droplets. More specifically, the droplets contain paramagnetic nanoparticles coated with an anti- human Fc nanobody, a fluorescently labeled anti-human Fc detection reagent, and a fluorescently labeled soluble antigen (in this case either the full-length extracellular domain or the S1-RBD domain of the SARS-CoV-2 Spike protein (see FIG.2). The droplets are then sorted using a microfluidic droplet sorter by applying a series of selection criteria including exclusion of droplets containing dead cells and selection of droplets showing positive signals for both IgG secretion and antigen specificity (FIG.2). This strategy enables sorting out antigen-specific, IgG-secreting cells, from the rest of the PBMC population (FIG.2). Cells in the sorted drops are released by emulsion breaking, and plated into individual wells, e.g., of a 384-well plate. The sequence of the cognate VH and VL variable regions were then recovered through amplification using gene-specific primers followed by next generation sequencing (NGS). Using this approach, thousands of sorted hits were recovered, which were then analyzed bioinformatically. Bioinformatic analysis of the resultant antibody repertoire identified high antibody diversity in terms of V-gene usage and CDR3 length. Non-redundant VH/VL paired sequences were filtered for serious residue liabilities that would disqualify them as potential therapeutics, and clustered into families of presumed ancestral recombination and a single representative of each family chosen for synthesis and functional characterization With this approach, screens for multiple samples were performed, from convalescent patients for antigen specificity. Samples analyzed yielded hundreds of VH/VL pairs representing over one hundred non-redundant pairs. The droplet-based assay performed on patient samples was able to detect ~1% of the IgG secreting cells specific to the antigen. It was observed that sequences of the light chains exhibit substantially less amino-acids difference with respect to germline, compared to the sequences of the heavy chains. The complementary approach (schematics shown in FIG.3) used to identify SARS- CoV2-specific antibodies 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 VH and VL chains. The mass spectra peptide data of the VH or VL of affinity-purified antibodies covering the CDR3 region were used to unambiguously map back to full-length VH-VL paired sequences from the repertoire database. Based on titer information, one sample was selected from convalescent patient to interrogate with this approach. From this sample, a total of 6.02 million reads yielded 22,135 VH and 86,886 VL unique sequences, of which 1180 were unique VH/VL pairs. The antibodies purified from serum showed a ~150 kDa band for patient samples but not for the healthy donor control (data not shown). After manual verification of CDR3 peptide spectra, a total of 52 VH and 25 VL sequences with different CDR3 were identified. The CDR3 sequences for VH chains demonstrate significant degree of diversity. The broad diversity of the CDRH3 is not only required for covering different epitopes but also required for identifying antibodies with a specific functional properties, as in this case, neutralizing the virus or blocking the virus’ entry into the cell by blocking the S protein-ACE2 interaction. In summary, by analyzing those sequences for their pairing information, a total of 80 unique antibodies belonging to 31 different CDRH3 groups were retrieved and synthesized. For example, 2 consecutive peptides (LNSVTAADTAVYYCATPHTR (SEQ ID NO: 230) and WGPDYWGQGTLVTVTVSSPSTK (SEQ ID NO: 231)) covering heavy chain F22a1-VH0’s full CDRH3 with almost all fragment y ions matched. The paired LC F22a1-VL0 was also identified with high confidence mass spectra with CDRL3 peptide TEDEADYYCQSYDGDNLVFGGGTK (SEQ ID NO: 232). Identification of both paired VH and VL further improved the confidence for the identity of the antibody. The binding and the neutralization activities of the synthesized antibodies (synthesized based on DNA sequences) were further confirmed. One antibody, C1S5-2A2A (obtained elsewhere), as a reference positive control S1- binding antibody, demonstrated sub nanomolar affinity binding to SARS-CoV-2 spike protein and the blocking activity against spike protein to ACE2, both in ELISA and FACS assays. Meanwhile, another reference antibody C1S2-6A6A is specific for the S2 stalk protein. With the complementary approach, two antibodies showed potent binding and blocking as predicted (FIG. 4). Specifically, for S1-specific binding, the reference antibody C1S5-2A2A has an EC50 of 0.31 nM, while the newly identified antibody H13S1-8A9A has EC50 of 3.67 nM. For S2- specific binding, the reference antibody C1S2-6A6A has an EC50 of 1.71 nM, while the newly identified antibodies H13S1-9A10A, H13S1-14A14A, and H13S4-8A8A has EC50 of 1.57 nM, 5.32 nM, and 20.58 nM, respectively. S2 binder binning assay was also performed using standard technology, and the results were shown in FIG.8. It appears that the H13S1-9A10A antibody binds to a distinct epitope on S2, while H13S4-8A8A and H13S9.1-7A7A bind closely related epitopes that somewhat overlap with that of the reference antibody C1S2-6A6A. Knowledge of such binning assays can be used to design bi-specific antibodies, such as those exemplified in FIG.9, that have bi-specific binding affinity towards both S1 and S2 portions of the S protein. The complementary approach demonstrated that it can identify anti-viral antibodies from recovered patients at both protein and DNA level with matching sequences. Further, binding and blocking capabilities were also demonstrated from synthesized antibodies based on their DNA sequences. From the bioinformatic analysis data, the variable region gene encoding the heavy and light chains were synthesized, then cloned into a vector with a selected constant region to produce IgG1 antibodies. In summary, this example shows successful identification of multiple SARS-CoV-2 S protein binders using two approaches, the direct screening as well as the LC-MS and single cell sequencing coupled approach. The antibodies can effectively bind to SARS-CoV-2 with EC50 10-9 to 10-10 M, where some bind to S1 glycoprotein and at least one antibody binds to S2 protein, showing the diversity of mAb being identified. The identified binders can also block S1 binding to Vero E6 cell line with IC5010-9 -10-10 M (FIG.6). The neutralizing activities of those antibodies against pseudovirus and live virus were also confirmed. Potent neutralizing activities were observed, with IC50 values ranging from 0.53 to 5.8 nM for neutralization of pseudovirus entry and < 0.06 nM for neutralization of live SARS- CoV-2 virus entry (data not shown). Overall, this approach has been demonstrated to efficiently and rapidly identify patient- derived antibodies against a specific antigen (such as the SARS-CoV-2 Spike protein, including S1 and S2 portions), and use the sequences of the identified strong binders to construct synthetic monoclonal antibodies to treat disease caused by the antigen-associated pathogen (such as SARS- CoV-2 mediated infection). While not all neutralizing antibodies can be used as therapeutic antibodies for patient treatment, some of them can be used prophylactically to prevent virus infection. Moreover, certain binding antibodies can be used in combination with the current vaccine approaches, even though they may not have sufficient