Anti-SARS-cov-2-spike glycoprotein antibodies and antigen-binding fragments

Abstract

To provide antibodies and antigen-binding fragments thereof that bind specifically to a coronavirus spike protein and methods of using such antibodies and fragments for treating or preventing viral infections (e.g., coronavirus infections), and also provide neutralizing human antigen-binding proteins that specifically bind to SARS-CoV-2-S, for example, antibodies or antigen-binding fragments thereof.SOLUTION: An isolated host cell is provided, the host cell comprising a first polynucleotide encoding a heavy chain variable region (HCVR) of an antibody or an antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising a specific amino acid sequence, and a second polynucleotide encoding a light chain variable region (LCVR) of the antibody or the antigen-binding fragment.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Sequence Listing An official copy of the Sequence Listing will be submitted electronically via EFS-Web concurrently with the specification as an ASCII Sequence Listing having the filename "10753WO01-Sequence.txt," a creation date of June 25, 2020, and a size of approximately 922,462 bytes. The Sequence Listing contained in this ASCII document is a part of the specification and is incorporated herein by reference in its entirety.

[0002] The present invention relates to antibodies and antigen-binding fragments that specifically bind to coronavirus spike proteins, and methods for treating or preventing coronavirus infection with such antibodies and fragments. [Background technology]

[0003] Newly identified viruses, such as coronaviruses, can be difficult to treat because they are not well characterized. The emergence of these newly identified viruses highlights the need for the development of new antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that causes the severe acute respiratory disease COVID-19. SARS-CoV-2 was first identified from an outbreak in Wuhan, China. As of March 20, 2020, the World Health Organization reported 209,839 confirmed cases in 168 countries, regions, or provinces, resulting in 8,778 deaths. Clinical features of COVID-19 include fever, dry cough, and fatigue, and the disease can lead to respiratory failure and death.

[0004] To date, there are no vaccines or therapeutics to prevent or treat SARS-CoV-2 infection. Given the ongoing threat to human health, there is an urgent need for preventive and therapeutic antiviral therapies to control SARS-CoV-2. Because the virus uses its spike glycoprotein for interaction with the cellular receptor ACE2 and the serine protease TMPRSS2 for entry into target cells, this spike protein represents an attractive target for antibody therapy. In particular, fully human antibodies that specifically bind to the SARS-CoV-2 spike protein (SARS-CoV-2-S) with high affinity and inhibit viral infectivity may be important for the prevention and treatment of COVID-19. Summary of the Invention

[0005] There is a need for neutralizing therapeutic anti-SARS-CoV-2 spike protein (SARS-CoV-2-S) antibodies and their use to treat or prevent viral infection. The present disclosure addresses this need, in part, by providing human anti-SARS-CoV-2-S antibodies, such as those in Table 1, and combinations thereof, including, for example, combinations with other therapeutic agents (e.g., anti-inflammatory agents, antimalarials, antivirals, or other antibodies or antigen-binding fragments), and methods of their use to treat viral infection.

[0006] The present disclosure provides neutralizing human antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof, that specifically bind to SARS-CoV-2-S.

[0007] In one aspect, the present disclosure provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to a coronavirus spike protein (CoV-S), wherein the antibody has the following characteristics: (a) a concentration of about 10 -9 EC less than M 50 (b) coronaviruses that bind to CoV-S and / or (c) comprising three heavy chain complementarity determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence having at least about 90% sequence identity with an HCVR of Table 1, and three light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) contained within a light chain variable region (LCVR) comprising an amino acid sequence having at least about 90% sequence identity with an LCVR of Table 1.

[0008] In some embodiments, the antibody or antigen-binding fragment comprises (a) an immunoglobulin heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of an antibody of Table 1, and / or (b) an immunoglobulin light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of an antibody of Table 1.

[0009] In some embodiments, the antibody or antigen-binding fragment comprises (a) a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to an HCVR sequence in Table 1, and / or (b) a light chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to an LCVR sequence in Table 1.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of a single antibody of Table 1. In some embodiments, the antibody or antigen-binding fragment comprises an immunoglobulin comprising the HCVR and LCVR of a single antibody of Table 1.

[0011] In one aspect, the present disclosure provides an antigen-binding protein that competes with any one of the antibodies or antigen-binding fragments described above or herein for binding to CoV-S.

[0012] In one aspect, the present disclosure provides an antigen binding protein that binds to the same epitope on CoV-S as, or an overlapping epitope on CoV-S as, any one of the antibodies or antigen-binding fragments described above or discussed herein.

[0013] In any of the various embodiments, the antibody or antigen-binding fragment may be multispecific.

[0014] In any of the various embodiments, the antibody or antigen-binding fragment may comprise one or more of the following properties: a) inhibiting coronavirus growth, b) binding to the surface of coronavirus, c) limiting the spread of coronavirus infection of cells in vitro, and d) protecting mice engineered to express human ACE2 or TMPRSS2 protein from death and / or weight loss caused by coronavirus infection.

[0015] In any of the various embodiments, the CoV-S is SARS-CoV-2-S.

[0016] In one aspect, the present disclosure provides a conjugate comprising an antibody or antigen-binding fragment bound to a CoV-S polypeptide described above or discussed herein. In some embodiments, the CoV-S is SARS-CoV-2-S.

[0017] In one aspect, the present disclosure provides an antibody or antigen-binding fragment as described above or discussed herein. The present invention provides a method for producing an antibody or antigen-binding fragment, the method comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favoring expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown. In some embodiments, the host cell is a Chinese hamster ovary cell.

[0018] In one aspect, the disclosure provides an antibody or antigen-binding fragment that is the product of the method discussed above.

[0019] In one aspect, the disclosure provides a polypeptide comprising: (a) CDR-H1, CDR-H2, and CDR-H3 of an HCVR domain of an antibody or antigen-binding fragment comprising an HCVR amino acid sequence set forth in Table 1; or (b) CDR-L1, CDR-L2, and CDR-L3 of an LCVR domain of an immunoglobulin chain comprising an LCVR amino acid sequence set forth in Table 1.

[0020] In one aspect, the disclosure provides polynucleotides encoding the polypeptides discussed above.

[0021] In one aspect, the present disclosure provides a vector comprising the polynucleotide discussed above.

[0022] In one aspect, the present disclosure provides a host cell comprising the antibody or antigen-binding fragment or polypeptide or polynucleotide or vector described above or discussed herein.

[0023] In one aspect, the disclosure provides a composition or kit comprising an antibody or antigen-binding fragment as described above or herein, in association with an additional therapeutic agent.

[0024] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding protein, antibody, or antigen-binding fragment described above or discussed herein, a pharmaceutically acceptable carrier, and optionally an additional therapeutic agent. In some embodiments, the additional therapeutic agent is an antiviral agent or vaccine. In some embodiments, the additional therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an anti-malarial agent, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S. In some embodiments, the anti-malarial agent is chloroquine or hydroxychloroquine. In some embodiments, the anti-inflammatory agent is an antibody such as sarilumab, tocilizumab, or gimsilumab. In some embodiments, the additional therapeutic agent is a secondary antibody or antigen-binding fragment comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of Table 1.

[0025] In one aspect, the present disclosure provides a container or injection device comprising an antigen-binding protein, antibody or antigen-binding fragment, or composition as described above or discussed herein.

[0026] In one aspect, the present disclosure provides a method for treating or preventing a coronavirus infection in a subject in need thereof, comprising administering a therapeutically effective amount of an antigen-binding protein, antibody, or antigen-binding fragment as described above or herein. In some embodiments, the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, and MERS-CoV.

[0027] In some embodiments of the method for treating or preventing infection by a coronavirus, and the subject is administered one or more additional therapeutic agents. Optionally, the one or more additional therapeutic agents are an antiviral agent or a vaccine. Optionally, the one or more additional therapeutic agents are selected from the group consisting of an anti-inflammatory agent, an anti-malarial agent, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S. Optionally, the anti-malarial agent is chloroquine or hydroxychloroquine. Optionally, the anti-inflammatory agent is an antibody such as, for example, sarilumab, tocilizumab, or gimsilumab. In some embodiments, the additional therapeutic agent is a secondary antibody or antigen-binding fragment comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of Table 1. Other antibodies that can be used alone or in combination with each other, or in combination with one or more of the antibodies disclosed herein for use in the context of the methods of the present disclosure include, for example, LY-CoV555 (Eli Lilly), 47D11 (Wang et al Nature Communications Article No. 2251), B38, H4, B5, and / or H2 (Wu et al., 10.1126 / science.abc2241 (2020)), STI-1499 (Sorrento Therapeutics), VIR-7831, and VIR-7832 (Vir Biotherapeutics).

[0028] In one aspect, the present disclosure provides a method for administering an antibody or antigen-binding fragment described above or discussed herein to a subject, comprising injecting the antibody or antigen-binding fragment into the subject. In some embodiments, the antibody or antigen-binding fragment is injected into the subject subcutaneously, intravenously, or intramuscularly.

[0029] In any of the various embodiments described above or herein, the antibody or antigen-binding fragment comprises a VH3-66 or Vk1-33 variable domain sequence.

[0030] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, wherein the isolated antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 202, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 210.

[0031] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 204, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 206, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 208, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 212, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 214. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 210. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 210.

[0032] In one aspect, the disclosure provides an isolated antibody that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, wherein the isolated antibody comprises an immunoglobulin constant region and three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 202. and three light chain complementarity-determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 210.

[0033] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 204, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 206, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 208, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 212, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 214. In some embodiments, the isolated antibody comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 210. In some embodiments, the isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 216 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 218. Optionally, the immunoglobulin constant region is an IgG1 constant region. Optionally, the isolated antibody is a recombinant antibody. Optionally, the isolated antibody is multispecific.

[0034] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody as described above or discussed herein and a pharmaceutically acceptable carrier or diluent.

[0035] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. Optionally, the second therapeutic agent is selected from the group consisting of a second antibody or antigen-binding fragment thereof that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, an anti-inflammatory agent, an anti-malarial agent, and an antibody or antigen-binding fragment thereof that binds to TMPRSS2.

[0036] In some embodiments, the second therapeutic agent is a secondary antibody or antigen-binding fragment thereof that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832. Optionally, the secondary antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) comprised within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 646. Optionally, the secondary antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 642, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 499, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 644, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 648, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 650, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 652. Optionally, the secondary antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 646. In some cases, the second antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO:654 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:656.

[0037] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, wherein the isolated antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 640, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 646.

[0038] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 642, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 499, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 644, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 648, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 650, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 652. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 646. In some embodiments, the isolated antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 646.

[0039] In one aspect, the disclosure provides an isolated antibody that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, wherein the isolated antibody comprises an immunoglobulin constant region and three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 640, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 646.

[0040] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 642, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 499, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 644, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 648, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 650, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 652. In some embodiments, the isolated antibody comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 646. In some embodiments, the isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 654 and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 656. Optionally, the immunoglobulin constant region is an IgG1 constant region. Optionally, the isolated antibody is a recombinant antibody. Optionally, the isolated antibody is multispecific.

[0041] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody as described above or discussed herein and a pharmaceutically acceptable carrier or diluent.

[0042] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent, optionally selected from the group consisting of a second antibody or antigen-binding fragment thereof that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, an anti-inflammatory agent, an anti-malarial agent, and an antibody or antigen-binding fragment thereof that binds to TMPRSS2.

[0043] In some embodiments, the second therapeutic agent is a secondary antibody or antigen-binding fragment thereof that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832. Optionally, the secondary antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) comprised within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 210. Optionally, the secondary antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 204, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 206, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 208, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 212, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 55, an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 214. In some cases, the secondary antibody or antigen-binding fragment thereof comprises an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 202, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 210. In some cases, the secondary antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 216, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 218.

[0044] In various embodiments, any of the features or components of the embodiments described above or discussed herein can be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein can be combined with another related value described above or discussed herein to recite a range where those values ​​represent the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure. [Brief explanation of the drawings]

[0045] [Figure 1] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 2] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 3] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 4] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 5] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 6] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 7] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 8] Figure 1 shows ELISA blocking data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevent the spike protein from binding to its receptor ACE2. [Figure 9A]Figure 9 shows the V gene frequencies of heavy (X-axis) and light (Y-axis) chain pairs of isolated neutralizing antibodies against SARS-CoV-2 in VelocImmune® mice (Figure 9A, N=185) and convalescent human donors (Figure 9B, N=68). Circle shade and size correspond to the number of heavy and light chain pairs present in the repertoire of isolated neutralizing antibodies. Neutralization is defined as >70% at a 1:4 antibody dilution (approximately 2 μg / ml) in a VSV pseudoparticle neutralization assay. [Figure 9B] Figure 9 shows the V gene frequencies of heavy (X-axis) and light (Y-axis) chain pairs of isolated neutralizing antibodies against SARS-CoV-2 in VelocImmune® mice (Figure 9A, N=185) and convalescent human donors (Figure 9B, N=68). Circle shade and size correspond to the number of heavy and light chain pairs present in the repertoire of isolated neutralizing antibodies. Neutralization is defined as >70% at a 1:4 antibody dilution (approximately 2 μg / ml) in a VSV pseudoparticle neutralization assay. [Figure 10A] Neutralization potency is displayed. Figure 10A displays the neutralization potency of anti-SARS-CoV-2 spike mAbs. Serial dilutions of anti-spike mAb, IgG1 isotype control, and recombinant dimeric ACE2 (hACE2.hFc) were added to Vero cells along with pVSV-SARS-CoV-2-S-mNeon, and mNeon expression was measured as a readout of virus infectivity 24 hours post-infection. Data are graphed as percent neutralization relative to virus-only infected controls. [Figure 10B] Figure 10B displays the neutralization potency of individual anti-spike mAbs and mAb combinations against SARS-CoV-2-S virus in VeroE6 cells. [Figure 11]Epitope binning from a matrix of premix binding assays of various anti-SARS-CoV-2 mAbs is shown. Epitope binning was performed for nine anti-SARS-CoV-2 mAbs as described. Each graph had three phases (I, II, III). In phase I, anti-SARS-CoV-2 mAb (20 μg / ml) was loaded onto an anti-human Fc probe. In phase II, a human IgG1 blocking mAb solution (100 μg / ml) was loaded onto the anti-human Fc probe. In phase III, a solution of 100 nM of SARS-CoV-2 RBD-MMH premix complex for each 600 nM anti-SARS-CoV-2 mAb binding site was flowed over the mAb capture probe. [Figure 12] A 3D surface model of the spike protein RBD domain structure is displayed, showing the ACE2 interface and HDX-MS epitope mapping results. RBD residues protected by anti-SARS-CoV2-spike antibodies are shown with shading representing the degree of protection determined by HDX-MS experiments. The RBD structure is reproduced from PDB6M17. [Figure 13A] Figure 13A shows a 3.9 Å cryoEM map of the mAb10933+RBD+mAb10987 complex, shaded according to the chains in the refined model in Figure 13B. The RBD, heavy and light chains of mAb10933, and heavy and light chains of mAb10987 are identified. [Figure 13B] Figure 13A shows a 3.9 Å cryoEM map of the mAb10933+RBD+mAb10987 complex, shaded according to the chains in the refined model in Figure 13B. The RBD, heavy and light chains of mAb10933, and heavy and light chains of mAb10987 are identified. [Figure 14]Displaying cryoEM data statistics. Data collection and refinement statistics are reported for the mAb10987+mAb10933+SARS-CoV-2 RBD complex structure shown in Figure 13A and Figure 13B. DETAILED DESCRIPTION OF THE INVENTION

[0046] Before describing the methods of the present invention, it is to be understood that this invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

[0048] The terms "coronavirus" or "CoV" refer to any virus in the coronavirus family, including, but not limited to, SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to the newly emerged coronavirus identified as the cause of a severe outbreak in Wuhan, China, and has rapidly spread to other parts of the world. SARS-CoV-2 is also known as 2019-nCoV and Wuhan coronavirus. The viral spike protein binds to the human host cell receptor angiotensin-converting enzyme 2 (ACE2). The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for viral membrane fusion.

[0049] The term "CoV-S," also referred to as "S" or "S protein," refers to the spike protein of coronaviruses and can refer to specific S proteins, such as SARS-CoV-2-S, MERS-CoV S, and SARS-CoV S. The SARS-CoV-2 spike protein is a 1,273-amino acid type I membrane glycoprotein that assembles into trimers that constitute spikes or peplomers on the surface of enveloped coronavirus particles. This protein has two important functions: host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein. CoV-S binds to its cognate receptor through a receptor-binding domain (RBD) present in the S1 subunit. The amino acid sequence of the full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence provided in SEQ ID NO: 832. The term "CoV-S" includes protein variants of CoV spike proteins isolated from different CoV isolates, as well as recombinant CoV spike proteins or fragments thereof. The term also encompasses, for example, a CoV spike protein or fragment thereof linked to a signal sequence such as a histidine tag, mouse or human Fc, or ROR1.

[0050] As used herein, the term "coronavirus infection" or "CoV infection" refers to an infection caused by a coronavirus, such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term includes coronavirus respiratory tract infections, which often affect the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (renal failure and dysfunction), septic shock, and death in severe cases.

[0051] virus The present invention includes methods for treating or preventing viral infection in a subject. The term "virus" includes any virus whose infection in a subject is treatable or preventable by administration of an anti-CoV-S antibody or antigen-binding fragment thereof (e.g., a virus whose infectivity depends at least in part on CoV-S). In one embodiment of the present invention, a "virus" is any virus that expresses a spike protein (e.g., CoV-S). The term "virus" also includes CoV-S-dependent respiratory viruses, which are viruses that infect a subject's respiratory tissues (e.g., upper and / or lower respiratory tract, trachea, bronchi, lungs) and are treatable or preventable by administration of an anti-CoV-S antibody or antigen-binding fragment thereof. For example, in one embodiment of the present invention, viruses include coronaviruses, SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), SARS-CoV (severe acute respiratory syndrome coronavirus), and MERS-CoV (Middle East respiratory syndrome (MERS) coronavirus). Coronaviruses can include the genera alphacoronavirus, betacoronavirus, gammacoronavirus, and deltacoronavirus. In some embodiments, the antibodies or antigen-binding fragments provided herein are capable of binding to and / or neutralizing alphacoronavirus, betacoronavirus, gammacoronavirus, and / or deltacoronavirus. In certain embodiments, this binding and / or neutralization is consistent with the specific binding and / or neutralization of coronaviruses. It may be specific to a genus or a particular subgroup of a genus. "Viral infection" refers to the entry and multiplication of a virus into the body of a subject.

[0052] Coronavirus virions are spherical and approximately 125 nm in diameter. The most striking feature of coronaviruses is the club-shaped spikes emanating from the virion's surface. These spikes characterize the virion, giving it the appearance of a solar corona and prompting the name coronavirus. Within the virion envelope is a nucleocapsid. Coronaviruses possess a helically symmetric nucleocapsid, which is unusual for positive-sense RNA viruses but is far more common for negative-sense RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the coronavirus family. Initial attachment of the virion to a host cell is initiated by the interaction between the S protein and its receptor. The location of the receptor-binding domain (RBD) within the S1 region of the coronavirus S protein varies among viruses, with some possessing the RBD at the C-terminus of S1. The S protein / receptor interaction is a major determinant of coronavirus infection in host species and also governs the viral tissue tropism. Many coronaviruses utilize peptidases as cellular receptors. Following receptor binding, the virus must then access the host cell cytosol, which is generally achieved by acid-dependent proteolytic cleavage of the S protein by a cathepsin, TMPRRS2, or another protease, followed by fusion of the viral membrane with the cellular membrane.

[0053] Anti-CoV-S antibodies and antigen-binding fragments The present invention provides antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that specifically bind to CoV spike protein or an antigenic fragment thereof.

