Anti-SARS-COV-2 Spike Glycoprotein Antibody and Antigen-Binding Fragment

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.SOLUTION: The present disclosure provides a neutralizing human antibody- binding protein that binds specifically to SARS-CoV-2-S, such as an antibody and an antigen-binding fragment thereof.SELECTED DRAWING: None

Description

Technical Field

[0001] Sequence Listing The official copy of the sequence listing is electronically submitted via EFS-Web simultaneously with the specification as an ASCII-formatted sequence listing with the file name "10753WO01-Sequence.txt", creation date June 25, 2020, and size of approximately 922,462 bytes. The sequence listing contained in this ASCII-formatted document is part of the specification and is hereby incorporated by reference in its entirety into this specification.

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

Background Art

[0003] Newly identified viruses such as coronaviruses are difficult to treat because they are not fully characterized. The emergence of these newly identified viruses has highlighted 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, and as of March 20, 2020, the World Health Organization reported 209,839 confirmed cases and 8,778 deaths in 168 countries, regions, or territories. The clinical features of COVID-19 include fever, dry cough, and fatigue, and this disease can cause respiratory failure and death.

[0004] So far, there has been no vaccine or therapeutic agent for preventing or treating SARS-CoV-2 infection. Considering the continuous threat to human health, there is an urgent need for prophylactic and therapeutic antiviral therapies for SARS-CoV-2 control. This virus uses its spike glycoprotein for interaction with the cellular receptor ACE2 and serine protease TMPRSS2 for entry into target cells, so this spike protein is 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 to neutralize therapeutic anti-SARS-CoV-2-spike protein (SARS-CoV-2-S) antibodies and use them 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 of Table 1, and combinations thereof including, for example, other therapeutic agents (e.g., anti-inflammatory agents, anti-malarial agents, antiviral agents, or other antibodies or antigen-binding fragments), and methods of using them for treating viral infection.

[0006] The present disclosure provides a neutralizing human antigen-binding protein that specifically binds to SARS-CoV-2-S, such as an antibody or an antigen-binding fragment thereof.

[0007] In one aspect, the present disclosure provides an isolated recombinant antibody or an antigen-binding fragment thereof that specifically binds to a coronavirus spike protein (CoV-S), wherein the antibody has the following characteristics: (a) binds to CoV-S with an EC -9 of less than about 10 50 M, (b) coronavirus In the coronavirus-infected animal after administration to the infected animal, showing an increase in survival as compared to an equivalent coronavirus-infected animal without such administration, and / or (c) 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 the 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 the LCVR of Table 1, provided is an isolated recombinant antibody or an antigen-binding fragment thereof having one or more of the above.

[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 the antibody of Table 1, and / or (b) an immunoglobulin light-chain variable region comprising CDR-L1, CDR-L2, and CDR-L3 of the 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 the HCVR sequence of 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 the LCVR sequence of 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 for binding to CoV-S with any one of the antibodies or antigen-binding fragments described above or discussed herein.

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

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

[0014] In any of the various embodiments, the antibody or antigen-binding fragment can include one or more of the following properties: a) inhibiting the growth of coronavirus, b) binding to the surface of coronavirus, c) restricting the spread of in vitro cell coronavirus infection, 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, CoV-S is SARS-CoV-2-S.

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

[0017] In one aspect, the present disclosure provides an antibody or antigen-binding fragment described above or herein A method for producing the antibody or antigen-binding fragment, 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 for the 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 grows. In some embodiments, the host cell is a Chinese hamster ovary cell.

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

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

[0020] In one aspect, the present disclosure provides a polynucleotide encoding the polypeptide 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 an antibody or antigen-binding fragment or polypeptide or polynucleotide or vector discussed above or herein.

[0023] In one aspect, the present disclosure provides a composition or kit comprising an antibody or antigen-binding fragment discussed above or herein, in connection with a further therapeutic agent.

[0024] In one aspect, the present disclosure provides a pharmaceutical composition comprising an antigen-binding protein, antibody or antigen-binding fragment as described above or discussed herein, a pharmaceutically acceptable carrier, and optionally one or more additional therapeutic agents. In some embodiments, the additional therapeutic agent is an antiviral drug or a vaccine. In some embodiments, the additional therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an antimalarial agent, an antibody or antigen-binding fragment that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment that specifically binds to CoV-S. Optionally, the antimalarial agent is chloroquine or hydroxychloroquine. Optionally, the anti-inflammatory agent is an antibody such as sarilumab, tocilizumab, or gemtuzumab. 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 an injection device comprising an antigen-binding protein, antibody or antigen-binding fragment as described above or discussed herein, or a composition.