neutralization activities. The entire process, from patient blood collection, antigen-specific B-cell sorting, sequence recovery and sequencing, followed by bioinformatic analysis of the sequences, DNA synthesis and cloning to antibody expression and functional verification, can be finished in just under 2-3 weeks (e.g., 18 days). This proves to be a valuable tool to combat rapidly spreading infectious diseases such as SARS-CoV-2-mediated infection. Detailed methods used in this example are provided herein below as illustration, and are not meant to be limiting in any respect. Materials and Methods Titer measurements by ELISA: For ELISA, 384-well plates were coated overnight at 4°C degrees 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°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 (ThermoFisher 05-4220) at 0.2 µg/mL in 1X PBS with 0.05% Tween and 1% BSA) for 1 hour at room temperature. The plate was then washed again 4 times and developed in TMB substrate for 5 min before stopping the reaction with the stop solution. Flow cytometry: PBMC was thawed at 37°C, and 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 hours 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, eBioscience 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. Droplet-based sorting of antigen-specific B cells: The droplet assay was set up as described in (Gérard et al., 2020, supra). Briefly, two aqueous phases were prepared and then injected in a microfluidic device to generate 40 pL drops containing B cells and screening reagents. Aqueous phase I: the PBMC or B cell enriched sample was spun down and resuspended in DMEM supplemented with 0.2% Pluronic F68 (ThermoFisher 24040032), 50 mM HEPES pH 7.4 (LifeTechnologies), 10% HyClone Super Low IgG Defined Fetal Bovine serum (GE Healthcare), the NucGreen dead viability marker (ThermoFisher R37109), and 2% Pen/Strep (ThermoFisher), so as to achieve a λ (mean number of cells per droplet) of ~0.3 cell per drop. Aqueous phase II: Paramagnetic nanoparticles (Strep Plus, 300 nm, Ademtech) were coated with biotinylated VHH anti-human IgG-Fc (ThermoFisher 7103322500) as described (Eyer et al., 2017), and resuspended in working buffer containing goat anti human IgG-Fc (DyLight-650-labeled, Abcam ab98622) diluted to 50 nM and Spike-RBD (Sino biologicals# 40592-V05H) or full length spike protein (Sino biologicals#40589-V08B1), labeled with Dyligth550 using the Biorad Lynx labeling kit LNK231D550) diluted to 50 nM. After encapsulation the final concentration are half of the initial aqueous phase I and II concentrations. In addition to the B cell containing droplet condition, a positive control emulsion containing 10 nM ACE2-hFc (kactus cat# ACE-HM501) or 10 nM anti-SARS CoV-2 mAb (Sanyou # AHA001) and a negative control emulsion with 10 nM human IgG isotype (Thermo #31154). The controls were used to establish the assay and set antigen specificity gates for sorting. Droplet were sorted using a droplet surface acoustic wave based sorter (Franke, Braunmüller, Schmid, Wixforth, & Weitz, 2010) with the following gating strategy was the following: live cells were selected (absence of peaks in the Nucgreen channel), then the presence of IgG secreting cells was selected for, by selecting droplets that emit a signal in the detection reagent channel, then droplets emitting a signal in the antigen channel were selected, and finally, droplets were select if the peak emitted in the detection reagent and in the antigen channels are colocalized, confirming the accumulation of those two reagents on the beadline. The sorted droplets were broken by incubation for 10 min in 100 µL of DMEM supplemented with 5% serum Low IgG and followed by 100 µL of 1H,1H,2H,2H-Perfluoro-1- octanol (370533, Sigma) and finally centrifuged at 450 g for 10 min at 4°C to favor complete phase separation. Library preparation for antibody sequencing by single-cell barcoding for VH-VL Single-cell barcoding and library preparation for VH-VL sequencing was performed according to the method described in Gérard et al., 2020 (supra). Briefly, the reverse transcription of VH and VL mRNAs from individual cells in droplets was primed using barcoded primers carrying the T7-SBS12 sequences followed by a cell-specific barcode and the gene- specific primer (GSP) sequences complementary to heavy chain and light chain sequences. The resulting cDNAs from each single cell have a specific barcode, allowing cognate VH and VL pairs to be identified after NGS. After emulsion breaking and cDNA purification the barcoded VH and VL genes were amplified by nested PCR, with reverse primers priming sequentially on the T7 and on the Illumina SBS12 sequence of the barcode and forward primers priming sequentially on the leader and framework 1 sequences of the V gene. In the second PCR, the reverse primer appends the first part of the Illumina P7 and Illumina index sequences by priming on the SBS12 sequence and the forward primer appends the Illumina SBS3 sequence. A third PCR adds the P5 sequence by priming on the SBS3 sequences. Illumina MiSeq 2x300 were used for sequencing the amplified variable genes encoding heavy and light chains obtained. Plate based paired VH-VL sequencing was performed according to the method described in (Gérard et al., 2020, supra). Briefly, following emulsion breaking, cells were distributed into wells of a 384 well-plate using an acoustic liquid dispenser (Echo 550, Labcyte) at an average of one IgG-secreting cell per well. Each well of the plate contained lysis buffer, barcoded cDNA primers (with a unique barcode per row), reverse transcription buffer, dNTPs and reverse- transcriptase enzyme. After reverse transcription, the barcoded cDNAs were pooled by columns, purified, and the VH and VL genes amplified separately using a column barcode. After the first PCR all columns were pooled into one reaction for the VH and VL. Two more rounds of PCR were performed priming to the adaptor sequence. The resulting in amplicons possessing the Illumina adaptors P7-SBS12 followed by the Illumina index sequence on one end of the fragment and P5-SBS3 on the other end of the fragment. Sequencing was performed using Illumina MiSeq 2x300bp paired-end sequencing. Bioinformatics data processing Raw paired-end sequencing data from Illumina MiSeq v3 or Novaseq vX to identify paired or unpaired VH/VL sequences were processed as described in Gérard et al., 2020 (supra), but with the following modifications. Briefly, reads are filtered and trimmed for quality using the program trim_galore bioinformatics.babraham.ac.uk/projects/trim_galore, with a read quality threshold of 20 and a minimum length of 150. The full-length variable region is then recapitulated from each set of paired-end reads by merging the two reads using the program FLASH. Proprietary 3x11mer combinatorial barcode sequences were assigned by extracting each of the indices and comparing them to a white list of known indices allowing up to 2 mismatches per index. Sequences with no identifiable barcode were filtered out. Only barcodes containing a minimum of 4 reads were retained. Reads assigned to the same barcode are considered to come from the same cell. Consensus sequences for all distinct types of sequence assigned to the same barcode were created by first clustering them using a rapid DNA clustering algorithm MeShClust using a percent identity threshold of 93%. Non-redundant sequences within each cluster were then identified and if the top