[0054] The term "antibody," as used herein, refers to an immunoglobulin molecule (i.e., an "intact antibody molecule") comprising four polypeptide chains, two heavy chains (HC) and two light chains (LC), interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Exemplary antibodies include, for example, those listed in Table 1. Each heavy chain comprises a heavy chain variable region ("HCVR" or "V").H ") and heavy chain constant region (C H 1 domain, C H 2 domain, and C H Each light chain contains a light chain variable region ("LC"). VR or "V L ") and the light chain constant region (C L ) V H Area and V L The regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FRs). H and V Lcomprises three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain CDRs are sometimes referred to as HCDRs or CDR-H and are numbered as above (e.g., HCDR1, HCDR2, and HCDR3 or CDR-H1, CDR-H2, and CDR-H3). Similarly, the light chain CDRs are referred to as LCDRs or CDR-L and are numbered as LCDR1, LCDR2, and LCDR3 or CDR-L1, CDR-L2, and CDR-L3. In certain embodiments of the present invention, the FRs of an antibody (or antigen-binding fragment thereof) are identical to human germline sequences or are naturally or artificially modified. Exemplary human germline sequences include, but are not limited to, VH3-66 and Vk1-33. Thus, the present disclosure provides an anti-CoV-S antibody or antigen-binding fragment thereof (e.g., an anti-SARS-CoV-2-S antibody or antigen-binding fragment thereof) comprising the HCDR and LCDR sequences of Table 1 within the VH3-66 or Vk1-33 variable heavy or light chain region. The present disclosure further relates to IgKV4-1, IgKV1-5, IgKV1-9, IgKV1-12, IgKV3-15, IgKV1-16, IgKV1-17, IgKV3-20, IgLV3-21, IgKV2-24, IgKV1-33, IgKV1-39, IgLV1-40, IgLV1-44, IgLV1-51, IgLV3-1, IgKV1-6, IgLV2-8, IgKV3-11, IgLV2-11, IgLV2-14, a light chain selected from IgLV2-23, or IgLV6-57, and IgHV1-69, IgHV3-64, IgHV4-59, IgHV3-53, IgHV3-48, IgHV4-34, IgHV3-33, IgHV3-30, IgHV3-23, IgHV3-20, IgHV1-18, IgHV3-15, IgHV3-11, IgHV3-9, IgHV1-8, IgHV3-7, IgHV2-5, IgHV1 and an anti-CoV-S antibody or antigen-binding fragment thereof (e.g., an anti-SARS-CoV-2-S antibody or antigen-binding fragment thereof) comprising the HCDR and LCDR sequences of Table 1 in combination with a heavy chain selected from IgHV-2, IgHV2-70, IgHV3-66, IgHV5-51, IgHV1-46, IgHV4-39, IgHV4-31, IgHV3-30-3, IgHV2-26, or IgHV7-4-1. The present disclosure further relates to IgKV4-1, IgKV1-5, IgKV1-9, IgKV1-12, IgKV3-15, IgKV1-16, IgKV1-17, IgKV3-20, IgLV3-21, IgKV2-24, IgKV1-33, IgKV1-39, IgLV1-40, IgLV1-44, IgLV1-51, a light chain selected from IgLV3-1, IgKV1-6, IgLV2-8, IgKV3-11, IgLV2-11, IgLV2-14, IgLV2-23, or IgLV6-57, and a light chain selected from IgHV1-69, IgHV3-64, IgHV4-59, IgHV3-53, IgHV3-48, IgHV4-34, IgHV and IgHV3-33, IgHV3-30, IgHV3-23, IgHV3-20, IgHV1-18, IgHV3-15, IgHV3-11, IgHV3-9, IgHV1-8, IgHV3-7, IgHV2-5, IgHV1-2, IgHV2-70, IgHV3-66, IgHV5-51, IgHV1-46, IgHV4-39, IgHV4-31, IgHV3-30-3, IgHV2-26, or IgHV7-4-1.

[0055] Typically, both the heavy and light chain variable domains of immunoglobulins contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within relatively conserved framework regions (FRs). Generally, from the N-terminus to the C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In an embodiment of the present invention, the assignment of amino acids to each domain is based on the information in Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 th ed.;NIH Publ.No.91-3242(1991);Kabat(1978)Adv.Prot.Chem.32:1-75, Kabat,et al.,(1977)J.Biol.Chem.252:6609-6616,Chothia,et al.,(1987)J Mol.Biol.196:901-917, or according to the definition of Chothia, et al., (1989) Nature 342:878-883.

[0056] The present invention includes monoclonal anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" refers to a population of substantially homogeneous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for possible naturally occurring mutations that may be present in minor amounts. A "plurality" of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., except for possible naturally occurring mutations that may be present in minor amounts in the amino acid sequence, as discussed above) antibodies and fragments that is higher than would normally occur in nature, e.g., in the blood of a host organism such as a mouse or human.

[0057] In an embodiment of the invention, an anti-CoV-S antigen binding protein, e.g., an antibody or an anti- The antigen-binding fragment comprises, for example, a heavy chain constant domain of an IgA type (e.g., IgA1 or IgA2), an IgD type, an IgE type, an IgG type (e.g., IgG1, IgG2, IgG3, and IgG4), or an IgM type. In an embodiment of the invention, the antigen-binding protein, e.g., an antibody or antigen-binding fragment, comprises, for example, a kappa or lambda type light chain constant domain.

[0058] As used herein, the term "human" antigen-binding protein, such as an antibody, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences, whether grafted in human cells or non-human cells, e.g., mouse cells. See, e.g., US8502018, US6596541, or US5789215. Human mAbs of the invention may include, for example, amino acid residues in the CDRs, particularly CDR3, that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., mouse) are grafted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in the cells of non-human mammals. The term is not intended to include antibodies isolated from or produced in a human subject. See below.

[0059] The present invention includes anti-CoV-S chimeric antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of using them. As used herein, a "chimeric antibody" is an antibody having variable domains from a first antibody and constant domains from a second antibody, wherein the first and second antibodies are derived from different species. (See US 4,816,567, and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0060] The present invention includes anti-CoV-S hybrid antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods for their use. As used herein, a "hybrid antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, where the first and second antibodies are from different animals, or the variable domain (but not the constant region) is from the first animal. For example, the variable domain can be obtained from an antibody isolated from a human and expressed with a fixed constant region not isolated from that antibody. Exemplary hybrid antibodies are described in Example 1, which refer to PCR products derived from antibody heavy and light chain variable regions cloned into expression vectors containing heavy and light chain constant regions, respectively. Hybrid antibodies are synthetic and do not occur in nature because the variable and constant regions they contain are not isolated from a single natural source.

[0061] The term "recombinant" antigen-binding protein, such as an antibody or antigen-binding fragment thereof, refers to such molecules created, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice), or cell (e.g., CHO cell) expression systems, or non-human cell expression systems, or antibodies isolated from a recombinant combinatorial human antibody library. In some embodiments, recombinant antibodies share sequences with antibodies isolated from an organism (e.g., mouse or human) but have been expressed via recombinant DNA technology. Such antibodies may have post-translational modifications (e.g., glycosylation) that differ from antibodies isolated from the organism.

[0062] The recombinant anti-CoV-S antigen binding proteins, e.g., antibodies and antigen-binding fragments, disclosed herein can also be produced in an E. coli / T7 expression system. In this embodiment, nucleic acids encoding the anti-CoV-S antibody immunoglobulin molecules of the invention (e.g., those found in Table 1) can be inserted into a pET-based plasmid and expressed in an E. coli / T7 system. For example, the present invention includes methods for expressing an antibody or antigen-binding fragment thereof or an immunoglobulin chain thereof in a host cell (e.g., a bacterial host cell such as E. coli such as BL21 or BL21DE3), which comprises expressing T7 RNA polymerase in the cell, which also contains a polynucleotide encoding an immunoglobulin chain operably linked to a T7 promoter. For example, in an embodiment of the present invention, a bacterial host cell such as E. coli expresses a T7 RNA polymerase operably linked to a lac promoter. It contains a polynucleotide encoding an RNA polymerase gene, and expression of the polymerase and strands is induced by incubating the host cells with IPTG (isopropyl-beta-D-thiogalactopyranoside). See US 4,952,496 and US 5,693,489, or Studier & Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5;189(1):113-30.

[0063] There are several methods for producing recombinant antibodies known in the art. One example of a method for the recombinant production of antibodies is disclosed in US4816567.

[0064] Transformation can be by any known method for introducing polynucleotides (e.g., DNA or RNA, including mRNA) into host cells. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotide(s) in liposomes, lipid nanoparticle technology, biolistic injection, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors such as lentiviruses or adeno-associated viruses. Methods for transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. In some embodiments, an antibody or antigen-binding fragment thereof of the present disclosure can be introduced into a subject in the form of a nucleic acid (e.g., DNA or RNA, including mRNA) so that the subject's own cells produce the antibody. The present disclosure further provides modifications to the nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression against the CoV spike protein, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved antibody affinity or specificity.

[0065] Accordingly, the present invention includes recombinant methods for making anti-CoV-S antigen binding proteins, such as antibodies or antigen-binding fragments thereof, of the present invention, or immunoglobulin chains thereof, comprising: (i) introducing one or more polynucleotides (e.g., comprising any one or more nucleotide sequences of the sequences in Table 2) encoding the antigen binding protein, e.g., the light and / or heavy chains, or CDRs, of an immunoglobulin in Table 1 (where the polynucleotides are in a vector and / or integrated into a host cell chromosome and / or operably linked to a promoter); (ii) culturing host cells (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable for expression of the polynucleotides; and (iii) optionally isolating the antigen binding protein (e.g., antibody or fragment) or chain from the host cell and / or the medium in which the host cell is grown. For example, the polynucleotides can be expressed using, for example, a gene editing system (e.g., For example, integration into a host cell chromosome can be achieved by cleaving the chromosome using CRISPR (e.g., CRISPR-Cas9), TALEN, megaTAL, zinc finger, or Argonaute, followed by targeted insertion of a vector such as an adeno-associated virus (AAV). Targeted insertion can occur at a host cell locus, such as an albumin or immunoglobulin genomic locus. Alternatively, insertion can be performed at a random locus using a vector such as a lentivirus. When producing an antigen-binding protein (e.g., an antibody or antigen-binding fragment) comprising more than one immunoglobulin chain, for example, an antibody comprising two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of the chains in a single host cell can result in the association of the chains within, on the cell surface, or outside the cell, for example, when such chains are secreted to form an antigen-binding protein (e.g., an antibody or antigen-binding fragment). The methods include those in which only an immunoglobulin heavy chain or only an immunoglobulin light chain (e.g., any of those discussed herein, including mature fragments and / or variable domains thereof) is expressed. Such chains are useful, for example, as intermediates in the expression of antibodies or antigen-binding fragments comprising such chains. For example, the invention also includes anti-CoV-S antigen binding proteins, such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or including a variable domain or CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence set forth in Table 2, and a light chain immunoglobulin (or including a variable domain or CDRs thereof) encoded by a nucleotide sequence set forth in Table 2, which are the products of such production methods, and optionally, purification methods described herein. For example, in some embodiments, the product of this method is an anti-CoV-S antigen binding protein that is an antibody or fragment comprising an HCVR comprising an amino acid sequence set forth in Table 1 and an LCVR comprising an amino acid sequence set forth in Table 1, wherein the HCVR and LCVR sequences are selected from a single antibody listed in Table 1.In some embodiments, the product of this method is an anti-CoV-S antigen binding protein that is an antibody or fragment comprising HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences set forth in Table 1, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences set forth in Table 1, wherein the six CDR sequences are selected from a single antibody listed in Table 1. In some embodiments, the product of this method is an anti-CoV-S antigen binding protein that is an antibody or fragment comprising a heavy chain comprising the HC amino acid sequence set forth in Table 1, and a light chain comprising the LC amino acid sequence set forth in Table 1.

[0066] Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for the expression of anti-CoV-S antigen-binding proteins. Such host cells are well known in the art, and many are available from the American Type Culture Collection (ATCC). These host cells include, among others, Chinese hamster ovary (CHO) cells, NS0, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that can be used include insect cell lines (e.g., Spodoptera frugiperda or Trichoplusia ni), amphibian cells, bacterial cells, plant cells, and fungal cells. Fungal cells include, for example, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia koclamae, Pichia membranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri), Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia guercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica, Pichia sp., Saccharomyces cerevisiae, Saccharomyce s sp., Hansenula polymorpha, Kluyveromyces sp., Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium lucknowense, Fusarium sp., Fusarium gramineum, Fusarium venenatum, Physcomitrella patens, and Neurospora crassa. The present invention includes isolated host cells (e.g., CHO cells) containing antigen binding proteins such as those in Table 1, or polynucleotides encoding such polypeptides.

[0067] The term "specifically binds" refers to a binding activity that specifically binds to a protein as measured by a real-time label-free biolayer interferometry assay, e.g., at 25°C or 37°C, e.g., by an Octet® HTX biosensor, or by surface plasmon resonance, e.g., BIACORE™, or by lysis affinity ELISA. D CoV-, represented as A binding affinity for an antigen, such as the S protein (e.g., SARS-CoV-2-S), of at least about 10 -8 The present invention refers to those antigen binding proteins (e.g., mAbs) that specifically bind to CoV-S proteins.

[0068] As used herein, the terms "antigen-binding portion" or "antigen-binding fragment" of an antibody, or antigen-binding protein, and the like, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Non-limiting examples of antibody-binding fragments include: (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, and (iv) Fv fragments. Examples of antigen-binding fragments include (i) fragments, (ii) fragments of antibodies, (iii) fragments of antibodies, (iv) fragments of antibodies, (v) fragments of antibodies, (vi) fragments of antibodies, (v) fragments of antibodies, (vi) fragments of antibodies, (vii) fragments of antibodies, (v ... In one embodiment of the present invention, the antigen-binding fragment comprises three or more CDRs of an antibody of Table 1 (e.g., CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3).

[0069] Antigen-binding fragments of antibodies, in embodiments of the present invention, comprise at least one variable domain. A variable domain may be of any size or amino acid composition and generally comprises at least one CDR adjacent to or in-frame with one or more framework sequences. L V bound to the domain H In an antigen-binding fragment having a domain, V H Domains and V L The domains can be arranged relative to each other in any suitable configuration. For example, the variable region is a dimer, with the V H -V H , V H -V L or V L -V L Alternatively, the antigen-binding fragment of an antibody may comprise a monomeric V H or V L It may include a domain.

[0070] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found in an antigen-binding fragment of an antibody of the invention include: (i) V H -C H 1, (ii) V H -C H 2, (iii) V H -C H 3, (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3, (vii)V H -C L , (viii) V L -C H 1, (ix)V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2, (xii)V L -C H 1-C H 2-C H 3, (xiii)V L -C H 2-C H 3. and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be directly linked to each other or may be linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the present invention may be linked to each other and / or to one or more monomeric V H Or V L The variable domain and constant domain configurations may comprise homodimers or heterodimers (or other multimers) of any of the variable domain and constant domain configurations listed above in non-covalent association with the domains (e.g., by disulfide bond(s)).

[0071] Antigen-binding proteins (e.g., antibodies and antigen-binding fragments) can be monospecific or multispecific (e.g., bispecific). Multispecific antigen-binding proteins are discussed further herein.

[0072] In certain embodiments, the antibodies or antibody fragments of the invention can be conjugated to a moiety such as a ligand, or a therapeutic moiety ("immunoconjugate") such as an antiviral drug, a second anti-influenza antibody, or any other therapeutic moiety useful in treating a viral infection, e.g., an influenza virus infection. See below.

[0073] The present invention also provides complexes comprising an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment, discussed herein, complexed with an anti-CoV-S polypeptide or antigenic fragment thereof and / or a secondary antibody or antigen-binding fragment thereof (e.g., a detectably labeled secondary antibody) that specifically binds to the anti-CoV-S antibody or fragment. In embodiments of the invention, the antibody or fragment is in vitro (e.g., immobilized on a solid substrate) or within a subject's body. In embodiments of the invention, the CoV-S is in vitro (e.g., immobilized on a solid substrate), on the surface of a virus, or within a subject's body. Immobilized anti-CoV-S antibodies and antigen-binding fragments thereof covalently bound to an insoluble matrix material (e.g., glass or polysaccharides such as agarose or Sepharose, e.g., beads or other particles) are also part of the invention, and optionally the immobilized antibody is complexed with CoV-S or an antigenic fragment thereof, or a secondary antibody or fragment thereof.

[0074] "Isolated" antigen-binding proteins, antibodies or antigen-binding fragments thereof, polypeptides, polynucleotides, and vectors are at least partially free from other biological molecules from the cell or cell culture in which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen-binding fragments, lipids, carbohydrates, or other materials such as cellular debris and growth medium. Isolated antibodies or antigen-binding fragments may further be at least partially free from expression system components, such as biological molecules from host cells or their growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of pharmaceutical formulations that contain the antibody or fragment.

[0075] The term "epitope" refers to a specific antigen-binding site of an antigen-binding protein, e.g., an antigenic determinant (e.g., a CoV-S polypeptide) that interacts with the variable region of an antibody molecule, known as the paratope. A single antigen can have more than one epitope. Thus, different antibodies can bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. The term also refers to the region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and contain residues that directly contribute to the affinity of the interaction. Epitopes can be linear or conformational, i.e., can be composed of non-linear amino acids. In certain embodiments, epitopes can include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, can have specific three-dimensional structural and / or specific charge characteristics.

[0076] Methods for determining the epitope of an antigen-binding protein, such as an antibody or fragment or polypeptide, include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be used (Tomer (2000) Prot. Sci. 9:487-496). Another method (e.g., coversin) that can be used to identify amino acids in a polypeptide with which an antigen-binding protein (e.g., an antibody or fragment or polypeptide) interacts is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, the hydrogen / deuterium exchange method involves deuterium-labeling the protein of interest and then binding the antigen-binding protein, such as an antibody or fragment or polypeptide, to the deuterium-labeled protein. The CoV-S protein / antigen-binding protein complex is then transferred to water, and exchangeable protons within amino acids protected by the antibody complex undergo back-exchange from deuterium to hydrogen at a slower rate than exchangeable protons within amino acids not part of the interface. As a result, amino acids that form part of the protein / antigen-binding protein interface are able to retain deuterium and therefore exhibit a relatively large mass compared to amino acids not included in the interface. After dissociation of the antigen-binding protein (e.g., antibody or fragment or polypeptide), the target protein is subjected to protease cleavage and mass spectrometry, thereby revealing deuterium-labeled residues corresponding to the specific amino acids with which the antigen-binding protein interacts. See, e.g., Ehring (1999) Analytical Biochemistry 267:252-259; Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0077] As used herein, the term "compete" refers to an antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) that binds to an antigen (e.g., CoV-S) and inhibits or blocks the binding of another antigen-binding protein (e.g., an antibody or antigen-binding fragment thereof) to the antigen. The term also includes competition between two antigen-binding proteins, e.g., antibodies, in both orientations, i.e., a primary antibody that binds and blocks the binding of a secondary antibody, and vice versa. In certain embodiments, a first antigen-binding protein (e.g., an antibody) and a second antigen-binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different, but overlapping, epitopes, such that the binding of one inhibits or blocks the binding of the second antibody, e.g., through steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) can be measured by methods known in the art, for example, by real-time label-free biolayer interferometry assays. Epitope mapping (e.g., via alanine scanning or hydrogen-deuterium exchange (HDX)) can be used to determine whether two or more antibodies are not competing (e.g., on spike protein receptor-binding domain (RBD) monomers), are competing for the same epitope, or are competing but for diverse microepitopes (e.g., identified via HDX). In embodiments of the invention, competition between a first and a second anti-CoV-S antigen binding protein (e.g., antibody) is determined by the binding of a soluble CoV-S protein complexed with a second anti-CoV-S antigen binding protein (e.g., antibody). This is determined by measuring the ability of an immobilized first anti-CoV-S antigen binding protein (e.g., an antibody) (not initially complexed with CoV-S protein) to bind to the protein. A reduction in the ability of the first anti-CoV-S antigen binding protein (e.g., an antibody) to bind to the complexed CoV-S protein compared to the uncomplexed CoV-S protein indicates that the first and second anti-CoV-S antigen binding proteins (e.g., antibodies) compete with each other. The degree of competition can be expressed as a percentage of binding reduction. Such competition can be measured using real-time label-free biolayer interferometry assays, for example, with an Octet RED384 biosensor (Pall ForteBio Corp.), enzyme-linked immunosorbent assay (ELISA), or surface plasmon resonance (SPR).