[0026] In one aspect, the present disclosure provides a method for treating or preventing infection with a coronavirus in a subject in need thereof, the method comprising administering a therapeutically effective amount of an antigen-binding protein, antibody or antigen-binding fragment as described above or discussed 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 with a coronavirus Thereafter, the subject is administered one or more additional therapeutic agents. Optionally, the one or more additional therapeutic agents are antiviral agents or vaccines. Optionally, the one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, antimalarial agents, antibodies or antigen-binding fragments thereof that specifically bind to TMPRSS2, and antibodies or antigen-binding fragments thereof that specifically bind to CoV-S. Optionally, the antimalarial agent is chloroquine or hydroxychloroquine. Optionally, the anti-inflammatory agent is an antibody such as, for example, sarilumab, tocilizumab, or gemtuzumab. 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, 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 as described above or discussed herein to a subject's body, comprising injecting the antibody or antigen-binding fragment into the subject's body. In some embodiments, the antibody or antigen-binding fragment is injected subcutaneously, intravenously, or intramuscularly into the subject's body.

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

[0030] In one aspect, the present disclosure provides an isolated 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, wherein the isolated antibody or antigen-binding fragment comprises three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) 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.

[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 present 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, HCDR 2. and three heavy chain complementarity determining regions (HCDR1, HCDR2, and HCDR3) 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 (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 210, and provides an isolated antibody.

[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 the isolated antibody 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 secondary 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, an anti-inflammatory agent, an antimalarial 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 an 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 an 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. Optionally, the secondary antibody or an 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 an 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. Optionally, the secondary antibody or an 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 present disclosure provides an isolated antibody or an 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, wherein the isolated antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained 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) contained 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 a 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 a 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 a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 640 and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 646.

[0039] In one aspect, the present 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, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained 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) contained 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 present 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, a pharmaceutical composition further comprising a second therapeutic agent. Optionally, the second therapeutic agent is selected from the group consisting of 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, 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 an 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 an 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. Optionally, the secondary antibody or an 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, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 214. Optionally, the secondary antibody or an 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. Optionally, the secondary antibody or an 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 above or the embodiments discussed herein can be combined, and such combinations are included within the scope of the present disclosure. Any of the specific values discussed above or herein can be combined with another related value discussed above or herein to enumerate ranges representing the upper and lower limits of the ranges, and such ranges are included within the scope of the present disclosure.

Brief Description of the Drawings

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DETAILED DESCRIPTION OF THE INVENTION

[0046] Before explaining the method of the present invention, it should be understood that the present invention is not limited to such methods and conditions, as the specific methods and experimental conditions described may vary. It should also be understood that the terms used herein are for the purpose of describing only specific embodiments and are not intended to be limiting, as the scope of the present invention is 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 described herein. All publications mentioned herein are hereby incorporated by reference in their entirety.

[0048] The term "coronavirus" or "CoV" refers to any virus of the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to a newly emerged coronavirus identified as the cause of a severe outbreak in Wuhan, China, and has rapidly spread to other regions around the world. SARS-CoV-2 is also known as 2019-nCoV and Wuhan coronavirus. It binds to the human host cell receptor angiotensin-converting enzyme 2 (ACE2) via the viral spike protein. 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 type I membrane glycoprotein of 1273 amino acids that assembles into trimers that form spikes or peplomers on the surface of the enveloped coronavirus particle. 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 via the 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. This term also encompasses, for example, CoV spike proteins or fragments thereof linked to signal sequences such as histidine tags, mouse or human Fc, or ROR1.

[0050] As used herein, the terms "coronavirus infection" or "CoV infection" refer to infection with coronaviruses such as SARS-CoV-2, MERS-CoV, or SARS-CoV. This term includes coronavirus respiratory infections, which often infect 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 kidney dysfunction), septic shock, and death in severe cases.

[0051] virus The present invention includes a method for treating or preventing viral infection in a subject. The term "virus" includes any virus (e.g., a virus whose infectivity is at least partially dependent on CoV-S) whose infection in the body of a subject is treatable or preventable by administration of an anti-CoV-S antibody or an antigen-binding fragment thereof. In one embodiment of the present invention, the "virus" is any virus that expresses a spike protein (e.g., CoV-S). The term "virus" also includes CoV-S-dependent respiratory viruses that infect the respiratory tissues of a subject (e.g., the upper and / or lower respiratory tract, trachea, bronchi, lungs) and are treatable or preventable by administration of an anti-CoV-S antibody or an 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 alpha coronavirus, beta coronavirus, gamma coronavirus, and delta coronavirus. In some embodiments, the antibodies or antigen-binding fragments provided herein can bind to and / or neutralize alpha coronaviruses, beta coronaviruses, gamma coronaviruses, and / or delta coronaviruses. In certain embodiments, this binding and / or neutralization can be specific for a particular genus or a particular subgroup of a genus. "Viral infection" refers to the entry and proliferation of a virus in the body of a subject.