ranked sequence had more reads associated than all other sequences combined, then this sequence became the consensus. Otherwise, all non-redundant sequences were aligned using ClustalO and the most prevalent base at each position in the alignment is assigned to the consensus. Only consensus sequences made up from at least 4 reads were retained. All consensus sequences were characterized to identify the VH/VL amino acid sequence, CDRs and the closest germline V and J genes using VDJFasta (v2) and the latest database of human IgG germline V and J genes downloaded from IMGT (dated 2nd February 2020). For each barcode, the VH and VL sequences with the most reads associated were considered paired and the rest as orphans with a read threshold of 40 reads for reasons previously described in (Gérard et al., 2020). VH–VL pairs were assumed to derive from the same progenitor rearrangement event if they shared the same V and J gene assignments and had CDR3s of the same length. Clusters of ancestrally derived sequences were referred to as “antibody families.” Antibody selection for expression Antibodies were selected by prioritizing antibodies with germline mutations, larger family size, higher reads number and no major liabilities (e.g. cysteines). IgG repertoire database preparation The IgG repertoire was evaluated by directly encapsulating single B cells and performing barcoding RT as described in the previous paragraph. 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 ddH2O. 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°C for 1 hr. The denatured and reduced sample was then alkylated with 5 mM iodoacetamide for 30 mins followed by overnight trypsin (w/v=1:20) digestion at 37°C 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°C with the following gradients: 5%-30% ACN in 157 min; 30%-40% ACN in 15 min; 40%-90% ACN in 2min; 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°C 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 hr 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 hr at room temperature. The plate was then 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 1 hr and then added to Vero E6 cells (approximately 105 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. Example 2 Binding of Spike (S1 or S2) Proteins of SARS-CoV-2 The antibodies of the invention have been demonstrated to be able to bind to the spike protein (S1 or S2 domains) 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. FIG.4 middle panel illustrates a typical ELISA assay format useful for determining binding affinity of the subject antibodies against S1 or S2 domains. The left and right panels, respectively, shows representative antibodies that have high binding affinity towards S1 or S2. FIG.5A shows that two antibodies having the same VH sequence and closely related VL sequences - Ab-6 (H13S1-8A9A) and Ab-7 (H13S1-8A4A) - shared high sequence similarity to two other antibodies in the light chain CDR regions – H13S1-8A097A and H13S1-8A839A, and they all bind the S1 domain with nanomolar to subnanomalor affinity (FIG.5B). Interestingly, Ab-7 differs from Ab-6 only by 1 residue each in light chain CDR2 and CDR3 (see FIG.5A), yet its binding affinity increased by 4 fold (EC50 dropped from 3.7 nM of Ab-6 to 0.9 nM of Ab-7). Next, the blocking effects of the top antibodies on the binding of S1 (RBD) to Vero E6 cell line was analyzed by FACS analysis (FIG.6A). As expected, like the control Ab C1S5- 2A2A, Ab-7 (H13S1-8A4A), Ab-6 (H13S1-8A9A), and the related H13S1-8A839A all blocked S1 (RBD) binding to E6 at the nM range. Inhibition of soluble spike ECD binding to ACE2-expressing Vero-E6 cells were also tested. As S2-binder H13S4-8A8A concentration increased, partial inhibition of spike binding to ACE2 was observed. (FIG.6B). Binning assays were done by ELISA. Briefly, 384-well plates were coated with 2 µg/mL of tested antibodies in 30 µL 1xPBS at 4℃ overnight. After blocking with 2% BSA at room temperature for 1 hour, 40 µL of the competitor antibody-S2 protein mixture was added into a well and incubated at room temperature for 1 hour. The mixture was composed of 20 µL of 90 µg/mL competitor anti-S2 antibody or isotype control and 20µL of S2 spike protein with HIS tag at a determined EC80 concentration binding to the coated antibody. After washing, the remaining S2 spike protein binding to the coated antibody was detected by anti-His-HRP at 1/15000 dilution. Based on the result, Ab-3 (H13S4-8A8A) and Ab-1 (H13S9.1-7A7A) strongly competed with each other. C1S2-6A6A partially blocked the S2 protein binding to Ab-3 (H13S4-8A8A) and Ab-1 (H13S9.1-7A7A). Ab-3 (H13S4-8A8A), but not Ab-1 (H13S9.1-7A7A), strongly competed the S2 protein binding to C1S2-6A6A. Ab-5 (H13S1-9A10A) showed weak competition with the other tested anti-S2 antibodies. Summary of relative affinities to S2 protein competed against C1S2-6A6A are shown in FIG.7. Example 3 Pharmacological profile of the neutralizing antibodies The subject antibodies’ binding to S1/S2 domains is further profiled pharmacologically. A snapshot PK study at 1 h, 24 h and 72 h separately in wild-type (WT) mice shows good PK profile. Example 4 IgG4 Monoclonal Antibodies Have Comparable Activity This example demonstrates that certain subject antibodies with hIgG4 constant region (which does not engage or minimally engages FcγR), compared to their counterpart with the hIgG1 constant region, have comparable binding affinity for the SARS-CoV-2 S1 or S2 antigen, pseudovirus neutralization activity, and live virus neutralization activity. Such hIgG4 antibodies also have comparable favorable developability characteristics. For binding affinity assay, SPR (surface plasmon resonance) with anti-hIgG Fc immobilized on CM5 chip was used to capture several subject monoclonal antibodies with hIgG1 vs. hIgG4 constant region, respectively, in order to compare their respective binding affinity for the soluble S1 (for Ab-7) and S2 (for Ab-5) domain. “D614” refers to binding to the full length ectodomain of the S protein. “D614G” refers to binding to the full length ectodomain of the S protein carrying the D614G mutation. “RBD” refers to binding to the RBD domain. The pseudovirus neutralization functional assay was performed, in brief, by expressing SARS-CoV-2 S protein (either from the reference strain or from the D614G mutant strain) with GFP as a marker on VSVΔG (Delta-G VSV) to create a pseudovirus. This S protein-expressing VSVΔG pseudovirus could enter the Vero target cell, and the GFP fluorescent intensity inside the Vero cell is an indication for the extent of viral entry. Neutralization of pseudovirus entry by the subject antibodies can be determined based on the extent of GFP signal reduction inside the Vero cells. The concentrations of the antibodies required to achieve 50%, 80%, or 90% pseudovirus neutralization were determined based on GFP fluorescence reduction. Briefly, Delta-G-VSV (vesicular stomatitis virus) expressing SARS-CoV-2 S and GFP were inhibited with the respective test antibodies and then used to infect Vero cells. Expression of GFP from the Vero cells were measured to calculate the concentration of antibody needed to have 50%, 80%, or 90% inhibition or neutralization of the Delta-G-VSV expressing SARS-CoV S. IC501, IC801, and IC901 refer to concentration of antibody to result in 50%, 80%, and 90% inhibition, respectively. The results of the affinity and pseudovirus neutralization assays are summarized in the table below.