[0078] Binding competition between anti-CoV-S antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using a real-time label-free biolayer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.). For example, to determine competition between two anti-CoV-S monoclonal antibodies, an anti-CoV-S mAb can first be captured on an Octet biosensor chip (Pall ForteBio Corp., #18-5060) coated with an anti-hFc antibody by immersing the chip in a solution of anti-CoV-S mAb (hereafter referred to as "mAb1"). As a positive control for blocking, the antibody-captured biosensor chip can then be saturated with a known blocking isotype control mAb (hereafter referred to as "blocking mAb") by immersing it in a solution that blocks the mAb. To determine whether mAb2 competes with mAb1, the biosensor chip can then be submerged in a co-complex solution of a pre-incubated CoV-S polypeptide and a second anti-CoV-S mAb (hereafter referred to as "mAb2") for a period of time, and the binding of mAb1 to the CoV-S polypeptide can be determined. The biosensor chip can be washed with buffer between each step of the experiment. Real-time binding responses can be monitored during the course of the experiment, and the binding responses at the end of each step can be recorded.

[0079] For example, in an embodiment of the present invention, a competitive assay is performed at 25° C. and a pH of about 7, eg, 7.4, in the presence of, eg, buffer, salt, detergent, and non-specific protein (eg, bovine serum albumin).

[0080] Typically, antibodies or antigen-binding fragments of the invention, modified in some way, retain the ability to specifically bind to CoV-S, e.g., retain at least 10% of their CoV-S binding activity (compared to the parent antibody) when that activity is expressed on a molar basis. Preferably, antibodies or antigen-binding fragments of the invention retain at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the CoV-S binding affinity of the parent antibody. It is also intended that antibodies or antigen-binding fragments of the invention may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "function-conservative variants" of antibodies) that do not substantially alter their biological activity.

[0081] "Variants" of polypeptides such as immunoglobulin chains (e.g., mAb8021 V H , V L , HC, or LC, mAb8028 V H , V L , HC, or LC, or mAb 8029 V H , V L , HC, or LC) refer to a polypeptide containing an amino acid sequence that is at least about 70 to 99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a reference amino acid sequence described herein (e.g., SEQ ID NOs: 2, 10, 18, 20, 22, 30, 38, 40, 42, 50, 58, or 60) when the comparison is made using a BLAST algorithm, where the algorithm parameters are set to 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, The sequences are selected to maximize the match between them (e.g., expectation threshold: 10, word size: 3, maximum match in query range: 0, BLOSUM 62 matrix, gap costs: presence 11, extension 1, conditional composition score matrix adjustment).

[0082] A "variant" of a polynucleotide refers to a polynucleotide that contains a nucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to a reference nucleotide sequence described herein (e.g., SEQ ID NOs: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, or 59) when comparison is made using the BLAST algorithm, where the algorithm parameters are selected to maximize matches between the respective sequences over the entire length of the respective reference sequences (e.g., expectation threshold: 10, word size: 28, maximum match in query range: 0, match / mismatch score: 1, -2, gap cost: linear).

[0083] In embodiments of the invention, the anti-CoV-S antigen binding proteins, e.g., antibodies and antigen-binding fragments thereof, of the invention comprise a heavy chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to the HCVR amino acid sequences set forth in Table 1, and / or a light chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to the LCVR amino acid sequences set forth in Table 1.

[0084] In addition, variant anti-CoV-S antigen binding proteins can include polypeptides comprising an amino acid sequence described herein, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as, for example, missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. For example, the present invention includes antigen binding proteins comprising immunoglobulin light chain variants comprising an LCVR amino acid sequence set forth in Table 1, but with one or more such mutations, and / or immunoglobulin heavy chain variants comprising an HCVR amino acid sequence set forth in Table 1, but with one or more such mutations. In embodiments of the invention, the variant anti-CoV-S antigen binding protein comprises an immunoglobulin light chain variant comprising CDR-L1, CDR-L2, and CDR-L3 (wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more of such mutations (e.g., conservative substitutions)), and / or an immunoglobulin heavy chain variant comprising CDR-H1, CDR-H2, and CDR-H3 (wherein one or more (e.g., 1 or 2 or 3) of such CDRs have one or more of such mutations (e.g., conservative substitutions)). Substitutions may be in the CDRs, framework, or constant regions.

[0085] The invention further provides variant anti-CoV-S antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, comprising one or more variant CDRs described herein (e.g., any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3) that have at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity or similarity to the heavy and light chain CDRs of Table 1.

[0086] Embodiments of the present invention also include the corresponding V specifically described herein. H , V L, HC, or LC amino acid sequence and 70% or more (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the total Immunoglobulin V, including amino acid sequences with specific amino acid sequence identity or similarity. H and V L The present invention also includes variant antigen binding proteins, e.g., anti-CoV-S antibodies and antigen-binding fragments thereof, comprising the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 of such immunoglobulins, but the CDRs within the variant antigen binding proteins are not variants and comprise the CDR amino acid sequences set forth in Table 1. Thus, in such embodiments, the CDRs within the variant antigen binding proteins are not themselves variants.

[0087] Conservatively modified variant anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present invention. "Conservatively modified variants" or "conservative substitutions" refer to variants in which one or more amino acids in a polypeptide are replaced with other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure, and rigidity). Such changes can frequently be made without significantly destroying the biological activity of the antibody or fragment. Those skilled in the art will generally recognize that single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224(4)). th (See ED.) In addition, substitutions of structurally or functionally similar amino acids are unlikely to significantly destroy biological activity.

[0088] Examples of amino acid groups having side chains with similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45.

[0089] Function-conservative variants of anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present invention. Any of the variants of anti-CoV-S antibodies and antigen-binding fragments thereof (discussed herein) may be "function-conservative variants." Such function-conservative variants may also be characterized as conservatively modified variants in some cases. As used herein, "function-conservative variant" refers to a variant of an anti-CoV-S antibody or antigen-binding fragment thereof in which one or more amino acid residues have been altered without significantly altering one or more functional properties of the antibody or fragment. In embodiments of the present invention, a function-conservative variant anti-CoV-S antibody or antigen-binding fragment thereof of the present invention comprises a variant amino acid sequence and exhibits one or more of the following functional properties: inhibiting the proliferation of coronaviruses (e.g., SARS-CoV-2, SARS-CoV, and / or MERS-CoV) in ACE2- and / or TMPRSS2-expressing cells (e.g., Calu-3 cells); No significant binding to MDCK / Tet-on cells that do not express ACE2 and / or TMPRSS2; limiting the spread of coronavirus infection (e.g., by SARS-CoV-2, SARS-CoV, and / or MERS-CoV) in cells, e.g., Calu-3, in vitro; and / or Mice engineered to express human TMPRSS2 and / or ACE2 proteins were treated with coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, if or MERS-CoV) (e.g., mice are infected with an otherwise lethal dose of the virus, optionally in combination with a second therapeutic agent). Protecting mice engineered to express human TMPRSS2 and / or ACE2 proteins from weight loss caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) (e.g., mice are infected with a dose of virus that would otherwise cause weight loss, optionally in combination with a second therapeutic agent).

[0090] A "neutralizing" or "antagonist" anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment, refers to a molecule that inhibits the activity of CoV-S to any detectable extent, e.g., inhibits the ability of CoV-S to be cleaved by a protease such as TMPRSS2 or to bind to a receptor, such as ACE2, that mediates viral entry into or replication in a host cell.

[0091] Table 1 shows the heavy chain or V chain sequences as described below. H (or a variant thereof) and light chain Or V L (or a variant thereof), or its CDR (CDR-H1( or a variant thereof), CDR-H2 (or a variant thereof), and CDR-H3 (or a variant thereof) H and their CDRs (CDR-L1 (or variants thereof) ), CDR-L2 (or a variant thereof), and CDR-L3 (or a variant thereof) L refers to antigen-binding proteins such as antibodies and antigen-binding fragments thereof, including, for example, , immunoglobulin chains, variable regions, and / or CDRs comprise specific amino acid sequences as described below.

[0092] The antibodies described herein also include V H is wild-type IgG4 (e.g., residue 108 is S) ) or an IgG4 variant (e.g., residue 108 is P).

[0093] The antibodies and antigen-binding fragments of the present invention include immunoglobulin chains comprising the amino acid sequences described herein, as well as cellular and in vitro post-translational modifications to the antibodies. For example, the present invention includes antibodies and antigen-binding fragments thereof that specifically bind to CoV-S comprising the heavy and / or light chain amino acid sequences described herein (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3), as well as antibodies and fragments in which one or more amino acid residues are glycosylated, antibodies and fragments in which one or more Asn residues are deamidated, antibodies and fragments in which one or more residues (e.g., Met, Trp, and / or His) are oxidized, antibodies and fragments in which the N-terminal Gln is pyroglutamate (pyroE), and / or antibodies and fragments lacking the C-terminal lysine.

[0094] The amino acid and nucleotide sequences of exemplary anti-SARS-CoV-2 spike protein (SARS-CoV-2-S) antibodies are shown in the table of exemplary sequences below. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8] [Table 1-9]

[0095] Antibody administration The invention provides methods for administering an anti-CoV-S antigen binding protein of the invention, e.g., those in Table 1, comprising introducing the antigen binding protein into the body of a subject (e.g., a human). For example, the method comprises puncturing the subject's body with a syringe needle and injecting the antigen binding protein into the subject's body, e.g., into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0096] The invention provides a container (e.g., a plastic or glass vial with a cap or chromatography column, hollow bore needle, or syringe cylinder) containing an anti-CoV-S antigen binding protein of the invention, e.g., those in Table 1.

[0097] The present invention also provides an injection device comprising one or more antigen-binding proteins (e.g., antibodies or antigen-binding fragments) that specifically bind to CoV-S, e.g., those in Table 1, or a pharmaceutical composition thereof. The injection device may be packaged in a kit. An injection device is a device that introduces a substance into a subject's body via a parenteral route, e.g., intramuscularly, subcutaneously, or intravenously. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an auto-injector) that includes, for example, a cylinder or barrel for holding the fluid to be injected (e.g., comprising an antibody or fragment or pharmaceutical composition thereof), a needle for suturing the skin and / or blood vessel to inject the fluid, and a plunger for forcing the fluid out of the cylinder and through the bore of the needle. In one embodiment of the present invention, the injection device comprising an antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof, from a combination of the present invention, or a pharmaceutical composition thereof, is an intravenous (IV) injection device. Such a device may include an antigen-binding protein or pharmaceutical composition thereof within a cannula or trocar / needle that may be attached to tubing that may be attached to a bag or reservoir for holding fluid (e.g., saline) introduced into the subject's body through the cannula or trocar / needle. In one embodiment of the present invention, the antibody or fragment thereof or pharmaceutical composition thereof may be introduced into the device once the trocar and cannula are inserted into a subject's vein and the trocar is removed from the inserted cannula. IV devices can be inserted, for example, into a peripheral vein (e.g., hand or arm), the superior or inferior vena cava, or into the right atrium of the heart (e.g., central IV), or into the subclavian, internal jugular, or femoral vein, and advanced toward the heart until, for example, they reach the superior vena cava or right atrium (e.g., central venous line). In one embodiment of the present invention, the injection device is an auto-injector, jet injector, or external infusion pump. Jet injectors use a high-pressure, narrow jet of liquid to penetrate the epidermis. The antibody or fragment or pharmaceutical composition thereof is introduced into the subject's body via an external infusion pump. An external infusion pump is a medical device that delivers controlled amounts of an antibody or fragment or pharmaceutical composition thereof into a subject's body. External infusion pumps can be electrically or mechanically powered. Various pumps operate in different ways; for example, syringe pumps hold fluid in a syringe reservoir and a movable piston controls the fluid delivery, while elastomeric pumps hold fluid in a stretchable balloon reservoir and pressure from the balloon's elastic walls facilitates the fluid delivery. In peristaltic pumps, a set of rollers pinches a length of flexible tubing, forcing the liquid forward. Multichannel pumps can deliver fluid from multiple reservoirs at multiple rates.

[0098] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method can be used in the context of the present invention to generate human antibodies that specifically bind to CoV-S. To generate antibodies against CoV-S, an immunogen comprising any one of the following can be used: In certain embodiments of the present invention, the antibodies of the present invention are obtained from mice immunized with full-length native CoV-S, or with live-attenuated or inactivated virus, or with DNA encoding the protein or a fragment thereof. Alternatively, a CoV-S protein or a fragment thereof can be produced, modified, and used as an immunogen using standard biochemical techniques. In one embodiment of the present invention, the immunogen is a recombinantly produced CoV-S protein or a fragment thereof. In certain embodiments of the present invention, the immunogen can be a CoV-S polypeptide vaccine. In certain embodiments, one or more booster injections can be administered. In certain embodiments, the immunogen can be a recombinant CoV-S polypeptide expressed in E. coli or any other eukaryotic or mammalian cell, such as Chinese hamster ovary (CHO) cells.

[0099] Using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for producing monoclonal antibodies, high-affinity chimeric antibodies against CoV-S with human variable regions and mouse constant regions can first be isolated. VELOCIMMUNE® technology involves the generation of transgenic mice whose genomes contain human heavy chain variable regions and human light chain variable regions operably linked to endogenous mouse constant region loci, such that the mice produce antibodies containing the human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0100] Generally, VELOCIMMUNE® mice are challenged with an antigen of interest, and lymphocytes (such as B cells) are collected from the mice that express antibodies. Lymphocytes can be fused with myeloma cell lines to prepare immortalized hybridoma cell lines, which are then screened and selected to identify hybridoma cell lines that produce antibodies specific to the antigen of interest. DNA encoding the heavy and light chain variable regions can be isolated and linked to the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the light and heavy chain variable domains can be isolated directly from antigen-specific lymphocytes.

[0101] First, high-affinity chimeric antibodies with human variable regions and mouse constant regions are isolated. As in the experimental section below, the antibodies are then purified to obtain the desired characteristics, including affinity, selectivity, epitope, etc. The mouse constant regions are characterized and selected for their specificity. The mouse constant regions are replaced with the desired human constant regions to generate fully human antibodies of the invention, e.g., wild-type or modified IgG1 or IgG4. While the constant region selected can vary depending on the specific application, the high affinity antigen-binding and target specificity characteristics reside in the variable regions.

[0102] Anti-coronavirus spike protein antibodies containing Fc variants According to certain embodiments of the invention, there are provided anti-CoV-S antigen binding proteins, e.g., antibodies or antigen-binding fragments, comprising an Fc domain comprising one or more mutations that enhance or decrease antibody binding to, e.g., the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides an Fc domain comprising one or more mutations that enhance or decrease antibody binding to, e.g., the FcRn receptor, e.g., at acidic pH compared to neutral pH. H 2 or C HThe present invention also includes anti-CoV-S antibodies containing mutations in the 3 region, where the mutation(s) increase the affinity of the Fc domain for FcRn in acidic environments (e.g., endosomes with a pH ranging from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), 307 and / or 308 modifications (e.g., 308F or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0103] For example, the present invention provides 250Q and 248L (e.g., T250Q and M248L), 252Y, 254T, and 256E (e.g., M252Y, S254T, and T256E), 428L and 434S (e.g., M428L and N434S), 257I and 311I (e.g., P257I and Q311I), 257I and 434H (e.g., P257I and N434H), 376V and 434H (e.g., D376V and N434H), 307A, 380A, and 434A (e.g., T307A, E380A, and N434A), and 433K and 434F (e.g., H433K and N434F).

[0104] The VV ... H and / or V L Anti-CoV-S antigen-binding proteins, such as antibodies and Antigen-binding fragments are contemplated within the scope of the present invention.

[0105] The present invention also provides a method for treating a V H and chimeric heavy chain constant (C H ) region and an anti-CoV-S antigen binding protein, antibody, or antigen binding fragment, comprising a chimeric C H The area is 1 C of more than two immunoglobulin isotypes H This includes segments derived from the The antibodies of the invention are C-type antibodies derived from human IgG1 molecules, human IgG2 molecules, or human IgG4 molecules. H Human IgG1 molecules, human IgG2 molecules, combined with some or all of the three domains molecule, or C derived from human IgG4 molecules H 2. A website containing part or all of the domain La C HAccording to certain embodiments, the antibodies of the present invention may comprise a chimeric hinge region. Chimera C H For example, the chimeric hinge may include a human IgG1 hinge region, a human The chimeric hinge region may comprise an "upper hinge" amino acid sequence derived from a human IgG1 hinge region, a human IgG2 hinge region, or a human IgG4 hinge region (amino acid residues at positions 216-227 according to EU numbering) combined with a "lower hinge" sequence derived from a human IgG2 hinge region, or a human IgG4 hinge region (amino acid residues at positions 228-236 according to EU numbering). According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 or human IgG4 upper hinge and amino acid residues derived from a human IgG2 lower hinge. Chimeric C as described herein may be used in combination with a human IgG1 or human IgG4 hinge region. H Antibodies comprising the region are in particular embodiments In some embodiments, modified Fc effector functions can be exhibited without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., WO2014 / 022540).

[0106] immunoconjugate The present invention encompasses anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments, conjugated to another moiety, e.g., a therapeutic moiety ("immunoconjugate"), such as a toxoid, or an antiviral drug for treating influenza virus infection. In embodiments of the invention, the anti-CoV-S antibody or fragment is conjugated to any of the additional therapeutic agents described herein. As used herein, the term "immunoconjugate" refers to an antigen-binding protein, e.g., an antibody or antigen-binding fragment, chemically or biologically linked to a radioactive substance, cytokine, interferon, targeting or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antigen-binding protein may be linked to the radioactive substance, cytokine, interferon, targeting or reporter moiety, enzyme, peptide, or therapeutic agent at any position along the molecule, as long as it is capable of binding to its target (CoV-S). Examples of immunoconjugates include antibody-drug conjugates and antibody-toxin fusion proteins. In one embodiment of the present invention, the drug may be a second, different antibody that specifically binds to CoV-S. The type of therapeutic moiety that can be conjugated to an anti-CoV-S antigen-binding protein (e.g., an antibody or fragment) will depend on the condition to be treated and the desired therapeutic effect to be achieved. See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy," Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery," Controlled Drug Delivery Delivery(2 ndED.), Robinson et al. (eds.), pp. 623-53 (Marcel Dekker, Inc. 1987), Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, Monoclonal Antibodies 1984: Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985), “Analysis, Results, And Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, Monoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al. (eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “The Preparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immun See Vol. Rev., 62:119-58 (1982).

[0107] multispecific antibodies The present invention includes anti-CoV-S antigen binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as methods of use and methods of making such antigen binding proteins. The term "anti-CoV-S" antigen binding protein, e.g., antibody or antigen-binding fragment, includes multispecific (e.g., bispecific or biparatopic) molecules comprising at least one first antigen-binding domain that specifically binds to CoV-S (e.g., an antigen-binding domain from an antibody in Table 1) and at least one second antigen-binding domain that binds to a different antigen or epitope of CoV-S that is different from that of the first antigen-binding domain. In some embodiments, the first antigen-binding domain and the second antigen-binding domain are both selected from the antigen-binding domains in Table 1. In an embodiment of the invention, the first and second epitopes overlap. In another embodiment of the invention, the first and second epitopes do not overlap. For example, in embodiments of the invention, the multispecific antibody is a bispecific IgG antibody (e.g., IgG1 or IgG4) comprising a first antigen-binding domain that specifically binds to CoV-S, comprising the heavy and light immunoglobulin chains of an antibody of Table 1, and a second antigen-binding domain that specifically binds to a different epitope on CoV-S. In some embodiments, the bispecific IgG antibody (e.g., IgG1 or IgG4) comprises a first antigen-binding domain that specifically binds to CoV-S and a second binding domain that binds to a host cell protein, e.g., ACE2 or TMPRSS2.

[0108] The antibodies in Table 1 each have a CDR-H and CDR-L, V, H , and V L or a multi-specific amino acid sequence comprising an HC and an LC (including variants thereof as described herein). Includes heteromeric molecules, for example, antibodies or antigen-binding fragments.

[0109] In an embodiment of the invention, an antigen-binding domain that specifically binds to CoV-S that may be comprised in a multispecific molecule is (1) (i) a heavy chain variable domain sequence comprising the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences set forth in Table 1; and (ii) a light chain variable domain sequence comprising the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences set forth in Table 1; or (2) (i) a heavy chain variable domain sequence comprising an amino acid sequence set forth in Table 1; and (ii) a light chain variable domain sequence comprising an amino acid sequence set forth in Table 1; or (3) (i) a heavy chain immunoglobulin sequence comprising an amino acid sequence set forth in Table 1, and (ii) comprises a light chain immunoglobulin sequence comprising an amino acid sequence set forth in Table 1.