[0052] Coronavirus virions are spherical and have a diameter of approximately 125 nm. The most prominent feature of coronaviruses is the club-shaped spike projections that emanate from the surface of the virion. These spikes characterize the virion, give it the appearance of the solar corona, and prompt the name coronavirus. Inside the envelope of the virion is the nucleocapsid. Coronaviruses have a helically symmetric nucleocapsid, which is not common among positive-sense RNA viruses but is much more common among negative-sense RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the coronavirus family. The initial attachment of the virion to the host cell is initiated by the interaction between the S protein and its receptor. The site of the receptor-binding domain (RBD) within the S1 region of the coronavirus S protein varies among viruses, and some have the RBD at the C-terminus of S1. The S protein / receptor interaction is a major determinant for a coronavirus to infect host species and also governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as cell receptors. Following receptor binding, the virus must then access the cytoplasm of the host cell. This is generally achieved by acid-dependent proteolytic cleavage of the S protein by cathepsin, TMPRRS2, or another protease, followed by fusion of the viral membrane with the cell 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 a CoV spike protein or an antigenic fragment thereof.

[0054] As used herein, the term "antibody" refers to an immunoglobulin molecule (i.e., a "complete antibody molecule") comprising four polypeptide chains interconnected by disulfide bonds, two heavy chains (HC) and two light chains (LC), and multimers thereof (e.g., IgM). Exemplary antibodies include, for example, those listed in Table 1. Each heavy chain has a heavy chain variable region ("HCVR" or "VH ) and a heavy chain constant region (C H 1 domain, C H 2 domain, and C H 3 domain). Each light chain comprises a light chain variable region (「LC VR or 「V L ) and a light chain constant region (C L ). The V H region and the V L region can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with more conserved regions called framework regions (FR). Each V H and V LIt contains three CDRs and four FRs arranged from the amino terminus to the carboxy terminus in the order of FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy-chain CDRs may also be referred to as HCDR or CDR-H and are numbered as described above (e.g., HCDR1, HCDR2, and HCDR3 or CDR-H1, CDR-H2, and CDR-H3). Similarly, the light-chain CDRs are referred to as LCDR or CDR-L and are numbered LCDR1, LCDR2, and LCDR3, or CDR-L1, CDR-L2, and CDR-L3. In certain embodiments of the invention, the FRs of the antibody (or antigen-binding fragment thereof) are identical to human germline sequences or are modified naturally or artificially. Exemplary human germline sequences include, but are not limited to, VH3-66 and Vk1-33. Accordingly, the present disclosure provides anti-CoV-S antibodies or antigen-binding fragments thereof (e.g., anti-SARS-CoV-2-S antibodies or antigen-binding fragments thereof) that contain the HCDR and LCDR sequences of Table 1 within the VH3-66 or Vk1-33 variable heavy or light chain regions. The present disclosure further includes 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, Provided is an anti-CoV-S antibody or an antigen-binding fragment thereof (e.g., an anti-SARS-CoV-2-S antibody or an antigen-binding fragment thereof) that contains the HCDR and LCDR sequences of Table 1 within a combination of a light chain selected from IgLV2-23 or IgLV6-57 and a heavy chain selected from 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, IgHV1-2, 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 provides an anti-CoV-S antibody or an antigen-binding fragment thereof (e.g., an anti-SARS-CoV-2-S antibody or an antigen-binding fragment thereof) that contains the HCVR and LCVR sequences of Table 1 within a combination of a light chain selected from 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, IgLV2-23, or IgLV6-57 and a heavy chain selected from 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, IgHV1-2, IgHV2-70, IgHV3-66, IgHV5-51, IgHV1-46, IgHV4-39, IgHV4-31, IgHV3-30-3, IgHV2-26, or IgHV7-4-1.

[0055] Typically, the variable domains of both immunoglobulin heavy and light chains contain three hypervariable regions, also called complementarity-determining regions (CDRs), which are located within relatively conserved framework regions (FRs). Generally, from the N-terminus to the C-terminus, the variable domains of both the light and heavy chains include FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In embodiments of the present invention, the amino acid assignments to each domain are in accordance with the definitions of 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 Chothia, et al., (1989) Nature 342:878-883.

[0056] The present invention includes monoclonal anti-CoV-S antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, and monoclonal compositions comprising a plurality of isolated monoclonal antigen-binding proteins. As used herein, the term "monoclonal antibody" refers to a substantially homogeneous population of 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. Such "plurality" of monoclonal antibodies and fragments in a composition refers to a higher 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 than normally occurs in nature, e.g., in the blood of a host organism such as a mouse or a human.