Figure imgf000074_0001
Figure imgf000075_0001
Alternatively or in addition, authentic SARS-CoV-2 virus infection and blockage thereof can be assessed in a BSL-3 or BSL-4 laboratory with adequate safety measure, and the results can be compared with that of pseudovirus neutralization assay. Dual targeting bi-specific antibodies (e.g., the Ab-7/Ab-5 bi-specific antibody 8A4A/9A10A-scFv) can enhance blocking efficacy, limit escape mechanisms, and increase target selectivity via a strong avidity effect mediated by simultaneous binding to the virus spike protein at different epitopes. Bi-specific antibodies with an S1 binder/blocker (such as Ab-7 or 8A4A) together and an S2 binder (such as Ab-5 or 9A10A) can show potential synergetic effect in neutralization. Example 5 Antibody Dependent Phagocytosis, Complement Deposition, and Natural Killer Activation Antibodies were incubated with various cells to determine activity for dependent phagocytosis, complement deposition, and natural killer activation. The results are summarized in the table below.
Figure imgf000075_0002
Figure imgf000076_0001
The antibodies were assayed to determine binding affinity to FcγR and FcR molecules. The results are summarized in the table below.
Figure imgf000076_0002
Figure imgf000077_0001
Example 6 Developability Assay A series of developability assays are performed for the IgG4 version of lead antibodies, such as Ab-6 and Ab-7, including accelerated stability (2-3 mg/mL of Ab at 25 and 40°C in D- PBS, pH7.4, for up to 14 days); forced degradation (2-3 mg/mL of Ab at 25°C in 100 mM acetic acid at pH3.5, for up to 6 hours); and up to 5 freeze-thaw cycles (2-3 mg/mL of Ab). The results show that all samples are stable in the accelerated stability study; all antibodies show aggregation formation under the low pH stress condition; and all samples remain stable after 5 cycles of freeze-thaw. Example 7 Efficacy of antibodies against SARS-CoV-2 variants Live virus variant study Vero E6 cells are infected by the WuhanD614, BavPat D614G and other variants such as the UK B.1.1.7 (also named 20I/501Y.V1) SARS-CoV-2 variants and incubated with the subject antibodies with a series of two-fold dilutions in the 0.97-1000 ng/ml range in triplicate. Viral RNA in the supernatant is determined, and % of inhibition is calculated based on infected but untreated control. The results are shown in the table in Example 4. Additional tests are performed to compare the effectiveness of the subject antibody (i.e., Ab-5, Ab-6, and Ab-7, in IgG1 or IgG4 format) against the ACE2-Fc fusion, in various variant strains. Pseudovirus variant study Next, a pseudotyped lentiviral vector with SARS-CoV-2 S proteins as part of the envelope is constructed to mimic SARS-CoV-2 virus, which can infect target cells expressing hACE2. The neutralizing potency is then deduced by detecting the expression levels of luciferase reporter gene packaged into the lentiviral vector. Various variants of SARS-CoV-2 are tested in this experiment: SARS-CoV-2/Wild-type (WT), the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and the Lambda (C.37) variant. Those variants represent commonly isolated SARS-CoV-2 clinical isolates. Live virus study using Ab-6 and Ab-7 IgG1 and IgG4 The various SARS-CoV-2 strains described above at 100 TCID50 per 50 µL are each mixed with equal volume of culture medium containing serially Ab-6 and Ab-7 IgG1 and IgG4 formats diluted antibodies and incubated at 37°C for 1 hour, and then added to Vero E6 cells seeded in 96-well plates. After 48 hours of culture at 37°C, cells are fixed and processed for SARS-CoV-2 nucleocapsid protein (NP) and nuclei staining. % Inhibition is calculated by (total nuclei-infected cells)/ total nuclei × 100%. Fifty percent neutralization dose (ND50) and ninety percent neutralization dose (ND90) are calculated using 4-parameter non-linear regression with GraphPad Prism 8.0. The experiment is carried out in triplicate. Example 8 SARS-CoV-2 S protein variant binding by Ab-2 IgG4 The binding of the Ab-6 or Ab-7 IgG4 antibody to SARS-CoV-2 S protein variants (S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, A222V/D614G, the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and the Lambda (C.37) variant) is determined by enzyme linked immunosorbent assay (ELISA). The 384-well plates are coated with 20 nM of the different SARS-CoV-2 S protein RBD variants. The binding of Ab-2 IgG4 (12 concentrations obtained by 3-fold serial dilutions starting from 300 nM, in triplicate) to the SARS-CoV-2 S protein RBD (receptor binding domain) variants is detected by goat F(ab’)2 anti-human IgG (H+L)-HRP. The blocking activity of Ab-6 or Ab-7 IgG4 is determined by enzyme linked immunosorbent assay (ELISA). The 384-well plates are coated with 20 nM of the hACE2-mFc protein. SARS-CoV-2 S protein RBD/S1 variants (with His tag) at fixed concentration (binding EC90 of corresponding variant to hACE2-mFc) was pre-incubated with Ab-6 or Ab-7 IgG4 or isotype control at different concentrations (12 concentrations obtained by 3-fold serial dilutions starting from final concentration of 300 nM, in duplicates), before incubated with coated hACE2- mFc protein. The binding of SARS-CoV-2 S protein RBD/S1 variants (with His tag) to the hACE2-mFc protein is detected by HRP anti-6X His tag antibody. Additionally, binding affinities of Ab-6 or Ab-7 IgG4 to WT RBD or the above mutant RBD variants (Y453F, S477N, S494P, F490S, N439K, N501Y), an S1 variant (A222V/D614G), and other variants are evaluated using SPR (Biacore T200). Example 9 Absence of antibody-dependent enhancement (ADE) by Ab-6/7 IgG4 One potential hurdle for antibody-based therapeutics is the risk of exacerbating COVID- 19 severity via antibody-dependent enhancement (ADE). ADE has been documented to occur through two distinct mechanisms in viral infections: by enhanced