[0110] In an embodiment of the invention, the multispecific antibody or fragment comprises more than two different binding specificities (e.g., a trispecific molecule), e.g., one or more additional antigen-binding domains that are the same as or different from the first and / or second antigen-binding domain.

[0111] In one embodiment of the present invention, the bispecific antigen-binding fragment comprises a first scFv (e.g., V in Table 1) that has binding specificity for a first epitope (e.g., CoV-S). H and V L and a second sc having binding specificity for a second, different epitope. For example, in an embodiment of the invention, the first and second scFvs may be linked via a linker, e.g., a peptide linker (e.g., (GGGGS) n G such as (SEQ ID NO: 834) S linker), where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Other bispecific antigen-binding fragments include a F(ab)2 of a bispecific IgG antibody comprising the heavy and light chain CDRs of Table 1 and another antibody that binds to a different epitope. F(ab)2 of

[0112] Treatment method The present invention provides methods for treating or preventing a viral infection (e.g., a coronavirus infection) by administering a therapeutically effective amount of an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment (e.g., of Table 1), to a subject (e.g., a human) in need of such treatment or prevention.

[0113] Coronavirus infection can be treated or prevented in a subject by administering to the subject an anti-CoV-S antigen binding protein of the invention.

[0114] An effective or therapeutically effective amount of an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment (e.g., of Table 1), for treating or preventing a viral infection refers to the amount of antibody or fragment sufficient to alleviate one or more signs and / or symptoms of infection in a treated subject, whether by inducing regression or elimination of such signs and / or symptoms or by inhibiting the progression of such signs and / or symptoms. The amount administered may vary depending on the age and size of the subject, the target disease or condition, the route of administration, and the like. In an embodiment of the present invention, an effective or therapeutically effective amount of an antibody or antigen-binding fragment thereof of the present invention for treating or preventing a viral infection in, for example, an adult subject, is about 0.01 to about 200 mg / kg, e.g., up to 150 mg / kg. In one embodiment of the present invention, the dosage is up to about 10.8 or 11 grams (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 grams). The frequency and duration of treatment can be adjusted depending on the severity of the infection. In certain embodiments, the antigen-binding proteins of the present invention may be administered in an initial dose, followed by one or more secondary doses. In certain embodiments, the initial dose may be followed by administration of a second or multiple subsequent doses of the antibody or antigen-binding fragment thereof, in an amount that may be about the same as or less than the initial dose, with the subsequent doses being separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0115] As used herein, the term "subject" refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, in need of prevention and / or treatment of a disease or disorder, such as a viral infection or cancer. The subject may be suffering from or predisposed to developing a viral infection, such as influenza infection. Subjects predisposed to developing an infection or who may be at high risk of contracting an infection (e.g., coronavirus or influenza virus) include subjects with a weakened immune system due to an autoimmune disease, subjects undergoing immunosuppressive therapy (e.g., after an organ transplant), subjects suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), subjects with a form of anemia that depletes or destroys white blood cells, subjects undergoing radiation or chemotherapy, or subjects suffering from an inflammatory disorder. Furthermore, very young (e.g., under the age of 5) or elderly (e.g., over the age of 65) subjects are at high risk. Additionally, a subject may be at risk of contracting a viral infection due to proximity to a disease outbreak, for example, the subject lives in a densely populated city or in close proximity to a subject with confirmed or suspected viral infection, or employment choices, e.g., hospital workers, pharmaceutical researchers, travelers to, or frequent visitors to, affected areas.

[0116] "Treat" or "treating" refers to a subject having one or more signs or symptoms of a disease or infection, e.g., a viral infection, against which an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment of the invention (e.g., of Table 1), is effective when administered to the subject in an effective or therapeutically effective amount or dose (as discussed herein).

[0117] The present invention also encompasses the prophylactic administration of an anti-CoV-S antigen-binding protein, e.g., an antibody of the invention or an antigen-binding fragment thereof (e.g., of Table 1), to a subject at risk for viral infection to prevent such infection. Passive antibody-based immunoprophylaxis has proven to be an effective strategy for preventing subjects from acquiring viral infection. See, e.g., Berry et al., Passive broad-spectrum influenza immunoprophylaxis. Influenza Res. Treat.2014;2014:267594.Epub 2014 Sep 22, and Jianqiang et al., Passive immune neutralization strategies for prevention and See, control of influenza A infections, Immunotherapy. 2012 February;4(2):175-186; Prabhu et al., Antivir Ther. 2009;14(7):911-21, Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 hemagglutinin against lethal H5N1 influenza. "Prevent" or "preventing" means administering to a subject an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment of the invention (e.g., of Table 1), to inhibit symptoms of disease or infection (e.g., viral infection) in a subject, where the antigen binding protein is effective when administered to a subject in an amount or dose effective or therapeutically effective to do so (as discussed herein).

[0118] In one embodiment of the present invention, the sign or symptom of a viral infection in a subject is the survival or proliferation of the virus in the subject's body, for example, as determined by a viral titer assay (e.g., coronavirus growth in embryonated chicken eggs or a coronavirus spike protein assay). Other signs and symptoms of viral infection are discussed herein.

[0119] As noted above, in some embodiments, the subject may be a non-human animal, and the antigen-binding proteins (e.g., antibodies and antigen-binding fragments) discussed herein may be used in the animal context to treat and / or prevent disease in non-human animals (e.g., cats, dogs, pigs, cows, horses, goats, rabbits, sheep, etc.).

[0120] The present invention provides a method for treating or preventing a viral infection (e.g., a coronavirus infection) by administering a therapeutically effective amount of an anti-CoV-S antigen binding protein (e.g., of Table 1) to a subject, e.g., by injecting the protein into the subject's body, or for inducing regression or elimination of, or inhibiting the progression of, at least one sign or symptom of a viral infection (wherein the sign or symptom is secondary to the viral infection), such as: Fever or chills, ·cough, ·sore throat, runny or stuffy nose, ·sneeze, Muscle or body pain, ·headache, fatigue, ·vomiting, ·diarrhea, Respiratory tract infections, Chest discomfort, ·Difficulty breathing, bronchitis, and / or ·pneumonia, Methods are provided for doing so in a subject (eg, a human) in need thereof.

[0121] Combinations and Pharmaceutical Compositions To prepare pharmaceutical compositions of anti-CoV-S antigen-binding proteins, e.g., antibodies and antigen-binding fragments thereof (e.g., of Table 1), the antigen-binding proteins are mixed with a pharmaceutically acceptable carrier or excipient. See, e.g., Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984); Hardman, et al. (2001); Goodman, et al. (2002). and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY, Gennaro (2000) Remington: The Science and See Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. In an embodiment of the invention, the pharmaceutical composition is sterile. Such compositions are part of the present invention.

[0122] The scope of the present invention includes dried, e.g., lyophilized, compositions comprising anti-CoV-S antigen-binding proteins, e.g., antibodies or antigen-binding fragments thereof (e.g., of Table 1), or pharmaceutical compositions thereof (comprising a pharmaceutically acceptable carrier but substantially devoid of water).

[0123] In a further embodiment of the invention, the additional therapeutic agent administered to the subject, which is related to the anti-CoV-S antigen binding proteins disclosed herein, e.g., antibodies or antigen-binding fragments thereof (e.g., of Table 1), is selected from the group consisting of antibodies, antibodies, and antibodies described in Physicians' Desk Reference 2003 (Thomson Healthcare; 57 th The test is administered to the subject according to the following standard:

[0124] The method of administration can be varied and includes oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intra-arterial.

[0125] The present invention provides anti-CoV-S antigen binding proteins, such as antibodies or antigen-binding fragments thereof (e.g., For example, methods for administering an antigen-binding protein (e.g., in Table 1) are provided, which include introducing the protein into the body of a subject. For example, the method includes puncturing the subject's body with a syringe needle and injecting the antigen-binding protein into the subject's body, for example, into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0126] The present invention provides anti-CoV-S antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof (e.g., of Table 1), polypeptides (e.g., HC, LC, V, and V in Table 1). H , or V L ), or a polynucleotide (e.g., of Table 2), or a vector described herein, or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier, is provided.

[0127] In one embodiment of the present disclosure, an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof of the present invention (e.g., of Table 1), is administered in conjunction with one or more additional therapeutic agents. The additional therapeutic agents include, but are not limited to, an anti-inflammatory agent, an anti-malarial agent, a secondary antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and a secondary antibody or antigen-binding fragment thereof that specifically binds to CoV-S. In some embodiments, the anti-malarial agent is chloroquine or hydroxychloroquine. In some embodiments, the anti-inflammatory agent is an antibody such as sarilumab, tocilizumab, or gimsilumab. In some embodiments, the additional therapeutic agent is a secondary antibody or antigen-binding fragment disclosed herein, e.g., of Table 1. In certain embodiments, one, two, three, four, or more antibodies or antigen-binding fragments thereof of Table 1 can be administered in combination (e.g., simultaneously or sequentially). Specific combinations of antibodies in Table 1 are set forth in the table of exemplary antibody combinations below (e.g., each number representing a specific combination is mAb10989 and mAb10987 as combination 1, mAb10989 and mAb10934 as combination 2, etc.). In some embodiments, antibody combinations may be selected from those that bind to different epitope clusters. For example, certain antibodies described herein belong to the following epitope clusters: Cluster 1, mAb10987, mAb10922, mAb10936, and mAb10934; Cluster 2, mAb10989, mAb10977, and mAb10933; Cluster 3, mAb10920; Cluster 4, mAb10954, mAb10986, and mAb10964; and Cluster 5, mAb10984. Thus, combinations of two antibodies may be selected from, for example, cluster 1 and cluster 2, cluster 1 and cluster 3, cluster 1 and cluster 4, cluster 1 and cluster 5, cluster 2 and cluster 3, cluster 2 and cluster 4, cluster 2 and cluster 5, cluster 3 and cluster 4, cluster 3 and cluster 5, and cluster 4 and cluster 5.In some embodiments, the antibody that specifically binds to TMPRSS2 is H1H7017N, as described in International Patent Publication No. WO / 2019 / 147831. [Table 2]

[0128] In some embodiments, anti-CoV-S antigen-binding proteins (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) from different human donors can be combined. The present invention includes compositions comprising two (or more) anti-SARS-CoV-2-S antibodies or antigen-binding fragments comprising variable domains from human subjects, where the two (or more) antibodies or antigen-binding fragments are derived from different subjects (e.g., two different human subjects). Antibody variable regions derived from human B cells are discussed, for example, in Examples 1 and 2 (Table 3), and variable domains cloned from such B cells are combined with constant regions not from those B cells to generate hybrid antibodies. The sources (donors) of such antibody variable regions are shown in the table of exemplary human-derived antibody variable regions below. In some embodiments, a composition can include a combination of an antibody or antigen-binding fragment having a variable domain derived from donor 1 with an antibody or antigen-binding fragment having a variable domain derived from donor 2. In some embodiments, a composition can include a combination of an antibody or antigen-binding fragment having a variable domain derived from donor 1 with an antibody or antigen-binding fragment having a variable domain derived from donor 3. In some embodiments, a composition may comprise a combination of an antibody or antigen-binding fragment thereof having a variable domain from donor 2 and an antibody or antigen-binding fragment thereof having a variable domain from donor 3. In some embodiments, a composition may comprise a combination of mAb10987 from donor 1 (e.g., an antibody comprising the CDRs, variable regions, or heavy and light chain sequences shown in Table 1) and mAb10989 from donor 3 (e.g., an antibody comprising the CDRs, variable regions, or heavy and light chain sequences shown in Table 1). [Table 3]

[0129] In some embodiments, the additional therapeutic agent is an antiviral agent and / or a vaccine. As used herein, the term "antiviral agent" refers to any anti-infective drug or therapy used to treat, prevent, or ameliorate a viral infection in a subject. The term "antiviral agent" includes, but is not limited to, cationic steroid antibacterial agents, leupeptin, aprotinin, ribavirin, or interferon alpha 2b. Methods for treating or preventing a viral (e.g., coronavirus) infection in a subject in need of such treatment or prevention by administering an antibody or antigen-binding fragment of Table 1 in combination with an additional therapeutic agent are part of the present invention.

[0130] For example, in one embodiment of the invention, the additional therapeutic agent is a vaccine, e.g., a coronavirus vaccine. In one embodiment of the invention, the vaccine is an inactivated / killed virus vaccine, a live attenuated virus vaccine, or a viral subunit vaccine.

[0131] For example, in one embodiment of the present invention, the additional therapeutic agent is: [ka] [ka] See Shen et al. Biochimie 142:1-10 (2017).

[0132] In one embodiment of the present invention, the antiviral agent is an antibody or antigen-binding fragment that specifically binds to a coronavirus, e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV. Exemplary anti-CoV-S antibodies include H4sH15188P, H1H15188P, H1H15211P, H1H15177P, H4sH15211P, H1H15260P2, H1H15259P2, H1H15203P, H4sH15260P2, H4sH15231P2, H1H15237P2, H1H15208P ...4sH15231P2, H1H15237P2, H1H15208P, H4sH15231P2, H4sH15231P2, H4sH15231P2, H4sH15237P2, H4sH15208P, H4sH15231P2, H For example, the antibody or fragment includes, but is not limited to, a light chain immunoglobulin (e.g., V) comprising CDR-L1, CDR-L2, and CDR-L3 of any of the aforementioned anti-CoV-S antibodies. L or its light chain), and CDR-H1, CDR-H2, and and a heavy chain (e.g., V H or its heavy chain).

[0133] In certain embodiments of the invention, the additional therapeutic agent is not aprotinin, leupeptin, a cationic steroid antibacterial agent, an influenza vaccine (e.g., a killed, live, attenuated whole virus or subunit vaccine), or an antibody to influenza virus (e.g., an antihemagglutinin antibody).

[0134] The term "in connection with" refers to a component, an anti-CoV-S antigen-binding protein, e.g. It is noted that the antibodies or antigen-binding fragments thereof of the present invention can be formulated with another agent, e.g., in a single composition for simultaneous delivery, or can be formulated separately into two or more compositions (e.g., a kit). Each component can be administered to a subject at a time different from when the other components are administered; for example, each administration may be given non-concurrently, spaced apart over a given period of time (e.g., separately or sequentially). Furthermore, the separate components can be administered to a subject by the same route or by different routes (e.g., an anti-CoV-S antibody or antibody-binding fragment thereof).

[0135] kit Further provided are kits comprising one or more components including, but not limited to, an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment as discussed herein (e.g., Table 1), in association with one or more additional components, including, but not limited to, an additional therapeutic agent, as discussed herein. The antigen binding protein and / or additional therapeutic agent can be formulated in a pharmaceutical composition, as a single composition, or separately in two or more compositions, e.g., together with a pharmaceutically acceptable carrier.

[0136] In one embodiment of the invention, the kit comprises an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment thereof of the invention (e.g., of Table 1), or a pharmaceutical composition thereof, in one container (e.g., in a sterile glass or plastic vial) and an additional therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0137] In another embodiment, the kit comprises an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment thereof of the invention (e.g., of Table 1), or a pharmaceutical composition thereof, in a single common container, optionally in combination with one or more additional therapeutic agents formulated together in a pharmaceutical composition.

[0138] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device (e.g., an injection device) for performing such administration. For example, the kit can include one or more hypodermic needles or other injection devices described above that include an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment thereof of the invention (e.g., of Table 1).

[0139] The kit may include a package insert containing information about the pharmaceutical compositions and dosage forms in the kit. Generally, such information will assist patients and physicians in effectively and safely using the enclosed pharmaceutical compositions and dosage forms. For example, the following information about the combination of the present invention may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, side effects, overdosage, proper dosage and administration, methods provided, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0140] Diagnostic Uses of Antibodies Anti-CoV-S antigen binding proteins, such as antibodies of the invention or antigen-binding fragments thereof (e.g., of Table 1), can be used to detect and / or measure CoV-S in a sample. An exemplary assay for CoV-S can include, for example, contacting a sample with an anti-CoV-S antigen binding protein of the invention, where the anti-CoV-S antigen binding protein is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CoV-S from the sample. The presence of the anti-CoV-S antigen binding protein complexed with CoV-S indicates the presence of CoV-S in the sample. Alternatively, an unlabeled anti-CoV-S antibody can be used in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be 3 H, 14 C. 32 P, 35 S, or 125 Radioactive isotopes such as I, The antigen-binding protein may be a fluorescent or chemiluminescent moiety, such as fluorescein isothiocyanate or rhodamine, or an enzyme, such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CoV-S in a sample include neutralization assays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence-activated cell sorting (FACS). Accordingly, the present invention includes methods for detecting the presence of spike protein polypeptides in a sample, comprising contacting the sample with an anti-CoV-S antigen-binding protein and detecting the presence of the CoV-S / anti-CoV-S antigen-binding protein, wherein the presence of a complex indicates the presence of CoV-S.

[0141] The anti-CoV-S antigen binding proteins of the invention (e.g., of Table 1) can be used in Western blot or immunoprotein blot procedures to detect the presence of CoV-S or its fragments in a sample. Such procedures form part of the present invention and include, for example, the following steps: (1) Providing a membrane or other solid substrate containing a sample to be tested for the presence of CoV-S, e.g., optionally transferring proteins from the sample to be tested for the presence of CoV-S (e.g., from PAGE or SDS-PAGE electrophoretic separation of proteins in the sample) to the membrane or other solid substrate using methods known in the art (e.g., semi-dry blotting or tank blotting), and contacting the membrane or other solid substrate to be tested for the presence of CoV-S or a fragment thereof with an anti-CoV-S antigen binding protein of the invention.

[0142] Such membranes can take the form of, for example, nitrocellulose or vinyl-based (e.g., polypolyylidene fluoride (PVDF)) membranes onto which proteins to be tested for the presence of CoV-S have been transferred (e.g., after electrophoretic separation within the gel) in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. Prior to contacting the membrane with the anti-CoV-S antigen-binding protein, the membrane is optionally blocked with, for example, non-fat dry milk, to bind to non-specific protein-binding sites on the membrane. (2) washing the membrane one or more times to remove unbound anti-CoV-S antigen-binding protein and other unbound substances; and (3) Detecting bound anti-CoV-S antigen-binding proteins.

[0143] Detection of bound antigen-binding protein indicates that CoV-S protein is present on the membrane or substrate and in the sample. Detection of bound antigen-binding protein can be by binding the antigen-binding protein with a detectably labeled secondary antibody (anti-immunoglobulin antibody) and then detecting the presence of the secondary antibody label.

[0144] The anti-CoV-S antigen binding proteins (e.g., antibodies and antigen-binding fragments (e.g., of Table 1)) disclosed herein can also be used for immunohistochemistry. Such methods form part of the invention and include, for example, the following: (1) contacting a tissue to be tested for the presence of a CoV-S protein with an anti-CoV-S antigen binding protein of the invention; and (2) detecting antigen-binding proteins on or within tissues;

[0145] If the antigen binding protein itself is detectably labeled, it can be detected directly. Alternatively, the antigen binding protein can be bound by a detectably labeled secondary antibody, and the label then detected. [Example]

[0146] The following examples are presented to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric pressure.

[0147] Example 1: Generation of human antibodies against the SARS-CoV-2 spike protein (SARS-CoV-2-S) Human antibodies against the SARS-CoV-2 spike protein (SARS-CoV-2-S) were generated in VELOCIMMUNE® mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions or human immunoglobulin heavy and lambda light chain variable regions. Each mouse was immunized with a vector expressing the SARS-CoV-2-S receptor-binding domain (RBD) (amino acids 1-1273 of NCBI accession number (MN908947.3), SEQ ID NO: 832) followed by SARS-CoV-2-S vector or SARS-CoV-2-S protein. Antibody immune responses were monitored by SARS-CoV-2-S-specific immunoassays. When the desired immune response was achieved, splenocytes were harvested and fused with mouse myeloma cells to maintain their viability and form hybridoma cell lines. Hybridoma cell lines were screened and selected to identify cell lines producing SARS-CoV-2-S-specific antibodies. As described in U.S. Patent No. 7,582,298 (specifically incorporated herein by reference in its entirety), anti-SARS-CoV-2-S antibodies were isolated directly from antigen-positive mouse B cells without fusion to myeloma cells. Using this method, fully human anti-SARS-CoV-2-S antibodies (i.e., antibodies with human variable and constant domains) were obtained.