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

[0058] As used herein, the term "human" antigen-binding protein, such as an antibody, includes an antibody having variable and constant regions derived from human germline immunoglobulin sequences, whether in human cells or transplanted into non-human cells, such as mouse cells. See, for example, US8502018, US6596541, or US5789215. The human mAbs of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by in vitro random or site-directed mutagenesis or by somatic mutations in vivo), for example, in the CDRs, particularly CDR3. 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 transplanted onto human FR sequences. The term includes antibodies recombinantly produced in non-human mammals or in 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, such as antibodies and antigen-binding fragments thereof, and methods of using the same. As used herein, a "chimeric antibody" is an antibody having a variable domain from a primary antibody and a constant domain from a secondary antibody, wherein the primary and secondary antibodies are from different species. (US4816567, 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, such as antibodies and antigen-binding fragments thereof, and methods of using the same. As used herein, a "hybrid antibody" is an antibody having a variable domain from a primary antibody and a constant domain from a secondary antibody, where the primary and secondary antibodies are from different animals, or the variable domain (not the constant region) is from a first animal. For example, the variable domain can be obtained from an antibody isolated from a human and expressed with a constant domain that is not isolated from that antibody. Exemplary hybrid antibodies are described in Example 1 and refer to PCR products derived from antibody heavy chain variable regions and light chain variable regions cloned into expression vectors containing heavy chain constant regions and light chain constant regions, respectively. Hybrid antibodies are synthetic and not found in nature because the variable and constant regions contained therein 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 a molecule made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA techniques, including, for example, DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammalian (including transgenic non-human mammals, such as transgenic mice), or cell (e.g., CHO cell) expression systems, or antibodies isolated from recombinant combinatorial human antibody libraries. In some embodiments, the recombinant antibody shares a sequence with an antibody isolated from an organism (e.g., a mouse or a human), but has been expressed via recombinant DNA techniques. Such an antibody can have post-translational modifications (e.g., glycosylation) different from those of an antibody isolated from an organism.

[0062] The recombinant anti-CoV-S antigen-binding proteins disclosed in this specification, such as antibodies and antigen-binding fragments, can also be produced in an E. coli / T7 expression system. In this embodiment, the nucleic acid encoding the anti-CoV-S antibody immunoglobulin molecule of the present invention (e.g., as found in Table 1) can be inserted into a pET-based plasmid and expressed in the E. coli / T7 system. For example, the present invention includes a method for expressing an antibody or its antigen-binding fragment or its immunoglobulin chain in a host cell (e.g., a bacterial host cell such as E. coli, such as BL21 or BL21DE3), which includes expressing T7 RNA polymerase in a cell that also includes 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 includes a polynucleotide encoding a T7 RNA polymerase gene, and the expression of the polymerase and the chain is induced by incubating the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). See US4952496 and US5693489, 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 generating recombinant antibodies known in the art. An example of a method for the recombinant production of antibodies is disclosed in US4816567.

[0064] Transformation can be by any known method for introducing a polynucleotide (e.g., including DNA, RNA, mRNA) into a host cell. 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 the polynucleotide(s) into liposomes, lipid nanoparticle technology, particle bombardment 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 lentivirus or adeno-associated virus. 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, the antibodies or antigen-binding fragments thereof of the present disclosure can be introduced into a subject in nucleic acid form (e.g., including DNA, RNA, mRNA) such 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 affinity or specificity of the antibody.

[0065] Accordingly, the present invention is a recombinant method for producing an anti-CoV-S antigen-binding protein such as an antibody or an antigen-binding fragment thereof, or an immunoglobulin chain thereof, of the present invention, comprising: (i) introducing an antigen-binding protein, for example, a light chain and / or a heavy chain of an immunoglobulin of Table 1, or one or more polynucleotides encoding CDRs (for example, comprising any one or more nucleotide sequences of the sequences of Table 2) (wherein the polynucleotide is in a vector and / or integrated into the host cell chromosome and / or operably linked to a promoter), (ii) culturing a host cell (for example, CHO or Pichia or Pichia pastoris) under conditions favorable for the expression of the polynucleotide, and (iii) optionally, isolating the antigen-binding protein (for example, an antibody or fragment) or chain from the host cell and / or the medium in which the host cell grows. For example, the polynucleotide may be, for example, a gene editing system (e.g For example, after cleaving a chromosome using CRISPR (e.g., CRISPR-Cas9), TALEN, megaTAL, zinc finger, or Argonaute), a vector such as adeno-associated virus (AAV) can be inserted targeting and integrated into the host cell chromosome. Targeted insertion can occur at host cell loci such as, for example, the albumin or immunoglobulin genomic loci. Alternatively, the insertion can be performed at random loci using a vector such as, for example, a lentivirus. When producing an antigen-binding protein (e.g., an antibody or antigen-binding fragment) containing more than one immunoglobulin chain, e.g., an antibody containing two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of the chains in a single host cell results in association of the chains intracellularly or on the cell surface or extracellularly such that, for example, an antigen-binding protein (e.g., an antibody or antigen-binding fragment) is formed when such chains are secreted. The method includes 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 their variable domains) is expressed. Such chains are useful, for example, as intermediates in the expression of an antibody or antigen-binding fragment containing such chains. For example, the present invention also includes a heavy chain immunoglobulin (or including its variable domain or its CDR) encoded by a polynucleotide containing the nucleotide sequence set forth in Table 2, and a light chain immunoglobulin (or including its variable domain or its CDR) (which is a product of such a production method) encoded by the nucleotide sequence set forth in Table 2, an anti-CoV-S antigen-binding protein such as an antibody and its antigen-binding fragments, and optionally, a purification method 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 containing an HCVR containing the amino acid sequence set forth in Table 1 and an LCVR containing the amino acid sequence set forth in Table 1, and 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 thereof 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 thereof 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 containing 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 can be obtained 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, simian 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 cells from humans, mice, rats, dogs, monkeys, pigs, goats, cows, horses, and hamsters. Other cell lines that can be used are 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 Yeast and filamentous fungal cells are included, including 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 affinity for an antigen such as the S protein (e.g., SARS-CoV-2-S) of at least about 10 D expressed as K M when 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 soluble affinity ELISA. The present invention includes antigen-binding proteins that specifically bind to a CoV-S protein. -8 By "antigen-binding portion" or "antigen-binding fragment" of an antibody, or the term antigen-binding protein, and the like, as used herein, is meant any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen and forms a complex. Non-limiting examples of antibody-binding fragments include (i) Fab fragments, (ii) F(ab’)