antibody-mediated virus uptake into Fc gamma receptor IIa (FcγRIIa)-expressing phagocytic cells leading to increased viral infection and replication, or by excessive antibody Fc-mediated effector functions or immune complex formation causing enhanced inflammation and immunopathology. Both ADE pathways can occur when non-neutralizing antibodies or antibodies at sub-neutralizing levels bind to viral antigens without blocking or clearing infection. Raji cells, originally derived from a Burkitt’s lymphoma patient, have been shown to facilitate SARS-CoV-1 infection in the presence of anti-S-protein immune serum. Thus, this FcγRII-bearing human B lymphoblast cell line is used to study the antibody-dependent viral entry of SARS-CoV-2 as an indicator of ADE. Briefly, Raji cells are seeded in 96-well plates. Antibodies at different concentrations are pre-incubated with the SARS-CoV-2 pseudo virus encoding wild-type spike protein and luciferase. The mixture of antibody and pseudo virus are then added to plated Raji cells. The plates are incubated and pseudovirus infection of Raji cells was quantified by measuring luciferase activity. Example 10 Pharmacokinetics of Ab-6/7 IgG4 in rhesus monkeys Systemic circulation of antibody A total of 9 Rhesus monkeys are included for efficacy study of Ab-6/7 IgG4. Monkeys are allocated into 3 groups (three per group) receiving a single intravenous infusion of isotype control at 50 mg/kg (group 1), Ab-6/7 IgG4 at 10 mg/kg (group 2) and Ab-6/7 IgG4 at 50 mg/kg (group 3) one day after intratracheal inoculation of SARS-CoV-2 at 1 × 105 TCID50. Serum samples are collected once daily from 0-7 days post infection (d.p.i.). The concentration of Ab-2 IgG4 in plasma samples is determined using validated ELISA methods. Briefly, plates are coated at 4°C overnight with anti-human (h) IgG (quantification of total hIgG) or SARS-CoV-2 S protein S1 subunit recombinant protein (quantification of unbound Ab-6/7 IgG4), before incubated with rhesus monkey plasma collected during Ab-6/7 IgG4 Study. The concentration of plasma antibodies is detected by HRP conjugated anti-hIgG antibody. Pharmacokinetics and immunogenicity study A study is conducted to evaluate serum pharmacokinetics (PK) and immunogenicity following a single IV infusion administration of Ab-2 IgG4 in naïve male and female cynomolgus monkeys. On Day 1 of the study, 3 male and 3 female cynomolgus monkeys are administered a single 10 mg/kg dose of Ab-2 IgG4 by IV infusion (60 minutes; 4 mL/kg). Blood is collected and processed for PK, anti-drug antibody (ADA), hematology, and clinical chemistry evaluations (data not shown). The concentration of Ab-6/7 IgG4 and ADA, is monitored in serum for up to 56 days (1345 hours) after beginning of infusion and are provided below. Safety is also monitored based on clinical observations and hematology and clinical chemistry evaluations. Example 11 In vivo efficacy of Ab-6/7 IgG4 in rhesus monkeys The efficacy of Ab-6/7 IgG4 to treat SARS-CoV-2 infection is evaluated in Rhesus monkeys. On Day 0, 3 groups of 3 Rhesus monkeys (2 females and 1 male per group) are infected by intratracheal (IT) inoculation with SARS-CoV-2 at 1 × 105 TCID50/animal. On Day 1, following confirmation of infection via oropharyngeal swab, a single treatment with isotype control antibody (50 mg/kg) or Ab-6/7 IgG4 at 10 or 50 mg/kg was administered by IV infusion. Health status and infection are monitored via body temperature, body weight, hematology, and blood chemistry analyses from samples collected prior to viral challenge (pi0d) and on Days 1 (blood swabs and feces collected before Ab-2 IgG4 administration) 2 3 4 5 6 and 7 SARS- CoV-2 viral load is evaluated in blood; oropharyngeal, nasal and rectal swabs; and feces using RT-qPCR method. The lungs are X- rayed on Days 0 (prior to infection), 3, and 6. One monkey from each group is euthanized 5, 6 and 7 days post-infection and selected organs (lungs [6 lobes, trachea, left and right bronchia], spleen, pulmonary hilar lymph node, liver, and kidney) are processed, stained with hematoxylin and eosin [H&E] and Masson's trichrome staining, and associated pathological changes were evaluated microscopically. Serum samples are collected prior to viral challenge (pi0d) and on Days 1 (before Ab-2 IgG4 administration), 2, 3, 4, 5, 6, and 7, for evaluation of Ab-6/7 IgG4 levels. The findings for animal health (body weight, body temperature), viral load, X-rays, and microscopic evaluations of lung tissues are reported, as well as pharmacokinetics results. Materials and Methods 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°C 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 105 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 pseudovirus 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 8x104 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°C, 5% CO2 for 48h, half-maximal inhibitory concentration (IC50) 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 10050% tissue-culture infectious doses (TCID50) or infected with a mixture of viruses and diluted antibodies (incubated together for 1 hr) are cultured at 37°C for 48 hours, 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 are 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 or Alexa Fluor® 488-conjugated goat anti-mouse IgG antibody as the secondary antibody. Cell nuclei were stained using Hoechst 33258 at room temperature for 10 minutes. Images were taken under an inverted fluorescence microscope (Nikon). In some experiments, numbers of nuclei and cells infected with viruses are counted using an Operetta CLSTM system. % inhibition is calculated by (total nuclei-infected cells)/ (total nuclei) x 100%. Fifty percent neutralization dose (ND50) and ninety percent neutralization dose (ND90) are calculated using 4-parameter non-linear regression with GraphPad Prism 8.0.