[0148] Antibody variable regions were also isolated from human blood samples. Whole blood was collected from patients 3–4 weeks after a laboratory-confirmed PCR-positive test for SARS-CoV-2 and symptomatic COVID-19 disease. Red blood cells were lysed using an ammonium chloride-based lysis buffer (Life Technologies), and B cells were enriched by negative selection. Single B cells that bound the SARS-CoV-2 spike protein were isolated by fluorescence-activated cell sorting (FACS). The isolated B cells were plated in a single well and mixed with antibody light and heavy variable region-specific PCR primers. cDNA from each single B cell was synthesized via reverse transcriptase (RT) reaction. Each resulting RT product was then divided and transferred to two corresponding wells for subsequent antibody heavy and light chain PCR. One set of the resulting RT products was first amplified by PCR using a 5' degenerate primer specific to the antibody heavy variable region leader sequence or a 5' degenerate primer specific to the antibody light chain variable region leader sequence and a 3' primer specific to the antibody constant region to form amplicons. The amplicons were then amplified again by PCR using a 5' degenerate primer specific to antibody heavy variable region framework 1 or a 5' degenerate primer specific to antibody light chain variable region framework 1 and a 3' primer specific to the antibody constant region to generate amplicons for cloning. The PCR products derived from the antibody heavy and light chains were cloned into expression vectors containing the heavy and light constant regions, respectively, thereby generating hybrid antibody expression vectors. The expression vectors expressing the full-length heavy and light chain pairs were transfected into CHO cells to produce antibody proteins for testing.

[0149] The biological properties of exemplary antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0150] Example 2: Amino acid and nucleotide sequences of heavy and light chain variable regions Table 1 shows the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs, as well as the heavy and light chain sequences, of exemplary anti-SARS-CoV-2-S antibodies. The corresponding nucleic acid sequence identifiers are listed in Table 2. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

[0151] The antibodies disclosed herein have fully human variable regions, but can have mouse constant regions (e.g., mouse IgG1 Fc or mouse IgG2 Fc (a or b isotype)) or human constant regions (e.g., human IgG1 Fc or human IgG4 Fc). As will be recognized by those skilled in the art, an antibody with a particular Fc isotype can be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc can be converted to an antibody with a human IgG4, etc.), but in any event, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Tables 1 and 2 will remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar, regardless of the nature of the constant domains.

[0152] The variable regions of antibodies from VELOCIMMUNE® mouse and human samples were sequenced by next-generation sequencing, and the repertoires of heavy and light chain pairs were identified (Figures 10A and 10B). The major lineage of VI antibodies utilized VH3-53 paired with VK1-9, VK1-33, or VK1-39, while human-derived antibodies utilized VH3-66 paired with VK1-33 or VH2-70 paired with VK1-39. Further analysis of the overlaid sequences showed a strong overlap in the isolated kappa chain repertoire between VI and human-derived antibodies. The lambda chain repertoires did not overlap well, which may be due to the inclusion of only two lambda mice in this study. The average CDR lengths of the heavy chains were similar between VI and human-derived antibodies, with average lengths of 13 and 14.5 amino acids, respectively. The average kappa CDR length was identical for V1 and human-derived antibodies at 9 amino acids, and was close for lambda chains with average lengths of 11.1 and 10.6 amino acids, respectively. The availability of humanized mouse- and human-derived antibodies increased the diversity of V genes, allowing the subsequent identification of non-competing antibodies.

[0153] As noted above, antibodies were obtained from hybridomas generated from VELOCIMMUNE® mice, by direct isolation from antigen-positive VELOCIMMUNE® mouse B cells, or derived from variable regions cloned from antigen-positive human B cells. A summary of these sources is shown in Table 3. [Table 6-1] [Table 6-2]

[0154] Example 3: Characterization of hybridoma supernatants by binding ELISA An ELISA binding assay was performed to identify antibody supernatants that bound to the SARS-CoV-2 spike protein receptor-binding domain (RBD). A protein consisting of the SARS-CoV-2 RBD (amino acids 319-541) expressed with a 6x histidine tag and two C-terminal myc epitope tags (SARS-CoV-2-S-RBD-mmH, NCBI accession number MN908947.3) was coated onto a 96-well plate in PBS buffer at 1 μg / ml overnight at 4°C. Nonspecific binding sites were then blocked using a 0.5% (wt / vol) solution of BSA in PBS. Antibody supernatants or medium alone were diluted 1:40 or 1:50 in PSA + 0.5% BSA blocking buffer and transferred to the washed microtiter plate. After 1 hour of incubation at room temperature, the wells were washed, and plate-bound supernatant was detected with either a goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson Immunoresearch) or an anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson Immunoresearch). Plates were then developed using TMB substrate solution (BD Biosciences) according to the manufacturer's recommendations, and absorbance was measured at 450 nm using a VictorX5 plate reader.

[0155] The ability of anti-SARS-CoV-2-S antibodies to bind to the receptor-binding domain of SARS-CoV-2-S (SARS-CoV-2-S-RBD) was assessed using a binding ELISA with SARS-CoV-2-S-RBD-mmH protein coated onto microplates, as described above. Single-point antibody supernatant binding to SARS-CoV-2-S-RBD-mmH coated onto 96-well microtiter plates was detected with HRP-conjugated anti-hFc or anti-mFc antibodies.

[0156] The binding results for the three studies are summarized in Table 4. SARS-CoV-2 binding signals (absorbance 450 nm) are shown, with a media-only background provided as a negative reference for each experiment. Samples marked IC (indeterminate) are reported without a value due to experimental anomalies on the plate. The tested supernatants demonstrated substantial binding to SARS-CoV-2 S-RBD, as shown compared to the media-only control. [Table 7-1] [Table 7-2]

[0157] Example 4: Antibodies that bind to virus-like particles expressing SARS-CoV-2-S To investigate the ability of a panel of anti-SARS-CoV-2-S monoclonal antibodies to bind to the SARS-CoV-2 spike glycoprotein, an in vitro binding assay was developed utilizing virus-like particles (VLPs) expressing the SARS-CoV-2 spike protein on an electrochemiluminescence-based detection platform (MSD).

[0158] To transiently express the SARS-CoV-2 spike protein (NCBI accession number MN908947.3, amino acids 16-1211, SEQ ID NO: 833), vesicular stomatitis virus (VSV) lacking glycoprotein G (VSV delta G) was pseudotyped with the SARS-CoV-2 spike protein (VSV-SARS-CoV-2-S) and produced in HEK293T cells. As a negative binding control, VSV delta G was pseudotyped with the VSV G protein (VSV-G).

[0159] The experiment was performed as follows: The two types of VLPs described above were diluted in PBS and seeded onto a 96-well carbon electrode plate (Multi-Array High Bind Plate, MSD) and incubated overnight at 4°C to allow VLP adhesion. Nonspecific binding sites were blocked with 2% (wt / vol) BSA in PBS for 1 hour at room temperature. Antibody-containing supernatants generated from SARS-CoV-2-immune or infected human sera were added to the plate-bound particles, along with a medium-only control diluted 1:10 or 1:20 in 1x PBS + 0.5% BSA buffer. The plate was then incubated with shaking for 1 hour at room temperature, after which the plate was washed with 1x PBS and unbound antibody was removed using an AquaMax2000 plate washer (MDS Analytical Technologies). Plate-bound antibodies were detected for 1 hour at room temperature using SULFO-TAG™-conjugated anti-human IgG antibodies (Jackson Immunoresearch) or SULFO-TAG™-conjugated anti-mouse IgG antibodies (Jackson Immunoresearch). After washing, plates were developed with read buffer (MSD) according to the manufacturer's recommended procedure, and luminescence signals were recorded on a SECTOR Imager 600 (Meso Scale Development) instrument. Direct binding signals (in RLU) were captured, and the ratio of SARS-CoV-2-S-expressing VLPs to irrelevant VLPs was calculated.

[0160] The ability of anti-SARS-CoV-2-S monoclonal antibodies to bind SARS-CoV-2-S-expressed VLPs compared to binding to irrelevant VSV-expressed VLPs was assessed using an immunobinding assay as described above. Single-point binding to VLPs immobilized on 96-well High Bind plates (MSD) was assessed using antibody supernatant dilutions of 1:10 or 1:20. The VLPs were run at 4°C, allowed to bind for 1 hour, and detected using SULFO-TAG™-conjugated anti-human IgG or anti-mouse IgG antibodies. The binding signal from electrochemiluminescence was recorded on a Sector Imager 600 (MSD). The RLU values ​​of antibody binding to the VLPs were determined. The ratio was calculated by comparing the binding signal of SARS-CoV-2-S-expressing VLPs to that of control VLPs.

[0161] The binding results from three experiments are summarized in Table 5. The signal observed from SARS-CoV-2-S-expressing VLPs indicates binding, and comparison with the negative VLPs provides a relative background. The medium-only sample provides a baseline signal for secondary antibody binding to samples without supernatant. 46 antibodies specifically bound SARS-CoV-2-S-expressing VLPs more than fourfold higher than the medium-only samples (20–35 RLU), exhibiting binding signals ranging from 85 to 13,600 RLU. The ratios of SARS-CoV-2-S-expressing VSV:VSV-VLPs (negative control) ranged from 1.1 to 22.7, with many exhibiting high background levels for VSV-VLPs. The ratio of 0.9 for mAb 11002 may be due to the low concentration of monoclonal antibody in the supernatant samples. [Table 8-1] [Table 8-2] [Table 8-3]

[0162] Example 5: Antibody Neutralization of VSV-SARS-CoV-2-S Pseudovirus Infectivity To investigate the ability of a panel of anti-SARS-CoV-2-S monoclonal antibodies to neutralize SARS-CoV-2, an in vitro neutralization assay utilizing a VSV-SARS-CoV-2-S pseudovirus was developed.

[0163] VSV pseudotyped viruses were generated by transiently transfecting 293T cells with a plasmid encoding the SARS-CoV-2 spike protein, as described above. Cells were plated at 1.2 × 10 cells per plate in DMEM complete medium 1 day before transfection with 15 μg / plate of spike protein DNA using 125 μL of Lipofectamine LTX, 30 μL of PLUS Reagent, and up to 3 mL of Opti-Mem. 7 Cells were seeded in a 15 cm plate. After 24 h, the cells were washed with 10 mL of PBS and resuspended in 0.1% VSVΔ in 10 mL of Opti-Mem. G:mNeon The virus was incubated on the cells for 1 hour. The cells were then overlaid with 20 mL of infection medium and washed three times with 10 mL of PBS before being incubated for 24 hours at 37°C, 5% CO2. The supernatant was collected on ice in a 250 mL centrifuge tube, centrifuged at 3000 rpm for 5 minutes to pellet any cellular debris, aliquoted on ice, and frozen at -80°C. Infectivity was tested on Vero cells before use in the neutralization assay. This material is designated VSV-SARS-CoV-2-S.

[0164] VSV-SARS-CoV-2-S neutralization assay On day 1, Vero cells were seeded at 80% confluence in a T225 flask. To seed the cells, the medium was removed from the cells, the cells were washed with 20 mL of PBS (Gibco: 20012-043), 5 mL of TrypLE was added, and the cells were incubated at 37°C for approximately 5 minutes until detached. 5 mL of complete DMEM was added to inactivate the trypsin, and the cells were dispersed by pipetting up and down. To count the resuspended cells, 20,000 Vero cells were seeded into 100 μL of prewarmed complete DMEM per well of a 96-well black polystyrene microplate (Corning: 3904).

[0165] On day 2, VSV-SARS-CoV-2-S was thawed on ice and diluted 1:1 in infection medium.

[0166] In a V-bottom 96-well plate, dilutions of each supernatant were made in 60 ul of infection medium. For the negative control, 60 μL of diluted conditioned medium was added to the wells. 60 μL of diluted VSV-SARS-CoV-2-S was added to all wells except the medium control wells. 60 μL of infection medium was then added to those wells. The pseudoviruses were then incubated with the supernatant dilutions at room temperature for 30 minutes. The medium was removed from the Vero cell plates, and 100 μL of the supernatant / pseudovirus mixture was transferred to the cells. The plates were incubated at 37°C and 5% CO2 for 24 hours. The 1:4 and 1:20 ratios were used. The final supernatant dilution, and in some samples 1:100, was used to assess neutralization of the VSV-SARS-CoV-2-S pseudovirus.

[0167] On day 3, after 24 hours of incubation, the supernatant was removed from the cell wells and replaced with 100 μL of PBS. The plates were then read on a SpectraMax i3 with a MiniMax Imaging Cytometer.

[0168] The ability of anti-SARS-CoV-2-S antibodies to neutralize VSV-based SARS-CoV-2-S-expressing pseudotyped viruses was evaluated using a neutralization fluorescent focus assay. The binding results for the three assays are summarized below. The neutralization potency of the antibodies at each dilution is expressed as a percentage compared to the mock supernatant control. All antibodies demonstrated neutralization capacity, and antibodies showing higher neutralization potential, especially in the set of antibodies evaluated at 1:100, may represent stronger neutralization capacity. [Table 9-1] [Table 9-2] [Table 9-3]

[0169] Example 6: Characterization of antibodies in antibody-dependent cell-mediated toxicity surrogate assays The ability of antibodies targeting the spike protein of SARS-CoV-2 to interact with FcγR3a, an Fc receptor prominently expressed on natural killer (NK) cells that elicits antibody-dependent cellular cytotoxicity (ADCC), was measured in a surrogate bioassay using antibody-bound reporter and target cells. 176 Val allotype receptor (Jurkat / NFAT-Luc / hFcγR3a 176 We used Jurkat T cells engineered to express the reporter gene luciferase under the control of the transcription factor NFAT (NFAT-Luc) along with Val (Val). Target cells were engineered Jurkat T cells expressing human CD20 (used as a positive control for a human IgG1 antibody targeting CD20) and the full-length SARS-CoV-2 spike protein controlled by a doxycycline-inducible promoter. The reporter cells were incubated with the target cells, and engagement of FcγR3a via the Fc domain of the human IgG1 antibody bound to the target cells activated the transcription factor NFAT in the reporter cells, driving luciferase expression, which was then measured via a luminescent readout.

[0170] Jurkat T cells were engineered to constitutively express full-length human CD20 (amino acids M1-P297 of NCBI accession number NP_690605.1), the Tet3G transactivator protein (cloned using the Takara pEF1α-Tet3G vector, catalog number 631167), and the doxycycline-inducible full-length SARS-CoV-2 spike protein (amino acids M1-T1273 of NCBI accession number YP_009724390.1). Engineered Jurkat / Tet3G / hCD20 / SARS-CoV2 spike protein-expressing cells were selected for high spike protein expression and then maintained in RPMI + 10% Tet-free FBS + P / S / G + 500 μg / ml G418 + 1 μg / ml puromycin + 250 μg / ml hygromycin growth medium.

[0171] Jurkat T cells express high-affinity human FcγR3a 176 Together with the Val allotype receptor The cells were engineered to stably express a nuclear factor-activated T cell (NFAT) luciferase reporter construct (amino acids M1-K254 of NCBI accession number P08637 VAR_003960). The engineered reporter cells were maintained in RPMI 1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 500 μg / ml G418 growth medium.

[0172] 36 hours prior to the start of the surrogate ADCC assay, 5 x 10 5 1μg / ml of target cells RPMI + 10% Tet-free FBS containing ml of doxycycline (Sigma) +P / S / G cell culture medium. The day before the experiment, reporter cells were cultured at 7.5 × 10 in RPMI 1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 500 μg / ml G418 growth medium. 5 The cells were split to a density of 100 cells / ml.

[0173] Briefly, on the day of the experiment, target and reporter cells were transferred into assay medium (RPMI + 10% Tet-free FBS + P / S / G) at a 3:2 ratio (3 × 10 4 / 2 x 10 target cells per well 4 / well reporter cells) in a 384-well white microplate The plate was then titered with 1000 ng / mL of IgG1-containing CD20 antibody, followed by the addition of various concentrations of anti-SARS-CoV-2-S antibody supernatants. To normalize the detected ADCC activity of the anti-SARS-CoV-2-S antibody supernatants, a positive control (human IgG1-containing CD20 antibody) sample and a negative control sample containing no antibody were included on each plate. The plate was then incubated at 37°C / 5% CO2 for 5 hours. After incubation, cells were lysed by adding an equal volume of ONE-Glo™ (Promega) reagent and luciferase activity was detected. Emitted light was captured in relative light units (RLU) on a multilabel plate reader Envision (PerkinElmer), and data were analyzed and normalized using the following equation:

number

[0174] The ability of anti-SARS-COV-2-S antibodies to activate the FcγR3a receptor was examined using Jurkat / N FAT-Luc / FcγR3a as a reporter cell line. 176 Val) The antibodies were evaluated in a surrogate ADCC assay using Jurkat / hCD20 / SARS-COV2 spike as target cells. Each antibody tested contained an IgG1 domain.

[0175] Table 7 summarizes the results, showing the raw luciferase activity and calculated % of the positive control. A range of % ADCC activity was observed, indicating activation of FcγR3a by the antibody supernatants. All samples showed some measure of surrogate ADCC activity, with 10 of the antibody supernatants exhibiting surrogate ADCC activity superior to that observed with the positive control. [Table 10-1] [Table 10-2]

[0176] Example 7: Anti-SARS-CoV-2-S antibody binding specificity assay To determine the binding of anti-SARS-COV-2-S antibodies to a panel of antigens, a Luminex binding assay was performed. In this assay, antigens were either amine-coupled or captured by streptavidin onto Luminex microspheres as follows: approximately 1,000 10,000 MagPlex microspheres (Luminex Corp., MagPlex Microspheres, catalog numbers MC10000 and MC12000) were vortexed in 500 μL of 0.1 M NaPO, pH 6.2 (activation buffer). The microspheres were resuspended, centrifuged, and the supernatant removed. Because the microspheres are light-sensitive, they were protected from light. The microspheres were resuspended in 160 μL of activation buffer, and the carboxylic acid groups (-COOH) were activated by adding 20 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog number 24525) followed by 20 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog number 22980) at 25 °C. After 10 min, the pH of the reaction was lowered to 5.0 by adding 600 μL of 50 mM MES, pH 5 (coupling buffer). The microspheres were vortexed and centrifuged, and the supernatant removed. The activated microspheres were immediately mixed with 500 μL of 25 μg / mL protein antigen or streptavidin in coupling buffer and incubated at 25°C for 2 hours. The coupling reaction was stopped by adding 50 μL of 1 M Tris-HCl, pH 8.0, and the microspheres were vortexed, centrifuged, and washed three times with 800 μL of PBS 0.005% (Tween 20 0.05%) to remove uncoupled proteins and other reaction components. The microspheres were resuspended at 10 million microspheres / mL in 1 mL of PBS 2% BSA 0.05% sodium azide. For streptavidin-mediated antigen capture, 500 μL of 12.5 μg / mL biotinylated protein in PBS was added to the streptavidin-coupled microspheres and incubated at 25°C for 1 hour. The microspheres were vortexed, centrifuged, washed three times with 800 μL of PBS, and then blocked with 500 μL of 30 mM biotin (Millipore-Sigma, catalog number B4501) in 0.15 M Tris pH 8.0. The microspheres were incubated for 30 minutes, then vortexed, centrifuged, and washed three times with 800 μL of PBS. The microspheres were resuspended in 1 mL of PBS 2% BSA 0.05% sodium azide at 10 million microspheres / mL.