[0068] fragments, (iii) Fd fragments, (iv) Fv fragments 2 , (v) single-chain Fv (scFv) molecules (wherein a variable heavy chain and a variable light chain are joined by a linker), (vi) single-domain antibodies, (vii) tandem single-domain antibodies, and (viii) multispecific antibodies formed from antibody fragments. Examples of the minimal recognition units include a fragment, (v) a single-chain Fv (scFv) molecule, (vi) a dAb fragment, and (vii) amino acid residues mimicking the hypervariable regions of an antibody (e.g., an isolated complementarity-determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain-deleted antibodies, chimeric antibodies, CDR-grafted antibodies, diabodies, triabodies, tetra-bodies, mini-bodies, nanobodies (e.g., as defined in WO08 / 020079 or WO09 / 138519) (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunopharmaceuticals (SMIPs), and other engineered molecules such as shark variable IgNAR domains are also included within the expression "antigen-binding fragment" as used herein. In one embodiment of the present invention, the antigen-binding fragment comprises three or more CDRs of the antibody of Table 1 (e.g., CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3).

[0069] The antigen-binding fragment of an antibody, in embodiments of the present invention, comprises at least one variable domain. The variable domain can be of any size or amino acid composition and generally comprises at least one CDR flanked by or in-frame with one or more framework sequences. V L domain bound to V H In an antigen-binding fragment having a V H domain and a V L domain, the V H -V H and V H -V L or V L -V L dimers can be included. Alternatively, the antigen-binding fragment of an antibody can comprise a monomeric V H or V L domain.

[0070] In certain embodiments, an antigen-binding fragment of an antibody can include at least one variable domain covalently attached to at least one constant domain. Non-limiting exemplary conformations of variable and constant domains that can be found within the antigen-binding fragments of the antibodies of the present 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 Linclude. In any three-dimensional arrangement of the variable domain and the constant domain, including any of the exemplary three-dimensional arrangements listed above, the variable domain and the constant domain may be directly linked to each other or may be linked by a complete or partial hinge region or linker region. The hinge region can consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that provide a flexible or semi-flexible bond between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragments of the antibodies of the present invention can be non-covalent bonds (e.g., by disulfide bond(s)) with each other and / or with one or more monomeric V H or V L domains, and can include homodimers or heterodimers (or other multimers) of any of the variable domain three-dimensional arrangements and constant domain three-dimensional arrangements listed above.

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

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

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

[0074] An "isolated" antigen-binding protein, antibody or antigen-binding fragment thereof, polypeptide, polynucleotide, and vector is at least partially free of other biological molecules from the cells 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 cell debris and growth media. An isolated antibody or antigen-binding fragment may also be at least partially free of expression system components such as biological molecules from the host cell or its growth media. Generally, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffer, or salts, or components of a pharmaceutical formulation containing the antibody or fragment).

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

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

[0077] As used herein, the term "competing" refers to an antigen-binding protein (e.g., an antibody or an 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 an antigen-binding fragment thereof) to the antigen. The term 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) can bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) can bind to different, e.g., overlapping, epitopes, and one binding inhibits or blocks the binding of a secondary antibody, e.g., via steric hindrance. Competition between antigen-binding proteins (e.g., antibodies) can be measured by methods known in the art, e.g., by a real-time label-free biolayer interferometry assay. 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 a spike protein receptor binding domain (RBD) monomer), competing for the same epitope, or 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., an antibody) is determined by a soluble CoV-S protein complexed with the second anti-CoV-S antigen-binding protein (e.g., an antibody). It is determined by measuring the ability of an immobilized first anti-CoV-S antigen-binding protein (e.g., an antibody) that binds to a protein (initially not complexed with the CoV-S protein). A decrease in the ability of a first anti-CoV-S antigen-binding protein (e.g., an antibody) to bind to the complexed CoV-S protein as compared to the uncomplexed CoV-S protein indicates that the first and second anti-CoV-S antigen-binding proteins (e.g., antibodies) compete. The degree of competition can be expressed as the rate of decrease in binding. Such competition can be measured using a real-time label-free biolayer interferometry assay, for example, with an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assay), or SPR (surface plasmon resonance).