Claims

WHAT IS CLAIMED IS: 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 (e.g., the Spike or S protein responsible for ACE2 binding) of SARS-CoV-2, and wherein said monoclonal antibody comprises: (1) (1a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 1, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 2, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 3; and, (1b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 4, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 5, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 6; and/or (2) (2a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 11, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 12, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 13; and, (2b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 14, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 15, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 16; and/or (3) (3a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 21, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 22, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 23; and, (3b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 24, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 25, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 26; and/or (4) (4a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 31, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 12, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 33; and (4b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 34 or SEQ ID NO: 115, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 35, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 36; and/or (5) (5a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 51, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 12, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 53; and, (5b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 54, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 55, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 56; and/or (6) (6a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (6b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 65, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 66; and/or (7) (7a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (7b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 75, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 76; and/or (8) (8a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (8b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64 a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 75, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 66; and/or (9) (9a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 63; and, (9b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 65, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 96; and/or (10) (10a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (10b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 14, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 15, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 106; and/or (11) (11a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (11b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 114, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 115, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 106; and/or (12) (12a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (12b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 124, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 15, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 106; and/or (13) (13a) a heavy chain variable region (HCVR), comprising a HCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 101, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 102, and a HCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 103; and, (13b) a light chain variable region (LCVR), comprising a LCVR CDR1 sequence comprising the amino acid of SEQ ID NO: 134, a LCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 115, and a LCVR CDR3 sequence comprising the amino acid of SEQ ID NO: 136. optionally, said isolated monoclonal antibody is not naturally occurring; and/or, optionally further comprising a signal peptide sequence at the N-terminus of said HCVR and/or at the N-terminus of said LCVR.
2. The isolated monoclonal antibody or antigen-binding fragment thereof of claim 1, wherein: (1) (1A) the HCVR sequence comprising the amino acid of SEQ ID NO: 7; and/or, (1B) the LCVR sequence comprising the amino acid of SEQ ID NO: 8, or, (2) (2A) the HCVR sequence comprising the amino acid of SEQ ID NO: 17; and/or, (2B) the LCVR sequence comprising the amino acid of SEQ ID NO: 18, or, (3) (3A) the HCVR sequence comprising the amino acid of SEQ ID NO: 27; and/or, (3B) the LCVR sequence comprising the amino acid of SEQ ID NO: 28, or, (4) (4A) the HCVR sequence comprising the amino acid of SEQ ID NO: 37; and/or, (4B) the LCVR sequence comprising the amino acid of SEQ ID NO: 38, or, (5) (5A) the HCVR sequence comprising the amino acid of SEQ ID NO: 57; and/or, (5B) the LCVR sequence comprising the amino acid of SEQ ID NO: 58, or, (6) (6A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (6B) the LCVR sequence comprising the amino acid of SEQ ID NO: 68, or, (7) (7A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (7B) the LCVR sequence comprising the amino acid of SEQ ID NO: 78, or, (8) (8A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (8B) the LCVR sequence comprising the amino acid of SEQ ID NO: 88, or, (9) (9A) the HCVR sequence comprising the amino acid of SEQ ID NO: 67; and/or, (9B) the LCVR sequence comprising the amino acid of SEQ ID NO: 98, or, (10) (10A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (10B) the LCVR sequence comprising the amino acid of SEQ ID NO: 109, or, (11) (11A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (11B) the LCVR sequence comprising the amino acid of SEQ ID NO: 119, or, (12) (12A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (12B) the LCVR sequence comprising the amino acid of SEQ ID NO: 129, or, (13) (13A) the HCVR sequence comprising the amino acid of SEQ ID NO: 107; and/or, (13B) the LCVR sequence comprising the amino acid of SEQ ID NO: 139.
3. The isolated monoclonal antibody or antigen-binding fragment thereof according to claim 1 or 2, wherein said monoclonal antibody has: (1) (1a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 7; and/or, (1b) a light chain sequence comprising the amino acid of SEQ ID NO: 8, or, (2) (2a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 17; and/or, (2b) a light chain sequence comprising the amino acid of SEQ ID NO: 18, or, (3) (3a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 27; and/or, (3b) a light chain sequence comprising the amino acid of SEQ ID NO: 28, or, (4) (4a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 37; and/or, (4b) a light chain sequence comprising the amino acid of SEQ ID NO: 38, or, (5) (5a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 57; and/or, (5b) a light chain sequence comprising the amino acid of SEQ ID NO: 58, or, (6) (6a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (6b) a light chain sequence comprising the amino acid of SEQ ID NO: 68, or, (7) (7a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (7b) a light chain sequence comprising the amino acid of SEQ ID NO: 78, or, (8) (8a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (8b) a light chain sequence comprising the amino acid of SEQ ID NO: 88, or, (9) (9a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 67; and/or, (9b) a light chain sequence comprising the amino acid of SEQ ID NO: 98, or, (10) (10a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (10b) a light chain sequence comprising the amino acid of SEQ ID NO: 109, or, (11) (11a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (11b) a light chain sequence comprising the amino acid of SEQ ID NO: 119, or, (12) (12a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (12b) a light chain sequence comprising the amino acid of SEQ ID NO: 129, or, (13) (13a) a heavy chain sequence comprising the amino acid of SEQ ID NO: 107; and/or, (13b) a light chain sequence comprising the amino acid of SEQ ID NO: 139, or,
4. The isolated monoclonal antibody or antigen-binding fragment thereof according to any one of claims 1-3, wherein: (a) the isolated monoclonal antibody is a human antibody, a CDR-grafted antibody, or a resurfaced antibody; (b) the isolated monoclonal antibody is a bi-specific antibody, optionally the bi- specific antibody comprises the HCVR CDR1, CDR2, and CDR3 and LCVR CDR1, CDR2, and CDR3 of any one of a first antibody of claim 1(1) – 1(13), and the HCVR CDR1, CDR2, and CDR3 and LCVR CDR1, CDR2, and CDR3 of any one of a second antibody of claim 1(1) – 1(13), wherein the first and the second antibodies are different or bind to different (preferably non-overlapping) epitopes; optionally, (i) said first antibody is specific for S1 (such as Ab-1, Ab-6, or Ab-7), and said second antibody is specific for S2 (such as Ab-3, Ab-5), and/or, (ii) said bi-specific antibody comprises an antigen-binding fragment (such as an scFv, Fab, or Fab’ fragment) of one of said first antibody and said second antibody, fused to the light chain (or heavy chain) of the other of said first antibody and said second antibody), and/or, (c) the antigen-binding fragment thereof is an Fab, Fab’, F(ab’)2, Fd, single chain Fv or scFv, disulfide linked Fv, V-NAR domain, IgNar, intrabody, IgGΔCH2, minibody, F(ab’)3, tetrabody, triabody, diabody, single-domain antibody, DVD-Ig, Fcab, mAb2, (scFv)2, or scFv-Fc. 5. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-4, wherein said monoclonal antibody or antigen-binding fragment thereof: (i) binds to the S1 or S2 glycoprotein of SARS-CoV-2; (ii) binds the SARS-CoV-2 antigen with a Kd of less than about 5 nM, 2 nM, 1 nM, 0.