[0177] Microspheres of different proteins and biotinylated proteins were mixed at 2700 beads / ml, and 75 μL of microspheres per well were seeded into a 96-well ProcartaPlex flat-bottom plate (ThermoFisher, catalog number EPX-44444-000) and mixed with 25 μL of each anti-SARS-CoV-2 supernatant containing antibody. The samples and microspheres were incubated at 25°C for 2 hours and then washed twice with 200 μL of DPBS containing 0.05% Tween 20. To detect the level of antibody binding to individual microspheres, 100 μL of 2.5 μg / mL R-phycoerythrin-conjugated goat F(ab')2 anti-human kappa (Southern Biotech, catalog no. 2063-09) in blocking buffer (for antibodies with mouse Fc regions) or 100 μL of 1.25 μg / mL R-phycoerythrin Affinipure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment specific (Jackson Immunoresearch, catalog no. 115-116-072) in blocking buffer (for antibodies with human Fc regions) was added and incubated for 30 minutes at 25°C. After 30 minutes, samples were washed twice with 200 μL of wash buffer and resuspended in 150 μL of wash buffer. Plates were read with Luminex FlexMap 3D® (Luminex Corp.) and Luminex xPonent® software version 4.3 (Luminex Corp.) The SARS-CoV-2 proteins used in the assay were as follows: RBD_(R319-F541).mmh:SEQ ID NO:829 RBD_(R319-F541).mFc: SEQ ID NO: 830 RBD_(R319-F541).hFc): SEQ ID NO: 831

[0178] Luminex binding results are shown in Tables 8 and 9 as median fluorescence intensity (MFI) signal intensities. The results show that 46 anti-SARS-CoV-2-S antibody supernatants specifically bound to the SARS-CoV-2-S RBD protein. These results also show that five of these antibodies cross-reacted with the SARS-CoV-2-S spike RBD protein, exhibiting binding signals greater than 1000 MFI. [Table 11-1] [Table 11-2] [Table 12-1] [Table 12-2] [Table 13-1] [Table 13-2] [Table 14]

[0179] Example 8: Anti-SARS-CoV-2-S antibody diversity assay A binding assay was performed to determine the binding profile of anti-SARS-COV-2-S antibodies. In this assay, antigens were amine-coupled as described in the Luminex binding assay above. Briefly, approximately 9 million MagPlex microspheres (Luminex Corp., MagPLex Microspheres, catalog numbers MagPLex MC10000 and MC12000) for 16 different bead regions were resuspended by vortexing in 500 μL of 0.1 M NaPO4, pH 6.2, and centrifuged. The supernatant was removed by centrifugation. The microspheres were resuspended in 160 μL of activation buffer. The carboxylic acid groups (-COOH) were activated by adding 20 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog number 24525) followed by 20 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog number 22980) at 25 °C. After 10 min, the pH of the reaction was lowered to 5.0 by adding 600 μL of 50 mM MES, pH 5 (coupling buffer), and the microspheres were vortexed and centrifuged to remove the supernatant. The activated microspheres were immediately mixed with 500 μL of 20 μg / mL SARS-CoV-2 spike protein (RBD) (R319-F541)-mmH in coupling buffer and incubated at 25 °C for 2 h. The coupling reaction was quenched by adding 50 μL of 1 M Tris-HCl, pH 8.0, and the microspheres were vortexed, centrifuged, and washed three times with 100 μL of PBS. The microspheres were resuspended in 250 μL of PBS at 9 million microspheres / mL.

[0180] Fifteen of the 16 microsphere regions containing amine-conjugated proteins were modified for the binning assay as follows: the microspheres were washed twice with 5% DMSO in PBS, and 500 μl of chemical or enzyme solution was dissolved according to the manufacturer's recommendations and added to the amine-conjugated microspheres at 10 nM. This was then vortexed and incubated for 2 hours at room temperature with rotation. The microspheres were washed three times with 2% BSA in PBS. The microspheres were resuspended in 1 mL of PBS at 9 million microspheres / mL.

[0181] Protein-modified and intact microspheres were mixed at 2700 beads / ml, and 75 μL of the microspheres were seeded into a 96-well ProcartaPlex 96-well flat-bottom plate (ThermoFisher, catalog number EPX-44444-000) and mixed with 25 μL of each anti-SARS-CoV-2-S supernatant-containing antibody. The samples and microspheres were incubated at 25°C for 2 hours and then washed twice with 200 μL of DPBS containing 0.05% Tween 20. To detect antibody binding levels to individual microspheres, 100 μL of 2.5 μg / mL R-phycoerythrin-conjugated goat F(ab')2 anti-human kappa (Southern Biotech, catalog no. 2063-09) in blocking buffer (for antibodies with hFc) or 100 μL of 1.25 μg / mL R-phycoerythrin Affinipure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment specific (Jackson Immunoresearch, catalog no. 115-116-072) in blocking buffer (for antibodies with mFc) or 100 μL of 1.25 μg / mL R-phycoerythrin anti-His (Biolegend, catalog no. 362603) in blocking buffer (ACE-2 control, R&D, catalog no. 933-ZN) was added and incubated for 30 minutes at 25°C. After 30 minutes, samples were washed twice with 200 μl of wash buffer and resuspended in 150 μL of wash buffer. Plates were read with FlexMap 3D® (Luminex Corp.) and Luminex xPonent® software version 4.3 (Luminex Corp.).

[0182] The Luminex binning results are shown in Table 10 as median fluorescence intensity (MFI) signal strength. To determine clusters, the data were normalized to intact protein (unmodified microspheres) and clustered. The 46 anti-SARS-CoV-2 antibodies were classified into nine clusters with two or more antibodies, and 11 antibodies were classified as a single node. Clusters were assigned based on these results of hierarchical clustering and dendrograms. These results indicate that the 46 anti-SARS-CoV-2-S antibody supernatants have diverse binding properties and profiles, suggesting a collection of antibodies that bind to different epitopes of the SARS-CoV-2 spike protein. [Table 15-1] [Table 15-2] [Table 16-1] [Table 16-2]

[0183] Example 9: Biacore binding kinetics of anti-SARS-CoV-2-S monoclonal antibodies Equilibrium dissociation constants (K D ) is a real-time surface plasmon resonance-based biomarker assay using BioCal e Determined using a T200 / Biacore8K biosensor. All binding studies were performed at 25 °C in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v Surfactant Tween-20, pH 7.4 (HBS-ET). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with a mouse anti-human Fc-specific mAb or a rabbit anti-mouse Fcγ monoclonal antibody (GE, catalog no. BR-1008-38) to capture anti-SARS-CoV-2 antibodies. Binding studies were performed with the human SARS-CoV-2 RBD ectodomain expressed with a C-terminal myc-myc-hexahistidine tag (SARS-COV-2 RBD-MMH), the SARS-CoV-2 RBD ectodomain expressed with a C-terminal mouse IgG2a (SARS-COV-2 RBD-mFc), or the SARS-CoV-2 RBD ectodomain expressed with a C-terminal human IgG1 (SARS-COV-2 RBD-hFc). A single concentration of SARS-CoV-2 RBD-MMH (100 nM), SARS-CoV-2 RBD-mFc (50 nM), or SARS-CoV-2 RBD-hFc (50 nM) prepared in HBS-ET running buffer was injected for 1.5 minutes at a flow rate of 30 μL / min. Dissociation of the various SARS-CoV-2 RBD reagents bound to the antibody was monitored for 2 minutes in HBS-ET running buffer. At the end of each cycle, the SARS-CoV-2 RBD antibody capture surface was regenerated using either a 10-second injection of 20 mM phosphate for mouse anti-human Fc-specific monoclonal antibody surfaces or a 40-second injection of 10 mM glycine, HCl, pH 1.5 for rabbit anti-mouse Fcγ-specific polyclonal antibodies. The association rate (K) was measured. a ) and dissociation rate (K dThe binding dissociation equilibrium constant (K) was determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using BiaEvaluation software v3.1 or Biacore Insight Evaluation software v2.0 or curve fitting software. D ) and dissociation half-life (t1 / 2) was calculated from the kinetic velocity as follows:

number

[0184] The binding kinetic parameters of different SARS-CoV-2 monoclonal antibodies binding to different anti-SARS-CoV-2 RBD reagents of the invention at 25°C are shown in Tables 11 and 12. [Table 17-1] [Table 17-2] [Table 17-3] [Table 18-1] [Table 18-2] [Table 18-3]

[0185] Example 10: Characterization of anti-SARS-CoV-2-S monoclonal antibodies by blocking ELISA An ELISA-based blocking assay was developed to determine the ability of anti-SARS-CoV2-S antibodies to block the binding of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to human angiotensin-converting enzyme 2 (hACE2).

[0186] The SARS-CoV-2 protein used in the experiment consisted of the C-terminus (SARS-CoV-2 The antibody consisted of the receptor binding domain (RBD) portion of the SARS-CoV-2 spike protein (amino acids Arg319 to Phe541) expressed in the Fc portion of human IgG1 (RBD-hFc, see NCBI accession number MN908947.3). The human ACE2 protein used in the experiments was purchased from R&D Systems and consisted of amino acids Glutamine 18 to Serine 740 with a C-terminal 10X histidine tag (hACE2-His, NCBI accession number Q9BYF1).

[0187] The experiment was performed as follows: Monoclonal anti-Penta-His antibody (Qiagen) was coated onto a 96-well microtiter plate at 1 μg / ml in PBS overnight at 4°C. The hACE2-His receptor was added at 0.2 μg / ml in PBS and allowed to bind for 2 hours at room temperature. Nonspecific binding sites were then blocked using a 0.5% (wt / vol) solution of BSA in PBS. In other microtiter plates, a fixed amount of 10 pM or 15 pM (as shown in Table 13) of SARS-CoV-2 RBD-hFc protein was combined with the antibody diluted 1:10 or 1:20 in PBS + 0.5% BSA. After 1 hour of incubation, these antibody-protein complexes were transferred to the hACE2-His-coated microtiter plate. After 1.5 hours of incubation at room temperature, the wells were washed, and plate-bound SARS-CoV-2 RBD-hFc protein was detected with a goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson). Plates were then developed using TMB substrate solution (BD Biosciences, catalog no. 555214) according to the manufacturer's recommendations, and absorbance was measured at 450 nm using a VictorX5 plate reader.

[0188] Data analysis was performed by calculating the percent signal reduction for immobilized SARS-CoV-2-S RBD-hFc concentrations in the presence and absence of antibody. In the calculation, the binding signal for a given SARS-CoV-2-S RBD-hFc sample without antibody on each plate was referenced as 100% binding or 0% blocking, and the percent signal reduction for SARS-CoV-2 The baseline signal of the medium sample in the absence of RBD-hFc alone was referenced as 0% binding or 100% blocking.

[0189] The ability of anti-SARS-CoV-2-S antibodies to block SARS-CoV-2-S RBD binding to human ACE2 was assessed using a blocking ELISA format: single-point test antibody supernatant blockade of 10 pM or 15 pM SARS-CoV-2S RBD-hFc binding to hACE2-His presented on anti-His antibody coated on 96-well microtiter plates was detected with HRP-conjugated anti-hFc antibody.

[0190] Blockage results for the three assays are summarized in Table 13. SARS-CoV-2-S binding signal (450 nm) and % blockage calculated in G are shown. Varying blockage was observed in the test samples. For samples with NAs shown in columns 6 and 7, plate correction values ​​are included in columns 4 and 5 because the data were consistent with a single plate switch occurring in these samples. 43 of the 46 antibody supernatants blocked 50% or more of SARS-CoV-2-S RBD-hFc binding to plate-coated human ACE2, 16 of which blocked 90% or more of the signal. [Table 19-1] [Table 19-2] [Table 19-3]

[0191] Example 11: Epitope Mapping of Anti-SARS-CoV-2-S Monoclonal Antibodies Against the Spike Glycoprotein by Hydrogen-Deuterium Exchange Mass Spectrometry Hydrogen-deuterium exchange mass spectrometry (HDX-MS) was performed to determine the amino acid residues of the SARS-CoV-2 spike protein receptor binding domain (RBD (amino acids R319-F541)) that interact with mAb10989, mAb10987, mAb10934, mAb10933, mAb10920, mAb10922, mAb10936, mAb10954, mAb10964, mAb10977, mAb10984, and mAb10986. A general description of the HDX / MS method is provided in Ehring (1999) Analytical Biochemistry 267(2):252-259, and Engen and Smith (201) Anal. Chem. 73:256A-265A.

[0192] HDX-MS experiments were performed on an integrated HDX / MS platform, consisting of a Leaptec HDX PAL system for deuterium labeling and quenching, a Waters Acquity I-Class (Binary Solvent Manager) for sample digestion and loading, a Waters Acquity I-Class (Binary Solvent Manager) for analytical gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0193] The labeling solution was prepared in PBS buffer in D2O at pH 7.0 (10 mM LiCl). For deuterium labeling, 10 μL of RBD protein or RBD protein premixed with each of the 12 antibodies listed above was incubated with 90 μL of DO labeling solution at 20°C. Samples were incubated twice at various time points. For mAb10989, mAb10987, mAb10934, and mAb10933, the time points were 0 min (non-deuterated control), 5 min, and 10 min. For mAb10920, mAb10922, mAb10936, mAb10954, mAb10964, mAb10977, mAb10984, and mAb10986, the time points were 0 min (non-deuterated control) and 10 min. The deuteration reaction was quenched by adding 90 μL of pre-chilled quench buffer (0.5 M TCEP-HCl, 4 M urea, 0.5% formic acid) to each sample and incubating at 20°C for 90 seconds. The quenched samples were then transferred to a Leaptec HDX Injected into the PAL system for online pepsin / protease XIII digestion The digested peptides were trapped on a C18 column (2.1 mm x 5 mm, Waters) and separated on another C18 column (2.1 mm x 50 mm, Waters) at -5 °C with a 20 min gradient (for mAb10989, mAb10987, mAb10934, and mAb10933) or a 10 min gradient from 0% to 90% mobile phase B (mobile phase A: 0.5% formic acid and 4.5% acetonitrile in water; mobile phase B: 0.5% formic acid in acetonitrile) (for mAb10920, mAb10922, mAb10936, mAb10954, mAb10956, mAb10964, mAb10977, and mAb10984). Eluted peptides were analyzed by Thermo Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

[0194] LC-MS / MS data from undeuterated RBD protein samples were searched against a database containing the amino acid sequences of RBD proteins, pepsin, protease XIII, and their reverse sequences using the Byonic search engine (Protein Metrics). Search parameters were set as default, using nonspecific enzyme digestion and human glycosylation as common variable modifications. The list of identified peptides was then imported into HDExaminer software (version 3.1) to calculate the deuterium incorporation (D incorporation) and the difference in deuterium incorporation percentage (Δ%D) for all deuterated samples. The difference in deuterium incorporation percentage (Δ%D) was calculated as follows: Difference in deuterium uptake (ΔD) = D-uptake (RBD-mAb) - D-uptake (RBD only)

number

[0195] A total of 190 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10989 samples, representing 86.06% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. The peptide corresponding to amino acids 467-513 of the RBD (DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVL) (SEQ ID NO: 835) was significantly protected by mAb10989.

[0196] A total of 187 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10987 samples, representing 86.06% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. The peptide corresponding to amino acids 432-452 of the RBD (CVIAWNSNNLDSKVGGNYNYL) (SEQ ID NO: 836) was significantly protected by mAb10987.

[0197] A total of 188 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10934 samples, representing 86.06% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 432-452 (CVIAWNSNNLDSKVGGNYNYL) (SEQ ID NO: 836), 467-474 (DISTEIYQ) (SEQ ID NO: 837), and 480-513 (CNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVL) (SEQ ID NO: 838) of the RBD were significantly protected by mAb10934.

[0198] A total of 188 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10933 samples, representing 86.06% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. The peptide corresponding to amino acids 467-510 of the RBD (DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV) (SEQ ID NO: 839) was significantly protected by mAb10933.

[0199] A total of 75 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10920 samples, representing 83.27% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 471-486 (EIYQAGSTPCNGVEGF) (SEQ ID NO: 840) and 491-515 (PLQSYGFQPTNGVGYQPYRVVVLSF) (SEQ ID NO: 841) of the RBD were significantly protected by mAb10920.

[0200] A total of 86 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10922 samples, representing 87.25% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. The peptide corresponding to amino acids 432-452 of the RBD (CVIAWNSNNLDSKVGGNYNYL) (SEQ ID NO: 836) was significantly protected by mAb10922.

[0201] A total of 81 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10936 samples, representing 82.07% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 351-360 (YAWNRKRISN) (SEQ ID NO: 842), 432-452 (CVIAWNSNNLDSKVGGNYNYL) (SEQ ID NO: 836), 467-486 (DISTEIYQAGSTPCNGVEGF) (SEQ ID NO: 843), and 491-513 (PLQSYGFQPTNGVGYQPYRVVVL) (SEQ ID NO: 844) were significantly protected by mAb10936.

[0202] A total of 84 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10954, representing 87.25% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 400-422 (FVIRGDEVRQIAPGQTGKIADYN) (SEQ ID NO: 845), 453-486 (YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF) (SEQ ID NO: 846), and 490-515 (FPLQSYGFQPTNGVGYQPYRVVVLSF) (SEQ ID NO: 847) of the RBD were significantly protected by mAb10954.

[0203] A total of 109 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10964, representing 83.67% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 401-424 (VIRGDEVRQIAPGQTGKIADYNYK) (SEQ ID NO: 848) and 471-513 (EIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVL) (SEQ ID NO: 849) of the RBD were significantly protected by mAb10964.

[0204] A total of 78 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10977 samples, representing 87.25% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 351-364 (YAWNRKRISNCVAD) (SEQ ID NO: 850) and 471-486 (EIYQAGSTPCNGVEGF) (SEQ ID NO: 840) of the RBD were significantly protected by mAb10977.

[0205] A total of 88 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10984, representing 87.25% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 400-422 (FVIRGDEVRQIAPGQTGKIADYN) (SEQ ID NO: 845) and 453-486 (YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF) (SEQ ID NO: 846) of the RBD were significantly protected by mAb10984.

[0206] A total of 84 peptides from the RBD were identified from both the RBD alone and the RBD complexed with mAb10986 samples, representing 87.25% sequence coverage of the RBD. Peptides that showed a 5% or greater decrease in deuterium uptake upon mAb binding (i.e., a Δ%D value less than -5%, such as -6%, -10%) were defined as significantly protected. Peptides corresponding to amino acids 400-422 (FVIRGDEVRQIAPGQTGKIADYN) (SEQ ID NO: 845), 453-486 (YRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGF) (SEQ ID NO: 846), and 490-515 (FPLQSYGFQPTNGVGYQPYRVVVLSF) (SEQ ID NO: 847) of the RBD were significantly protected by mAb10986.

[0207] In summary, the majority of the neutralizing antibodies tested contact the RBD in a manner that overlaps with the RBD residues that make up the ACE2 interface. Furthermore, as shown in Figure 15, the antibodies can be grouped based on the patterns of contact with the RBD surface. The above data are also summarized in Tables 14-25. [Table 20] [Table 21] [Table 22] [Table 23] [Table 24] [Table 25] [Table 26] [Table 27] [Table 28] [Table 29] [Table 30] [Table 31]

[0208] Example 12: Neutralization of SARS-CoV-2 Wild-Type and Mutant Spike Proteins To test whether anti-SARS-CoV-2 spike protein antibodies could neutralize SARS-CoV-2 mutants, these antibodies were screened against a panel of VSV pseudotyped viruses expressing wild-type and mutant spike proteins. VSV pseudotyped viruses were generated by transiently transfecting 293T cells with a plasmid encoding the SARS-CoV-2 spike protein or the same plasmid containing nucleotide mutations encoding known variants of the SARS-CoV-2 spike protein amino acid sequence. All plasmids were confirmed by Sanger sequencing. Cells were plated at 1.2 × 10 per plate in DMEM complete medium (1000 mL DMEM, Gibco; 100 mL FBS, Gibco; 10 mL PSG, Gibco) 1 day before transfection with 15 μg / plate of spike DNA using 125 μL Lipofectamine LTX, 30 μL PLUS Reagent, and up to 3 mL Opti-Mem. 7 Cells were seeded onto a 15 cm plate. 24 hours after infection, cells were washed with 10 mL of PBS and resuspended in 0.1% VSVΔ in 10 mL of Opti-Mem. G:mNeon The virus was infected at an MOI of 1 The cells were incubated for 24 hours with gentle rocking every 10 minutes. The cells were washed three times with 10 mL of PBS, then covered with 20 mL of infection medium (1000 mL DMEM, Gibco; 10 mL sodium pyruvate, Gibco; 7 mL BSA, Sigma; 5 mL gentamicin, Gibco) and incubated at 37°C, 5% CO2 for 24 hours. The pseudovirus supernatant was collected on ice in a 250 mL centrifuge tube, centrifuged at 3000 rpm for 5 minutes to pellet any cellular debris, aliquoted on ice, and frozen at -80°C. The infectivity was tested on Vero cells before use in the neutralization assay. This material was used for VSVΔ G:mNeon / spike pseudovirus, or VSVΔ G:mNeon / spike_(mutant amino acid mutation) (e.g., VSVΔ G:mNeon / Spike_H49Y).