[0078] The 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 the competition between two anti-CoV-S monoclonal antibodies, first, the anti-CoV-S mAb can 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 the anti-CoV-S mAb (hereinafter referred to as "mAb1"). Then, as a positive control for blocking, the antibody-captured biosensor chip can be saturated with a known blocking isotype control mAb (hereinafter 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 is then immersed in a co-complex solution of a CoV-S polypeptide pre-incubated for a certain time and a second anti-CoV-S mAb (hereinafter referred to as "mAb2") to determine the binding of mAb1 to the CoV-S polypeptide. The biosensor chip can be washed with buffer between each step of the experiment. The real-time binding response can be monitored during the course of the experiment, and the binding response at the end of each step can be recorded.

[0079] For example, in embodiments of the present invention, the competition assay was performed at 25°C and a pH of about 7, e.g., 7.4, in the presence of, for example, buffer, salts, surfactants, and non-specific proteins (e.g., bovine serum albumin).

[0080] Typically, the antibody or antigen-binding fragment of the invention that is modified in some way retains the ability to specifically bind to CoV-S, and for example, when its activity is expressed on a molar basis, retains at least 10% of its CoV-S binding activity (when compared to the parental antibody). Preferably, the antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the CoV-S binding affinity as the parental antibody. It is also contemplated that the antibody or antigen-binding fragment of the invention may contain conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conserved variants" of the antibody) that do not substantially alter its biological activity.

[0081] A "variant" of a polypeptide such as an immunoglobulin chain (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) refers to a polypeptide that contains an amino acid 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 or similar to the reference amino acid sequence described herein (e.g., SEQ ID NO: 2, 10, 18, 20, 22, 30, 38, 40, 42, 50, 58, or 60) when the comparison is made by the BLAST algorithm, and the parameters of the algorithm are selected to obtain the maximum match between the respective sequences over the entire length of each reference sequence (e.g., expect value: 10, word size: 3, maximum match in query range: 0, BLOSUM 62 matrix, gap cost: existence 11, extension 1, conditional composition score matrix adjustment). Each is selected such that the maximum match is obtained between the respective sequences (e.g., expect value: 10, word size: 3, maximum match in query range: 0, BLOSUM 62 matrix, gap cost: existence 11, extension 1, conditional composition score matrix adjustment).

[0082] A "variant" of a polynucleotide, when the comparison is made by the BLAST algorithm, refers to a polynucleotide comprising 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 the reference nucleotide sequences described herein (e.g., SEQ ID NO: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, or 59), and the parameters of the algorithm are selected to obtain the maximum match between the respective sequences over the entire length of each reference sequence (e.g., expected threshold value: 10, word size: 28, maximum match in query range: 0, match / mismatch score: 1, -2, gap cost: linear).

[0083] In an embodiment of the present invention, the anti-CoV-S antigen-binding protein of the present invention, such as an antibody and its antigen-binding fragment, comprises 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 sequence described 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 sequence described in Table 1.

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

[0085] The present invention further provides a variant anti-CoV-S antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof, comprising, for example, one or more variant CDRs (e.g., any one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3) described herein 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 correspond to the specific V described herein H 、V L, an amino acid sequence of HC, or LC, and an amino acid sequence having an overall amino acid sequence identity or similarity of 70% or more (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more), including an immunoglobulin V and V H and V L , or a variant antigen-binding protein including HC and LC, such as an anti-CoV-S antibody and its antigen-binding fragment, also includes, but the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 of such immunoglobulins include the CDR amino acid sequences described in Table 1, not variants. Thus, in such embodiments, the CDRs within the variant antigen-binding protein are not variants themselves.

[0087] Conservatively modified variant anti-CoV-S antibodies and their antigen-binding fragments are also part of the present invention. "Conservatively modified variant" or "conservative substitution" refers to a variant having one or more substitutions of an amino acid in a polypeptide by another amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone structure, and rigidity, etc.). Such changes can be frequently made without significantly disrupting the biological activity of the antibody or fragment. Those skilled in the art generally recognize that a single amino acid substitution in a non-essential region of a polypeptide does not substantially change its biological activity (e.g., see Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4 th ED.). In addition, substitutions of structurally or functionally similar amino acids are less likely to significantly disrupt 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, conservative substitution is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 - 45.