5 nM, 0.2 nM, 0.1 nM, or 0.05 nM; (iii) binds to SARS-CoV-2 wild-type S protein and/or RBD/S1 variants selected from the group consisting of S477N, S494P, F490S, Y453F, N439K, N501Y, E484K, Q493R, A222V/D614G, the Alpha (B.1.1.7) variant, the Beta (B.1.351, B.1.351.2, B.1.351.3) variant; the Gamma (P.1, P.1.1, P.1.2) variant, the Delta (B.1.617.2, AY.1, AY.2) variant, the Eta (B.1.525) variant, the Iota (B.1.526) variant, the Kappa (B.1.617.1) variant, and the Lambda (C.37) variant, and/or, (iv) inhibits binding of the SARS-CoV-2 antigen (e.g., the S1 glycoprotein) to ACE2, optionally 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), and/or optionally 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). 6. The isolated monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-5, which: (i) 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; (ii) exhibits neutralizing activity against a pseudovirus of SARS-CoV-2 or a live SARS-CoV-2 virus with an IC50 value of less than 50 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 6 nM, less than 3 nM, less than 1 nM, less than 0.
6 nM or less than 0.5 nM; (iii) inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) at less than 50 nM, less than 30 nM, less than 20 nM, less than 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, less than 1 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; (iv) inhibits SARS-CoV-2 viral entry of a target cell (such as Vero E6 cell) with an IC50 of less than 50 nM, less than 30 nM, less than 20 nM, 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; (v) inhibits entry of wild-type SARS-CoV-2, and/or SARS-CoV-2 variants (e.g., WuhanD614, BavPat D614G, UK B.1.1.7, or South Africa B.1.351 strain, or a SARS-CoV-2 variant sharing one or more S1 protein mutations with the WuhanD614, BavPat D614G, UK B.1.1.7, and/or South Africa B.1.351 strain(s)) into a target cell; and/or, (vi) does not cause antibody-dependent enhancement (ADE).
7. The monoclonal antibody or antigen-binding fragment thereof of any one of claims 1-6, comprising a heavy chain constant region, wherein the heavy chain constant region is human IgG4, human IgG3 or human IgG2; optionally, the heavy chain constant region is human IgG4 which optionally comprises a YTE (M252Y/S254T/T256E) mutation and/or an LS (M428L/N434S) mutation.
8. 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 (e.g., the S protein responsible for ACE2 binding) of SARS- CoV-2, and wherein said monoclonal antibody comprises a heavy chain variable region (HCVR) comprising a HCVR CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 61, a HCVR CDR2 sequence comprising the amino acid of SEQ ID NO: 62, and a HCVR CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 63, and a light chain variable region (LCVR) comprising a LCVR CDR1 sequence comprising the amino acid sequence of SEQ ID NO: 64, a LCVR CDR2 sequence comprising the amino acid sequence of SEQ ID NO: 65, and a LCVR CDR3 sequence comprising the amino acid sequence of SEQ ID NO: 66, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises: (iii) an HCVR sequence comprising the amino acid sequence of SEQ ID NO: 67 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 67; and (iv) an LCVR sequence comprising the amino acid sequence of SEQ ID NO: 68 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 68; and/or, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG4, human IgG3, or human IgG2, preferably human IgG4.
9. 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 (e.g., the S protein responsible for ACE2 binding) of SARS- CoV-2, and wherein said monoclonal antibody comprises 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 a light chain variable region (LCVR) comprising a LCVR CDR1 sequence of SEQ ID NO: 64, a LCVR CDR2 sequence of SEQ ID NO: 75, and a LCVR CDR3 sequence of SEQ ID NO: 76, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises: (iii) an HCVR sequence of SEQ ID NO: 67 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 67; and (iv) an LCVR sequence of SEQ ID NO: 78 or having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity to SEQ ID NO: 78; and/or, optionally, the monoclonal antibody or antigen-binding fragment thereof comprises a heavy chain constant region of human IgG4, human IgG3, or human IgG2, preferably human IgG4.
10. The monoclonal antibody or antigen-binding fragment thereof of claim 8 or claim 9, comprising an IgG4 heavy chain (HC) sequence comprising SEQ ID NO: 67 or a heavy chain sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
11. 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 1-10 for binding to the same or substantially the same epitope (e.g., based on binning assay).
12. A mixture of two or more isolated monoclonal antibodies or antigen-binding fragments thereof of any one of claims 1-10, optionally, the proportion of each of said two or more isolated monoclonal antibodies or antigen-binding fragments thereof is substantially the same, or is different.
13. A polynucleotide encoding the heavy chain and/or the light chain, or the antigen-binding portion thereof, of any one of claims 1-11, optionally, the polynucleotide is codon optimized for expression in a human cell; and/or, optionally, the polynucleotide is in a vector, such as an expression vector (e.g., a mammalian expression vector, a yeast expression vector, an insect expression vector, or a bacterial expression vector), wherein the vector is optionally in a host cell that expresses said isolated monoclonal antibody or antigen-binding fragment thereof.
14. A pharmaceutical composition comprising the isolated monoclonal antibody or antigen- binding fragment thereof of any one of claims 1-11, or the mixture of claim 12, optionally the pharmaceutical composition is formulated for intravenous administration, or for inhalational or oral administration; and/or, optionally, the pharmaceutical composition is for treating a subject infected by SARS- CoV-2, and further comprises a pharmaceutically acceptable excipient or diluent.
15. A combination comprising the pharmaceutical composition of claim 14, and a second therapeutic agent effective to treat infection by SARS-CoV-2, optionally, 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.
16. 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 1-11, the mixture of claim 12; the polynucleotide of claim 13, or the pharmaceutical composition of claim 14, optionally, the method is for treating COVID-19 or a subject infected by SARS-CoV-2, wherein the method further comprises administering a second therapeutic agent; and/or, optionally, said 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.
17. A method of identifying an antibody specific for an antigen from a virus or a bacterium, from a B-cell population obtained from a subject having been infected by and recovering from infection by the virus or the bacterium, the method comprises: (i) obtaining a library of paired VH and VL antibody sequences from a B cell population obtained from the subject; and, (ii) obtaining amino acid sequences of fragments of antibodies specific for said antigen, wherein said antibodies are obtained (e.g., affinity purified) from a sample comprising said B cell population; thereby identifying the antibody specific for said antigen when said amino acid sequences obtained in (ii) match the paired VH and VL antibody sequences obtained in (i).