[0209] On day 1, Vero cells were seeded into a T225 flask to 80% confluence, washed with PBS (Gibco: 20012-043), detached from the flask by adding TrypLE, and inactivated trypsin by adding complete DMEM. 20,000 Vero cells were seeded into a 96-well black polystyrene microplate (Corning: 3904) in 100 μL of prewarmed complete DMEM per well. On day 2, VSVΔ G:mNeon The spiked pseudovirus was thawed on ice and diluted in infection medium. The antibodies were diluted in a 96-well U-bottom plate and diluted with 210 μl of infection medium to generate dilutions of each antibody at 2x assay concentration. 120 μL of diluted antibody was transferred to a new U-bottom plate, and medium and an IgG1 control antibody were added to each plate. 120 μL of diluted pseudovirus was added to all wells except the medium control wells, to which 120 μL of infection medium was added. The antibody-containing pseudovirus was incubated at room temperature for 30 minutes, after which the medium was removed from the Vero cells. 100 μL of the antibody / pseudovirus mixture was added to the cells, which were then incubated for 24 hours at 37°C in 5% CO2. On day 3, the supernatant was removed from the cell wells. The plate was read on a SpectraMax i3 with a MiniMax Imaging Cytometer.

[0210] In addition to testing for neutralization ability with non-replicating VSV-SARS-CoV-2-S virus, antibodies were also tested against SARS-CoV-2 virus. Monoclonal antibodies and antibody combinations were serially diluted in DMEM (Quality Biological) and supplemented with 10% (v / v) heat-inactivated fetal bovine serum (Sigma), 1% (v / v) penicillin / streptomycin (Gemini Bio-products), and 1% (v / v) L-glutamine (2 mM final concentration, Gibco) (VeroE6 medium) to a final volume of 250 μL. Next, 250 μL of VeroE6 medium containing SARS-CoV-2 (WA-1) (1000 PFU / mL) was added to each serum dilution and 250 μL of medium as an untreated control. The virus-antibody mixtures were incubated at 37°C for 60 minutes. After incubation, the virus titers of the mixtures were determined by plaque assay. Finally, 50% plaque reduction neutralization titer (PRNT50) values ​​(serum dilution at which plaque formation was reduced by 50% compared with untreated controls) were calculated using a four-parameter logistic curve fitted to the percent neutralization data (GraphPad software, La Jolla, CA).

[0211] The half-maximal inhibitory concentrations (IC50s) of individual monoclonal antibodies against a VSV-SARS-CoV-2 spike protein (S)-expressing pseudovirus encoding the Wuhan-Hu-1 (NCBI accession number MN908947.3) sequence of spike protein (S-wt) were measured in Vero cells (Table 26). Most of the antibodies showed neutralizing potency in the picomolar (pM) range, and some in the nanomolar (nM) range.

[0212] As previously reported, recombinant ACE2 mediates neutralization of VSV spike-pseudoparticles Although the potency was far inferior to that of monoclonal antibodies, with a more than 1000-fold decrease in potency compared to the best neutralizing mAb (Figure 10A). In addition, the potent neutralizing activity of mAb10987, mAb10989, mAb10933, and mAb10934 was confirmed in neutralization assays involving neutralization of SARS-CoV-2 in VeroE6 cells (Figure 10B). All neutralization assays produced similar potencies for the four mAbs (mAb10987, mAb10989, mAb10933, and mAb10934), with no combinations exhibiting synergistic neutralizing activity (Figure 10B). [Table 32-1] [Table 32-2]

[0213] Amino acid variants of the spike (S) protein were identified from over 700 publicly available SARS-CoV-2 sequences representing isolates circulating worldwide and cloned into VSV pseudoparticles. To assess the impact of each variant on the neutralization potency of monoclonal antibodies, neutralization assays were performed using pseudoparticles encoding the variants. Table 27 illustrates the relative neutralization potency of monoclonal antibodies against the variant-encoding pseudoparticles compared to the SARS-CoV-2 spike (S-wt) at a single concentration of 5 μg / ml. The percentage of neutralization against S-wt was obtained for each individual antibody and variant. With the exception of mAb10985 and the R408I variant, no antibodies showed a loss of neutralization potency at a concentration of 5 μg / ml. These data demonstrate the broad functional neutralization range of monoclonal antibodies against globally circulating SARS-CoV-2 spike variants.

[0214] To further investigate the impact of S protein variants on the neutralization potency of monoclonal antibodies, full neutralization curves were performed to determine the IC50 values ​​of the most potent neutralizing antibodies against a subset of variants localized within the receptor binding domain (RBD) of the S protein. Table 28 shows the IC50 neutralization values ​​for each variant pseudoparticle. Inherent variation of up to 3-fold can be observed between pseudoparticle neutralization assays and does not indicate a change in neutralization potency. These data demonstrate that the antibodies retain neutralization potency against a diverse panel of S protein RBD variants. [Table 33] [Table 34] [Table 35] [Table 36]

[0215] Example 13: Biacore binding kinetics of purified anti-SARS-CoV-2-S monoclonal antibodies Equilibrium dissociation constants (K) of various SARS-COV-2 RBD reagents binding to purified CHOt anti-SARS-COV-2 monoclonal antibody (mAb) D ) is a real-time surface The binding activity was determined using a plasmon resonance-based Biacore T200 / Biacore 8K biosensor. All binding studies were performed at 25°C and 37°C in a running buffer containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v detergent Tween-20, pH 7.4 (HBS-ET). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with a mouse anti-human Fc-specific mAb (Regeneron, mAb2567) to capture the anti-SARS-COV-2b mAb. Binding studies were performed with the human SARS-COV-2 RBD ectodomain expressed with a C-terminal myc-myc-hexahistidine (SARS-COV-2RBD-MMH) and the SARS-COV-2 RBD ectodomain expressed with a C-terminal mouse IgG2a (SARS-COV-2RBD-mFc). Using these reagents, we were able to test the ability of antibodies to bind to the monomeric and dimeric RBD peptides, respectively.

[0216] Different concentrations of hSARS-COV-2 RBD-MMH (90 nM-3.33 nM, 3-fold dilutions) and SARS-COV-2 RBD-mFc (30 nM-1.11 nM, 3-fold dilutions) prepared in HBS-ET running buffer were injected at a flow rate of 50 μL / min for 3 min. Dissociation of the various SARS-COV-2 RBD reagents bound to the mAb was monitored for 6–10 min in HBS-ET running buffer. At the end of each cycle, the SARS-COV-2 RBD mAb capture surface was transferred to a mouse anti-human Fc-specific mAb surface at 20 mM MgCl. The association rate (K a ) and dissociation rate (K d The binding dissociation equilibrium constant (K) was determined by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitation using BiaEvaluation software v3.1 or Biacore Insight Evaluation software v2.0 or curve fitting software.D ) and dissociation half-life (t1 / 2) from the kinetic rate as follows: It was calculated as follows:

number

[0217] The binding kinetic parameters of different SARS-COV-2 mAbs binding to different anti-SARS-COV-2 RBD reagents of the present invention at 25°C and 37°C are shown in Tables 29-32, respectively. [Table 37-1] [Table 37-2] [Table 38-1] [Table 38-2] [Table 39-1] [Table 39-2] [Table 40-1] [Table 40-2]

[0218] Example 14: Anti-SARS-CoV-2 antibodies block RBD binding to hACE2 as determined by ELISA An ELISA-based blocking assay was used to determine the ability of anti-SARS-CoV-2 antibodies to block the binding of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to its receptor, human angiotensin-converting enzyme 2 (hACE2).

[0219] The SARS-CoV-2 protein used in this assay consisted of the receptor-binding domain (RBD) portion of the SARS-CoV-2 spike protein (amino acids Arg319 to Phe541) expressed with the Fc portion of human IgG1 at the C-terminus (SARS-CoV-2 RBD-hFc). The human ACE2 protein used in the experiment was purchased from R&D Systems and tagged with a C-terminal 10X histidine tag (hACE2-His, NCBI accession number 1001). It is composed of amino acids Gln18 to Ser740 with the sequence number Q9BYF1.

[0220] The experiment was performed as follows: Monoclonal anti-Penta-His antibody (Qiagen) was coated onto a 96-well microtiter plate at 1 μg / ml in PBS overnight at 4°C. hACE2-His receptor was added at 0.2 μg / ml in PBS and allowed to bind for 2 hours at room temperature (RT). Nonspecific binding sites were then blocked using a 0.5% (wt / vol) solution of BSA in PBS. In another microtiter plate, a fixed amount of 100 pM SARS-CoV-2 RBD-hFc protein was combined with anti-SARS-CoV-2 antibodies and an isotype IgG1 antibody control at dilutions ranging from 0.0008 nM to 50 nM in PBS + 0.5% BSA. After 1 hour of incubation, the mixture was transferred to the hACE2-His coated microtiter plate. After 1.5 hours of incubation at room temperature, the wells were washed, and plate-bound SARS-COV2 was detected with a goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson). Plates were then developed using TMB substrate solution (BD Biosciences, #555214) according to the manufacturer's recommendations, and absorbance was measured at 450 nm using a VictorX5 plate reader.

[0221] Binding data were analyzed using a sigmoidal dose-response model within Prism™ software (GraphPad). Calculated IC50 values, defined as the concentration of antibody required to block 50% of SARS-CoV-2 RBD-hFc binding to plate-coated hACE2-His, were used as an indicator of blocking potency. Percent blocking was observed at the highest antibody concentration tested using this formula and was defined based on the background-corrected binding signal reported for all antibodies tested.

number

[0222] Antibodies that blocked 50% or less of binding at the highest concentration tested were classified as non-blockers and IC50 values ​​were not reported for those antibodies.

[0223] The ability of anti-SARS-CoV-2 antibodies to block SARS-CoV-2 RBD binding to human ACE2 was assessed using a blocking ELISA. In this assay, 100 pM of SARS-CoV-2 RBD-hFc was titrated with a wide range of concentrations of anti-SARS-CoV-2-S antibodies, and the inhibition of RBD binding to hACE2-His by the presence of antibodies was assessed. Plate-bound RBD-hFc was detected with an HRP-conjugated anti-hFc antibody.

[0224] The blocking IC50 and maximum blockade at the highest tested concentrations of anti-SARS-CoV-2-S antibodies are summarized in Table 33, and the blocking curves are shown in Figures 1-8. Of the 46 antibodies tested, 44 demonstrated antibody concentration-dependent blockade of RBD.hFc binding to hACE-2. IC50 values ​​ranged from 41 pM to 4.5 nM, and maximum blockade ranged from 55% to approximately 100% at the highest antibody concentrations tested. Two of the 46 antibodies tested did not demonstrate blocking activity under the assay conditions. As expected, an irrelevant isotype control antibody did not demonstrate blocking activity. [Table 41-1] [Table 41-2]

[0225] Example 15: Mutual competition between mAb10987, mAb10989, mAb10933, and mAb10934 mAb10987, mAb10989, mAb10933, and mAb10934 were examined in cross-competitive binding assays (Figure 11) to assess their potential to be combined to form antibody cocktails. Several pairs of non-competing mAbs with picomolar neutralizing potency were identified (e.g., mAb10987 and mAb0933).

[0226] Epitope binning of anti-SARS-CoV-2-S mAbs was performed using a ForteBio Octet HTX biolayer interferometer (Molecular Devices ForteBio) with a running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.4, and 1 mg / mL BSA. The assay was performed in a sandwich format using a premix containing mAbs competing with each other in a pairwise combinatorial manner for binding to the SARS-CoV-2 RBD-MMH protein using a ELISA kit (Agilent Technologies, LLC, Fremont, CA). The assay was performed at 30°C with continuous stirring at 1000 rpm. After obtaining an initial baseline with running buffer, 20 μg / mL of anti-COVID19 mAb was captured onto an anti-human Fc (AHC) biosensor chip for 300 seconds. To block any remaining free unsaturated binding sites on the AHC biosensor chip, all sensors were exposed to a blocking solution containing 100 μg / mL of irrelevant IgG1 for 240 seconds. Following this process, the biosensor was immersed for 300 seconds in a well containing 100 nM of the SARS-CoV-2 RBD-MMH protein premix and 600 nM of the anti-COVID19 mAb binding site of a second mAb. The binding response at each step was recorded, and the specific signal was normalized by subtracting the self-blocking mAb competition control from the dataset. Data analysis was performed by Epitope Binning was performed with Octet Data Analysis HT 10.0 software.

[0227] Comparing the cross-competition binding assay with the HDX-MS results described above provides structural insight into the mechanism by which non-competing antibody pairs can simultaneously bind to the RBD and thus be ideal partners for therapeutic antibody cocktails. mAb10987 and mAb10933 represent such an antibody pair. mAb10933 targets a spike-like loop region at one edge of the ACE2 interface. Within that region, the residues that show the most significant HDX protection by mAb10933 face upward, suggesting that the Fab region of mAb10933 binds to the RBD from the top, creating significant clashes between mAb10933 and ACE2. To avoid competition with mAb10933, mAb10987 only binds to the HDX-defined protected region from the front or bottom left (front view of mAb10987 in Figure 12). This is consistent with the neutralization data described above, as mAb10987 orients itself in a position likely to interfere with ACE2.

[0228] Example 16: Structure determination of antibody-binding spike proteins To better understand the binding of mAb10933 and mAb10987 to the spike protein RBD, structural analysis was performed via cryo-electron microscopy (cryo-EM). The Fab fragments of mAb10933 and mAb10987 were isolated using the FabALACTICA kit (Genovis). 600 μg of mAb10933 Fab and 600 μg of mAb10987 Fab were mixed with 300 μg of SARS-CoV-2-S RBD and incubated on ice for approximately 1 hour before being injected onto a Superdex 200 increasing gel filtration column equilibrated in 50 mM Tris pH 7.5, 150 mM NaCl. The peak fractions containing the mAb10933 Fab-mAb10987 Fab-RBD complex were collected and concentrated using a 10 kDa MWCO centrifugal filter. For cryoEM grid preparation, protein samples were diluted to 1.5 mg / mL and 0.15% PMAL-C8 amphipol was added. 3.5 μL of protein was deposited onto freshly plasma-cleaned UltrAufoil grids (1.2 / 1.3, 300 mesh). Excess solution was blotted using filter paper, and the grids were plunge-frozen in liquid ethane using a Vitrobot Mark IV. The cryoEM grids were transferred to a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan). Movies are available. The images were collected using an EPU (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.85 Å. A dose rate of 15 electrons per second per pixel was used, with each movie lasting 2 seconds, corresponding to a total dose of approximately 40 electrons per Ų.

[0229] All cryoEM data processing was performed using cryoSPARC v2.14.2. 2,821 movies were aligned using patch motion correction and patch CTF estimation. 2,197 aligned micrographs were selected for further processing based on the estimated defocus values ​​and CTF-fit resolution. An initial set of particles picked using a blob picker was subjected to 2D classification to generate templates for template picking. 989,553 particles picked by template picking were subjected to multiple rounds of 2D classification to remove particles containing unbound Fab and incomplete complexes. Ab initio reconstruction using three classes generated a single class containing 61,707 particles corresponding to the mAb10933 Fab-mAb10987 Fab-RBD complex. Heterogeneous refinement of this class of particles, followed by heterogeneous refinement, yielded a 3.9 Å resolution (FSC=0.143) map containing 48,140 particles that was used for model building. Onto this map, we manually placed models of the RBD (obtained from PDB code 6M17) and two Fabs (obtained from a previous antibody structure excluding the lambda light chain of mAb10987, obtained from PDB code 5U15). These models were then manually rebuilt using Coot and real-space aligned to the map using Phenix.

[0230] Confirming the above data, single-particle cryoEM of the complex of the SARS-CoV-2 spike RBD bound to the Fab fragments of mAb10933 and mAb10987 demonstrates that the two antibodies in this cocktail can simultaneously bind to different regions of the RBD (Figures 13A, 13B, and 14). A 3D reconstruction map of the complex at a nominal resolution of 3.9 Å shows that both Fab fragments bind to different epitopes on the RBD, confirming that they are non-competing antibodies. mAb10933 binds to the top of the RBD and overlaps extensively with the ACE2 binding site. In contrast, the epitope of mAb10987 is located to the side of the RBD, significantly distant from the mAb10933 epitope, with little or no overlap with the ACE2 binding site.

[0231] Example 17: Mutual competition between anti-SARS-CoV-2-S mAbs Binding competition between anti-SARS-CoV-S monoclonal antibodies (mAbs) was determined using a real-time label-free biolayer interferometry (BLI) assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05 v / v% surfactant Tween-20, 1 mg / mL BSA, pH 7.4 (HBS-EBT) buffer, with the plate shaking at a speed of 1000 rpm. To assess whether the two mAbs could compete with each other for binding to their respective epitopes on the SARS-COV-2-S RBD extracellular domain expressed with a C-terminal myc-myc-hexahistidine (SARS-COV-2 RBD-MMH), approximately 0.51 nm of SARS-COV-2-S RBD-MMH was first captured onto an Octet biosensor chip (Fortebio Inc, #18-5122) coated with an anti-penta-His antibody by immersing the biosensor chip in a well containing a 10 μg / mL solution of SARS-COV-2-S RBD-MMH for 1 minute. The biosensor chip with the captured SARS-COV-2-S RBD-MMH was then saturated with the first anti-SARS-CoV-2-S monoclonal antibody (hereafter referred to as mAb-1) by immersing the chip in a well containing a 50 μg / mL solution of mAb-1 for 5 minutes. The biosensor chip was then immersed in a well containing a 50 μg / mL solution of a second anti-SARS-CoV-2-S monoclonal antibody (hereafter referred to as mAb-2) for 5 minutes. The biosensor chip was immersed in HBS-ETB buffer between each step of the experiment. The real-time binding response was monitored throughout the entire experiment, and the binding response at the end of each step was recorded. The binding responses of mAb-1 and mAb-2 to the pre-complexed SARS-COV-2 RBD-MMH were compared to determine the competitive / non-competitive behavior of the different anti-SARS-CoV-2 monoclonal antibodies, as shown in Table 34. [Table 42-1]

Table 42-2

Table 42-3

Table 42-4

Table 42-5

Table 42-6

Table 42-7

Table 42-8

Table 42-9

Table 42-10

Table 42-11

Table 42-12

Table 42-13

Table 42-14

Table 42-15

Table 42-16

Table 42-17

Table 42-18

Table 42-19

Table 42-20

Table 42-21

Table 42-22

Table 42-23

Table 42-24

Table 42-25

Table 42-26

Table 42-27

Table 42-28

Table 42-29

Table 42-30

Table 42-31

Table 42-32

Table 42-33

Table 42-34

Table 42-35

[0232] Example 18: pH Sensitivity of Anti-SARS-CoV-2-S Monoclonal Antibody Binding to Monomeric SARS-CoV-2-S RBD Reagent Measured at 37°C Dissociation rate constants (K) of various anti-SARS-CoV-2-S monoclonal antibodies in pH 7.4, pH 6.0, and pH 5.0 buffers d ) is a real-time surface plasmon The binding activity was determined using a Biacore T200 biosensor based on SPR. All binding studies were performed at 37 °C using three running buffers: (i) PBS, 0.05% Tween-20 (v / v), pH 7.4 (PBS-T-pH 7.4), (ii) PBS, 0.05% Tween-20 (v / v), pH 6.0 (PBS-T-pH 6.0), and (iii) PBS, 0.05% Tween-20 (v / v), pH 5.0 (PBS-T-pH 5.0). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with a mouse anti-human Fc-specific mAb (Regeneron) to capture the anti-SARS-CoV-2-S monoclonal antibody. Binding studies were performed with the human SARS-COV-2-S RBD extracellular domain expressed with a C-terminal myc-myc-hexahistidine (SARS-COV-2 RBD-MMH). A single concentration of SARS-COV-2-S RBD-MMH (90 nM) prepared in PBS-T pH 7.4 buffer was injected for 3 minutes at a flow rate of 25 μL / min, followed by dissociation of bound SARS-COV-2-S RBD-MMH for 5 minutes in PBS-T pH 7.4, PBS-T pH 6.0, or PBS-T pH 5.0 running buffer.