[0089] Functionally 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) can be a "functionally conservative variant". Such functionally conservative variants may in some cases be characterized as conservatively modified variants. As used herein, "functionally 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 changed without significantly altering one or more functional properties of the antibody or fragment. In embodiments of the present invention, the functionally conservative variant anti-CoV-S antibody of the present invention or antigen-binding fragment thereof comprises a variant amino acid sequence and exhibits one or more of the following functional properties. · inhibiting the growth 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), · not significantly binding to MDCK / Tet-on cells that do not express ACE2 and / or TMPRSS2, · Limiting the spread of in vitro cell infection with coronaviruses, such as Calu-3 cells infected with (e.g., by SARS-CoV-2, SARS-CoV, and / or MERS-CoV), and / or · Protecting mice engineered to express human TMPRSS2 and / or ACE2 proteins from death caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) (e.g., optionally when combined with a second therapeutic agent, the mice would otherwise be infected with a lethal dose of the virus). · 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., optionally when combined with a second therapeutic agent, the mice would otherwise be infected with a dose of the virus that causes weight loss).

[0090] "Neutralizing" or "antagonist" anti-CoV-S antigen-binding proteins, such as antibodies or antigen-binding fragments, inhibit the activity of CoV-S to any detectable extent, e.g., by proteases such as TMPRSS2, or bind to receptors such as ACE2 that mediate viral entry into host cells or viral replication in host cells, and inhibit the ability of CoV-S to bind to such receptors.

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

[0092] The antibodies described herein also have V H wherein is fused to 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 containing the amino acid sequences described herein, as well as cellular and post-translational modifications to the antibodies. For example, the present invention provides antibodies and their antigen-binding fragments that specifically bind to CoV-S and contain the heavy chain and / or light chain amino acid sequences (e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3) described herein, 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 following table of exemplary sequences.

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] Administration of the antibody The present invention provides a method for administering an anti-CoV-S antigen-binding protein of the present invention, such as those in Table 1, the method comprising introducing the antigen-binding protein into the body of a subject (e.g., a human). For example, the method may include piercing the body of the subject with a syringe needle and injecting the antigen-binding protein into the body of the subject, such as into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0096] The present invention provides a container (e.g., a plastic or glass vial having a cap or chromatography column, a hollow bore needle, or a syringe cylinder) containing an anti-CoV-S antigen-binding protein of the present invention, such as 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, such as those in Table 1, or a pharmaceutical composition thereof. The injection device may be packaged in a kit. The injection device is a device for introducing a substance into the body of a subject via a parenteral route, such as intramuscular, subcutaneous, or intravenous. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition such as an auto-injector) comprising a cylinder or barrel for holding a fluid to be injected (e.g., containing an antibody or fragment or a pharmaceutical composition thereof), a needle for piercing the skin and / or blood vessel to inject the fluid, and a plunger for pushing the fluid out of the cylinder and through the hole of the needle. In one embodiment of the present invention, the injection device, or a pharmaceutical composition thereof, comprising an antigen-binding protein, such as an antibody or an antigen-binding fragment thereof, from the combination of the present invention is an intravenous (IV) injection device. Such a device may comprise an antigen-binding protein or a pharmaceutical composition thereof within a cannula or trocar / needle that can be attached to a tube that can be attached to a bag or reservoir for holding a fluid (e.g., saline) introduced into the body of the subject through the cannula or trocar / needle. In one embodiment of the present invention, the antibody or fragment thereof or a pharmaceutical composition thereof can be introduced into the device when the trocar and cannula are inserted into the vein of the subject and the trocar is removed from the inserted cannula. The IV device can be inserted, for example, into a peripheral vein (e.g., hand or arm), the superior vena cava or inferior vena cava, or within the right atrium of the heart (e.g., central IV), or the subclavian vein, internal jugular vein, or femoral vein, and advanced towards the heart until it reaches, for example, the superior vena cava or right atrium (such as a central venous line). In one embodiment of the present invention, the injection device is an auto-injector, a jet injector, or an external infusion pump. A jet injector uses a narrow, high-pressure liquid jet that penetrates the epidermis Thereafter, an antibody or fragment or a pharmaceutical composition thereof is introduced into the body of the subject. An external infusion pump is a medical device that delivers an antibody or fragment or a pharmaceutical composition thereof into the body of the subject in a controlled amount. The external infusion pump can be powered electrically or mechanically. Various pumps operate in various ways. For example, a syringe pump holds fluid in a syringe reservoir, and a movable piston controls the supply of the fluid. An elastomeric pump holds fluid in a stretchable balloon reservoir, and the pressure from the elastic wall of the balloon facilitates the supply of the fluid. In a peristaltic pump, a set of rollers pinches the length of a flexible tube to push the liquid forward. In a multi-channel pump, fluid can be supplied 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 produce human antibodies that specifically bind to CoV-S. To generate antibodies against CoV-S, an immunogen containing any one of the following can be used. In a particular embodiment of the present invention, the antibodies of the present invention are obtained from mice immunized with full-length native CoV-S, or attenuated or inactivated virus, or DNA encoding a protein or a fragment thereof. Alternatively, the CoV-S protein or a fragment thereof can be generated and modified using standard biochemical techniques and used as an immunogen. In one embodiment of the present invention, the immunogen is a recombinantly produced CoV-S protein or a fragment thereof. In a particular embodiment of the present invention, the immunogen can be a CoV-S polypeptide vaccine. In a particular embodiment, one or more booster immunizations can be administered. In a particular embodiment, 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] To generate monoclonal antibodies, high-affinity chimeric antibodies against CoV-S having human variable regions and mouse constant regions can first be isolated using the VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method. The VELOCIMMUNE® technology involves the generation of transgenic mice in which the genome, including the human heavy chain variable region and the human light chain variable region, is operably linked to the endogenous mouse constant region locus such that the mouse generates antibodies that, in response to antigen stimulation, contain human variable regions and mouse constant regions. DNA encoding the variable regions of the heavy and light chains of the antibody is isolated and operably linked to DNA encoding the human heavy chain constant region and the human light chain constant region. The DNA is then expressed in cells capable of expressing a fully human antibody.