18. The method of claim 17, wherein the virus is SARS-CoV-2, and the antigen is SARS- CoV-2 S (Spike) protein or N (Nucleocapsid) protein.
19. The method of claim 17 or 18, wherein the sample is a blood sample or plasma sample.
20. The method of any one of claims 17-19, wherein the B cell population is PBMCs isolated from a peripheral blood sample.
21. The method of any one of claims 17-20, wherein the B cell population comprises plasmablasts and memory B cells.
22. The method of any one of claims 17-21, wherein step (i) comprises: (1) generating a plurality of nanoliter scale droplets, each comprising: (a) one B cell from said B-cell population; (b) multiple (e.g., 1,000-1,500) co-encapsulated paramagnetic beads (e.g., colloidal nanoparticles) coated by a first non-specific antibody-binding molecule; and, (c) the antigen labeled by a first detectable label; (2) allowing, under a pre-determined condition, antibodies secreted by said one B-cell to bind said first non-specific antibody-binding molecule on said paramagnetic beads, and to bind said antigen when / if said antibodies are specific for said antigen; (3) passing the droplets through a magnetic field to aggregate said multiple paramagnetic beads, in order to concentrate the accumulative signal emitted by the first detectable label on the antigen bound by said antibodies over a background signal emitted by the first detectable label on the antigen unbound by said antibodies; and, (4) collecting droplets having significantly enhanced accumulative signal emitted by the first detectable label over the background signal; thereby identifying the antibody secreted by said one B-cell as being specific for said antigen.
23. The method of claim 22, wherein said plurality of nanoliter scale droplets are generated by a microfluidic device.
24. The method of claim 22 or 23, wherein the average size of said plurality of nanoliter scale droplets is about 30-1500 pL, about 40 pL, about 80 pL, about 125 pL, or about 1 nL.
25. The method of any one of claims 17-24, wherein the B-cell population is from a human or a non-human mammal (e.g., mouse, rat, rabbit).
26. The method of any one of claims 17-25, wherein the multiple co-encapsulated paramagnetic beads are in an amount sufficient to bind substantially all antibodies secreted by said one B cell.
27. The method of any one of claims 17-26, wherein said first non-specific antibody-binding molecule is biotin-labeled anti-human IgG-Fc (which biotin binds to streptavidin-coated paramagnetic beads), Protein G (which binds to immunoglobulin Fab and Fc regions, optionally, the Protein G lacks albumin-binding region), Protein A (which binds heavy chain Fc region and within the Fab region of human VH3 family), Protein A/G (which binds all subclasses of human IgG as well as IgA, IgE, IgM and to a lesser extent IgD, and all subclasses of mouse IgG but not mouse IgA, IgM or serum albumin), Protein L (which binds kappa light chain of all antibody classes including IgG, IgM, IgA, IgE, IgD as well as scFv and Fab fragments), or a species-specific antibody or antigen-binding fragment thereof (such as an anti-mouse κ light chain (Igκ) nanobody VHH).
28. The method of any one of claims 17-27, wherein the first detectable label is a fluorescent label (such as Alexa Fluor 488 and DayLight550).
29. The method of any one of claims 17-28, wherein the nanoliter scale droplets further comprise: (d) a second non-specific antibody-binding molecule labeled by a second detectable label, wherein said second non-specific antibody-binding molecule does not compete or interfere with binding by said first non- specific antibody-binding molecule; wherein in step (3), the accumulative signal emitted by the second detectable label (e.g., DayLight650) on said second non-specific antibody-binding molecule bound by said antibodies is reflective of the relative amount of said antibodies on aggregated paramagnetic beads.
30. The method of claim 29, wherein said second non-specific antibody-binding molecule is Protein G, Protein A, Protein A/G, Protein L, or a species-specific antibody or antigen- binding fragment thereof (e.g., Goat-anti-human IgG Fc).
31. The method of any one of claims 17-30, wherein the pre-determined condition is 15-60 min. at 37°C (under 5% CO2).
32. The method of any one of claims 17-31, wherein said multiple paramagnetic beads aggregate to form a geometric shape (such as a straight line) under the magnetic field.
33. The method of any one of claims 17-32, wherein said accumulative signal emitted by the first detectable label is a fluorescent signal emitted after laser excitation.
34. The method of any one of claims 17-33, wherein step (4) is carried out by an acoustic sorter device that generates a surface acoustic wave (SAW), or by fluorescence-activated dielectrophoretic sorting.
35. The method of any one of claims 17-34, further comprising (5) determining the sequences of the paired heavy and light chains (VH and VL) of each identified antibody.
36. The method of claim 35, wherein step (5) comprises compartmentalizing each B cell collected from the droplets in step (4) with a bead comprising a bead-specific nucleotide- based barcode and a reverse-transcription primer for initiating cDNA synthesis from mRNA encoding antibody heavy chain or antibody light chain.
37. The method of claim 36, wherein the primer is complementary to a heavy chain constant region coding sequence or a light chain constant region coding sequence, and the cDNA synthesized from the primer comprises heavy chain variable region or light chain variable region, or a CDR (such as CDR3) thereof.
38. The method of claim 36, wherein step (5) further comprises sequencing cDNA synthesized from the primer.
39. The method of claim 38, wherein cDNA sequencing is performed by next generation sequencing (NGS) (such as using an Illumina MiSeq sequencer with a 2 × 300 base pair sequencing flow chip).
40. The method of claim 38, further comprising cloning and expressing cDNA sequences in a host cell to produce said antibody specific for said antigen.
41. The method of claim 40, wherein the host cell is a CHO cell.
42. The method of claim 40 or 41, further comprising isolating and/or purifying said antibody.
43. The method of any one of claims 17-42, wherein in step (ii), IgM in said sample is first depleted before the remaining antibodies in said sample are affinity purified with said antigen, and the affinity-purified antibodies are digested with protease for sequencing analysis of the resulting fragments using mass spectrometry.
44. An antibody identified as specific for an antigen of SARS-CoV-2 by the method of any one of claims 18-43.
45. 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 18-43.
46. 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 18-43, and a pharmaceutically acceptable excipient or diluent.
47. 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 18-43, and a pharmaceutically acceptable excipient or diluent
48. A combination comprising the pharmaceutical composition of claim 46 or 47, and a second therapeutic agent effective to treat infection by SARS-CoV-2.
49. The combination of claim 48, 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.
50. A method of treating a subject infected by SARS-CoV-2, the method comprises administering a therapeutically effective amount of the antibody of claim 44 or 45, the pharmaceutical composition of claim 46 or 47, or the combination of claim 48 or 49.
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