[0233] Dissociation rate constants (k) for the four pH running buffers were determined by fitting the real-time binding sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software. dThe dissociation half-life (T1 / 2) and Do it K d was calculated from the values.

number

[0234] K of SARS-COV-2-S RBD-MMH binding to different anti-SARS-CoV-2-S monoclonal antibodies in PBS-T-pH 7.4 following dissociation at 37 °C in PBS-T-pH 7.4 and PBS-T-pH 6.0. d and t1 / 2 values ​​are shown in Table 35. Following dissociation at 37 °C in PBS-T-pH 7.4 and PBS-T-pH 5.0, the K values ​​of SARS-COV-2-S RBD-MMH binding to different anti-SARS-CoV-2-S monoclonal antibodies in PBS-T-pH 7.4 were d and t1 / 2 values ​​are shown in Table 36. Comparison of the dissociation half-life (t1 / 2) of SARS-COV-2 RBD-MMH in buffers of pH 7.4, pH 6.0, and pH 5.0. [Table 43-1] [Table 43-2] [Table 43-3] [Table 44-1] [Table 44-2] [Table 44-3]

[0235] Example 19: Anti-SARS-CoV-2-S antibodies that bind to virus-like particles To investigate the ability of a panel of anti-SARS-CoV-2 monoclonal antibodies to bind to the SARS-CoV-2 spike glycoprotein, an in vitro binding assay was developed utilizing vesicular stomatitis virus (VSV) pseudotyped with the SARS-CoV-2 spike protein on an electrochemiluminescence-based detection platform (MSD).

[0236] Pseudotyped vesicular stomatitis virus (VSV) virus-like particles (VLPs) were generated in HEK293T cells to transiently express the SARS-CoV-2 spike protein (accession number MN908947.3, amino acids 16-1211). VLPs expressing VSV alone were also generated as a negative binding control.

[0237] The experiment was performed as follows: VLPs from the two sources mentioned above were diluted with PBS and seeded onto a 96-well carbon electrode plate (Multi-Array High Bind Plate, MSD) and incubated overnight at 4°C to allow VLPs to adhere. Nonspecific binding sites were blocked with 2% (wt / vol) BSA in PBS for 1 hour at room temperature. Anti-SARS-CoV-2 antibodies and a non-binding human IgG1 control were diluted in PBS + 0.5% BSA at concentrations ranging from 0.0008 nM to 50 nM to the plate-bound cells. Antibody-free buffer was added twice, and the plate was incubated at room temperature for 1 hour with shaking. Next, the plate was washed with 1x PBS and washed with an AquaMax2000 plate washer (MDS). Unbound antibodies were removed using a ELISA kit (Jackson Immunoresearch). Plate-bound antibodies were detected using a SULFO-TAG™-conjugated anti-human IgG antibody (Jackson Immunoresearch) for 1 hour at room temperature. After washing, plates were developed with read buffer (MSD) according to the manufacturer's recommended procedure, and luminescence signals were recorded on a SECTOR Imager 600 (Meso Scale Development) instrument. Direct binding signals (in RLU) were captured for SARS-CoV-2-expressing VLPs and VSV-only VLPs.

[0238] The ability of anti-SARS-CoV-2-S monoclonal antibodies to bind to SARS-CoV-2-S-expressing VLPs compared to binding to irrelevant VSV-expressing VLPs was assessed using an immunobinding assay. Binding to VLPs immobilized on 96-well High Bind plates (MSD) was performed with a series of antibody dilutions, and bound antibody was detected using SULFO-TAG™-conjugated anti-human IgG. Binding signals from electrochemiluminescence were recorded with a Sector Imager 600 (MSD). RLU values ​​of antibody binding to VLPs were determined. All antibodies showed concentration-dependent binding, and the ratio of VSV-only binding to SARS-CoV-2-S-expressing VLPs was analyzed at 5.5 nM and 0.20 nM.

[0239] The binding results of two concentrations of anti-SARS-CoV-2-S mAbs to VSV / spike and VSV-only VLPs are summarized in Table 37. Of the 46 antibodies tested, 44 antibodies specifically bound to VSV / spike, with a ratio to VSV of 3 or greater at either concentration. At 0.2 nM antibody, the ratio of VSV / spike to VSV ranged from 3 to 56, and at 5 nM, the ratio ranged from 3 to 303. Two antibodies (mAb10998 and mAb11002) showed weak binding to VSV / spike VLPs, with a ratio to VSV VLP of less than 3, although at 5 nM the signal for VSV / spike was higher than for VSV. As expected, an irrelevant IgG1 isotype antibody showed minimal binding. [Table 45-1] [Table 45-2]

[0240] Example 20: Anti-SARS-CoV-2-S antibodies bind to spike protein-expressing cells To investigate the ability of a panel of anti-SARS-CoV-2-S monoclonal antibodies to bind to SARS-CoV-2-S-expressing cells, an in vitro binding assay-based detection platform (MSD) was developed that utilizes SARS-CoV-2-S-expressing cells with electrochemiluminescence.

[0241] Jurkat / Tet3G / hCD20 / Tet-3G-inducible cells were engineered to transiently express the SARS-CoV-2 spike protein (accession number MN908947.3, amino acids 16-1211, Jurkat / Tet3G / hCD20 / Tet-On 3G-inducible COVID-19 spike protein High Sorted) and sorted by flow cytometry to select for high expression of SARS-CoV-2 proteins. The parental Jurkat / Tet3G / hCD20 / Tet-3G was also included in the experiment as a negative binding control.

[0242] The experiment was performed as follows. Cells from the two lines described above were induced with 1 μg / ml doxycycline at 37°C for 36 hours, then harvested, spun down, washed with PBS, diluted with PBS, and seeded onto a 96-well carbon electrode plate (using a Multi-Array High Bind Plate (MSD)). The plate was incubated overnight at 4°C to allow cell adhesion. Nonspecific binding sites were blocked with 2% (wt / vol) BSA in PBS for 1 hour at room temperature. Anti-SARS-CoV-2 antibodies and non-binding human IgG1 controls were diluted in PBS + 0.5% BSA at concentrations ranging from 0.0008 nM to 50 nM. Antibody-free buffer was added twice to the plate, and the plate was incubated at room temperature for 1 hour with shaking. Next, the plate was washed with 1x PBS and washed using an AquaMax2000 plate washer (MDS Analytical Technologies). Unbound antibody was removed. Plate-bound antibody was detected using a SULFO-TAG™-conjugated anti-human IgG antibody (Jackson Immunoresearch) for 1 hour at room temperature. After washing, the plate was developed with read buffer (MSD) according to the manufacturer's recommended procedure, and the luminescent signal was recorded on a SECTOR Imager 600 (Meso Scale). The direct binding signal (RLU) was captured in SARS-CoV-2-S expressing cells and a negative control cell line.

[0243] The ability of anti-SARS-CoV-2 monoclonal antibodies to bind to SARS-CoV-2 spike protein-expressing cells compared to binding to parental cells was assessed using an immunobinding assay. Binding to immobilized cells on a 96-well high-binding plate (MSD) was performed with a series of antibody dilutions, and bound antibody was detected using SULFO-TAG™-conjugated anti-human IgG. Binding signals from electrochemiluminescence were recorded with a Sector Imager 600 (MSD). All antibodies showed concentration-dependent binding, and the ratio of spike-expressing cell binding to parental cell binding was analyzed at concentrations of 5.5 nM and 0.20 nM.

[0244] Table 38 summarizes the binding results of anti-SARS-CoV-2-S mAbs at two concentrations to spike protein-expressing cells and parental Jurkat cells. Of the 46 antibodies tested, 44 specifically bound to Jurkat / spike cells (Jurkat / Tet3G / hCD20 / Tet-On 3G-induced SARS-CoV-2 spike protein High Sorted cells) with a ratio of 4 or greater relative to parental cells at either concentration. At 0.2 nM, the ratios of binding signals for Jurkat / spike cells and parental cells ranged from 4 to 36, and at 5 nM, the ratios ranged from 4 to 63. Two antibodies (mAb10998 and mAb11002) showed weak binding to Jurkat / spike cells, with binding ratios to parental cells less than 4, but at 5 nM, the binding signal was higher for Jurkat / spike cells than for parental cells. As expected, an irrelevant IgG1 isotype antibody showed minimal binding. [Table 46-1] [Table 46-1] *****************

[0245] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent were specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by applicant to relate to every individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, even if such citation is not immediately adjacent to the specific statement incorporated by reference. The inclusion of specific statements incorporated by reference, if any, within this specification in no way weakens this general statement of incorporation by reference. The citation of references herein is not intended as an admission that the references are relevant prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

Claims

1. An isolated antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO:832, wherein the isolated antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO:202, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

210.

2. 2. The isolated antibody or antigen-binding fragment thereof of claim 1, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 204, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 206, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 208, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 212, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

214.

3. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO:

202.

4. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, comprising an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

210.

5. 3. The isolated antibody or antigen-binding fragment thereof of claim 2, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

210.

6. 1. An isolated antibody that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO:832, wherein the isolated antibody comprises an immunoglobulin constant region; three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO:202; and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

210.

7. 7. The isolated antibody of claim 6, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 204, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 206, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 208, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 212, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

214.

8. 7. The isolated antibody of claim 6, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

210.

9. 7. The isolated antibody of claim 6, wherein the isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 216 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

218.

10. 7. The isolated antibody of claim 6, wherein the immunoglobulin constant region is an IgG1 constant region.

11. The isolated antibody of claim 6, which is a recombinant antibody.

12. The isolated antibody of claim 6, which is multispecific.

13. A pharmaceutical composition comprising the isolated antibody of claim 6 and a pharmaceutically acceptable carrier or diluent.

14. 14. The pharmaceutical composition of claim 13, further comprising a second therapeutic agent.

15. 15. The pharmaceutical composition of claim 14, wherein the second therapeutic agent is selected from the group consisting of a second antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, an anti-inflammatory agent, an anti-malarial agent, and an antibody or antigen-binding fragment thereof that binds to TMPRSS2.

16. 15. The pharmaceutical composition of claim 14, wherein the second therapeutic agent is a second antibody or antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO:

832.

17. The pharmaceutical composition of claim 16, wherein the secondary antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

646.

18. The pharmaceutical composition of claim 17, wherein the secondary antibody or its antigen-binding fragment comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 642, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 499, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 644, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 648, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 650, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO:

652.

19. The pharmaceutical composition of claim 18, wherein the secondary antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

646.

20. 20. The pharmaceutical composition of claim 19, wherein the secondary antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 654 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

656.

21. An isolated antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO:832, wherein the isolated antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO:640, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

646.

22. 22. The isolated antibody or antigen-binding fragment thereof of claim 21, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 642, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 499, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 644, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 648, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 650, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

652.

23. 22. The isolated antibody or antigen-binding fragment thereof of claim 21, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO:

640.

24. 22. The isolated antibody or antigen-binding fragment thereof of claim 21, comprising an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

646.

25. 22. The isolated antibody or antigen-binding fragment thereof of claim 21, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

646.

26. 1. An isolated antibody that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO:832, wherein the isolated antibody comprises an immunoglobulin constant region; three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO:640; and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO:

646.

27. 27. The isolated antibody of claim 26, wherein HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 642, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 499, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 644, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 648, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 650, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO:

652.

28. 27. The isolated antibody of claim 26, comprising an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

646.

29. 27. The isolated antibody of claim 26, wherein the isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 654 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

656.

30. 27. The isolated antibody of claim 26, wherein the immunoglobulin constant region is an IgG1 constant region.

31. 27. The isolated antibody of claim 26, which is a recombinant antibody.

32. 27. The isolated antibody of claim 26, which is multispecific.

33. 27. A pharmaceutical composition comprising the isolated antibody of claim 26 and a pharmaceutically acceptable carrier or diluent.

34. 34. The pharmaceutical composition of claim 33, further comprising a second therapeutic agent.

35. 35. The pharmaceutical composition of claim 34, wherein the second therapeutic agent is selected from the group consisting of a second antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 832, an anti-inflammatory agent, an anti-malarial agent, and an antibody or antigen-binding fragment thereof that binds to TMPRSS2.

36. The second therapeutic agent is a SARS-CoV- comprising the amino acid sequence set forth in SEQ ID NO:

832.

35. The pharmaceutical composition of claim 34, wherein the antibody is a secondary antibody or antigen-binding fragment thereof that binds to the 2 spike protein.

37. The pharmaceutical composition of claim 36, wherein the secondary antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) contained within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

210.

38. The pharmaceutical composition of claim 37, wherein the secondary antibody or its antigen-binding fragment comprises an HCDR1 having the amino acid sequence set forth in SEQ ID NO: 204, an HCDR2 having the amino acid sequence set forth in SEQ ID NO: 206, an HCDR3 having the amino acid sequence set forth in SEQ ID NO: 208, an LCDR1 having the amino acid sequence set forth in SEQ ID NO: 212, an LCDR2 having the amino acid sequence set forth in SEQ ID NO: 55, and an LCDR3 having the amino acid sequence set forth in SEQ ID NO:

214.

39. 39. The pharmaceutical composition of claim 38, wherein the secondary antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 202 and an LCVR comprising the amino acid sequence set forth in SEQ ID NO:

210.

40. 40. The pharmaceutical composition of claim 39, wherein the secondary antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 216 and a light chain comprising the amino acid sequence set forth in SEQ ID NO:

218.

41. 1. An isolated, recombinant antibody or antigen-binding fragment thereof that specifically binds to a coronavirus spike protein (CoV-S), wherein the antibody has the following characteristics: (a) about 10 -9 EC less than M 50 Binding to CoV-S with (b) exhibiting increased survival in coronavirus-infected animals following administration to said coronavirus-infected animals compared to comparable coronavirus-infected animals without said administration; and / or (c) an isolated recombinant antibody or antigen-binding fragment thereof, comprising one or more of: three heavy chain complementarity determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) contained within a heavy chain variable region (HCVR) comprising an amino acid sequence having at least about 90% sequence identity with an HCVR of Table 1; and three light chain CDRs (CDR-L1, CDR-L2, and CDR-L3) contained within a light chain variable region (LCVR) comprising an amino acid sequence having at least about 90% sequence identity with an LCVR of Table 1.

42. (a) an immunoglobulin heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 of an antibody of Table 1, and / or 42. The antibody or antigen-binding fragment of claim 41, comprising: (b) an immunoglobulin light chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of an antibody of Table 1.

43. (a) a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to an HCVR sequence in Table 1, and / or 43. The antibody or antigen-binding fragment of claim 41 or 42, comprising: (b) a light chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to an LCVR sequence in Table 1.

44. 44. The antibody or antigen-binding fragment of any one of claims 41 to 43, wherein the antibody or antigen-binding fragment comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of a single antibody of Table 1.

45. 45. The antibody or antigen-binding fragment of any one of claims 41 to 44, comprising an immunoglobulin comprising the HCVR and LCVR of a single antibody of Table 1.

46. An antigen-binding protein that competes with the antibody or antigen-binding fragment of any one of claims 41 to 45 for binding to CoV-S.

47. An antigen-binding protein that binds to the same epitope on CoV-S as, or an overlapping epitope on CoV-S as, the antibody or antigen-binding fragment of any one of claims 41 to 46.

48. 48. The antibody or antigen-binding fragment of any one of claims 41 to 47, which is multispecific.

49. The following characteristics: (a) inhibiting the proliferation of coronaviruses; (b) binding to the surface of the coronavirus; (c) limiting the spread of coronavirus infection of cells in vitro; and (d) protecting mice engineered to express human ACE2 or TMPRSS2 protein from death and / or weight loss caused by coronavirus infection.

50. The antibody or antigen-binding fragment of any one of claims 41 to 49, wherein the CoV-S is SARS-CoV-2-S.

51. A complex comprising the antibody or antigen-binding fragment of any one of claims 41 to 50 bound to a CoV-S polypeptide.

52. 52. The complex of claim 51, wherein the CoV-S is SARS-CoV-2-S.

53. 51. A method for producing the antibody or antigen-binding fragment of any one of claims 41 to 50, comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into a host cell; (b) culturing the host cell under conditions favorable to expression of the one or more polynucleotides; (c) optionally isolating said antibody or antibody-binding fragment from said host cell and / or the medium in which said host cell is grown.

54. 54. The method of claim 53, wherein the host cell is a Chinese hamster ovary cell.

55. 55. An antibody or antigen-binding fragment that is the product of the method of claim 53 or 54.

56. (a) CDR-H1, CDR-H2, and CDR-H3 of the HCVR domain of an antibody or antigen-binding fragment comprising an HCVR amino acid sequence set forth in Table 1; or (b) a polypeptide comprising CDR-L1, CDR-L2, and CDR-L3 of the LCVR domain of an immunoglobulin chain comprising the LCVR amino acid sequence set forth in Table 1.

57. A polynucleotide encoding the polypeptide of claim 56.

58. A vector comprising the polynucleotide of claim 57.

59. A host cell comprising the antibody or antigen-binding fragment of any one of claims 41 to 50, or the polypeptide or polynucleotide or vector of any one of claims 55 to 58.

60. A composition or kit comprising the antibody or antigen-binding fragment of any one of claims 41 to 50 and 55 in association with a further therapeutic agent.

61. 56. A pharmaceutical composition comprising the antigen binding protein of any one of claims 41 to 50 and 55, a pharmaceutically acceptable carrier, and optionally a further therapeutic agent.

62. 62. The composition or kit of claim 60 or 61, associated with an additional therapeutic agent that is an antiviral agent or a vaccine.

63. The composition or kit of any one of claims 60 to 62, wherein the additional therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an anti-malarial agent, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S.

64. 64. The composition or kit of claim 63, wherein the antimalarial agent is chloroquine or hydroxychloroquine.

65. 64. The composition or kit of claim 63, wherein the anti-inflammatory agent is an antibody.

66. 66. The composition or kit of claim 65, wherein the antibody is sarilumab, tocilizumab, or gimsilumab.

67. 64. The composition or kit of claim 63, wherein the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of a secondary antibody of Table 1.

68. 64. A container or injection device comprising an antigen-binding protein or composition according to any one of claims 41 to 51, 55 and 60 to 63.

69. 56. A method for treating or preventing infection by a coronavirus in a subject in need thereof, the method comprising administering a therapeutically effective amount of an antigen binding protein of any one of claims 41 to 50 and 55.

70. 69. The method of claim 68, wherein the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, and MERS-CoV.

71. 71. The method of claim 69 or 70, wherein the subject is administered one or more additional therapeutic agents.

72. 72. The method of claim 71, wherein the one or more additional therapeutic agents is an antiviral agent or a vaccine.

73. The one or more additional therapeutic agents may be an anti-inflammatory agent, an anti-malarial agent, an anti-TMPRSS2 specific agent, and an antibody or antigen-binding fragment that specifically binds to CoV-S.

74. 74. The method of claim 73, wherein the antimalarial agent is chloroquine or hydroxychloroquine.

75. 74. The method of claim 73, wherein the anti-inflammatory agent is an antibody.

76. 76. The method of claim 75, wherein the antibody is sarilumab, tocilizumab, or gimsilumab.

77. 74. The method of claim 73, wherein the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of a secondary antibody of Table 1.

78. 56. A method for administering the antibody or antigen-binding fragment of any one of claims 41 to 50 and 55 into a subject, the method comprising injecting the antibody or antigen-binding fragment into the subject.

79. 79. The method of claim 78, wherein the antibody or antigen-binding fragment is injected into the subject subcutaneously, intravenously, or intramuscularly.

80. 80. The antibody or antigen-binding fragment, composition, kit, conjugate, polypeptide, polynucleotide, vector, cell, or method of any one of claims 41 to 79, wherein the antibody or antigen-binding fragment comprises a VH3-66 or Vk1-33 variable domain sequence.

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