[0100] Generally, VELOCIMMUNE® mice are loaded with the antigen of interest and lymphocytes (such as B cells) are recovered from the mice expressing the antibody. The lymphocytes can be fused with a myeloma cell line to prepare an immortalized hybridoma cell line, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific for the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and ligated 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, antigen-specific chimeric antibodies or DNA encoding the variable domains of the light and heavy chains can be isolated directly from antigen-specific lymphocytes.

[0101] First, high-affinity chimeric antibodies having human variable regions and mouse constant regions are isolated. As in the following experimental section, the antibodies have the desired characteristics, including affinity, selectivity, epitope, etc. It is characterized by and selected. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody of the present invention, such as wild-type or modified IgG1 or IgG4. The constant region selected can vary depending on the specific use, but the high-affinity antigen-binding characteristics and target-specificity characteristics reside in the variable region.

[0102] Antibody against coronavirus spike protein containing Fc variant According to certain embodiments of the present invention, there is provided an anti-CoV-S antigen-binding protein, such as an antibody or antigen-binding fragment, comprising an Fc domain containing one or more mutations that enhance or decrease antibody binding to the FcRn receptor, for example, at an acidic pH compared to a neutral pH. For example, the present invention relates to the C of the Fc domain H 2 or C HAn anti-CoV-S antibody comprising a mutation in the 3 domain, wherein the mutation(s) increase the affinity of the Fc domain for FcRn in an acidic environment (e.g., in an endosome with a pH in the range of about 5.5 to about 6.0). Such mutations can result in an increase in the serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at position 250 (e.g., E or Q), positions 250 and 428 (e.g., L or F), position 252 (e.g., L / Y / F / W or T), position 254 (e.g., S or T), and position 256 (e.g., S / R / Q / E / D or T), or modifications at position 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or position 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F, or N434Y]), or modifications at position 250 and / or 428, or modifications at position 307 or 308 (e.g., 308F, V308F), and position 434. In one embodiment, the modifications include modifications of 428L (e.g., M428L) and 434S (e.g., N434S), modifications of 428L, 259I (e.g., V259I), and 308F (e.g., V308F), modifications of 433K (e.g., H433K) and 434 (e.g., 434Y), modifications of 252, 254, and 256 (e.g., 252Y, 254T, and 256E), modifications of 250Q and 428L (e.g., T250Q and M428L), modifications of 307 and / or 308 (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 includes one or more pairs or groups of mutations selected from the group consisting of 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), and includes an anti-CoV-S antigen-binding protein comprising an Fc domain, such as an antibody or an antigen-binding fragment.

[0104] The V described herein, including any possible combination of the aforementioned Fc domain mutations H and / or V L comprising an anti-CoV-S antigen-binding protein, such as an antibody and its antigen-binding fragment, is contemplated within the scope of the present invention.

[0105] The present invention also includes an anti-CoV-S antigen-binding protein, antibody, or antigen-binding fragment comprising the V described herein H and a chimeric heavy chain constant (C H ) region, wherein the chimeric C H region includes segments derived from the C region of more than one immunoglobulin isotype. For example, the antibody of the present invention is a chimeric C H including a part or all of the C region of a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule, and a part or all of the C H region of a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule, and a part or all of the C H region of a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule, and a part or all of the C region of a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule, and a part or all of the C HAccording to certain embodiments, the antibodies of the 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 H Antibodies containing regions are disclosed in certain embodiments. In some embodiments, modified Fc effector functions may be exhibited without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., WO2014 / 022540).

[0106] immunoconjugate The present invention encompasses an anti-CoV-S antigen-binding protein, such as an antibody or antigen-binding fragment, conjugated to another moiety such as a toxoid, for example a therapeutic moiety (“immunoconjugate”), or an antiviral agent for treating influenza virus infection. In embodiments of the present 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 a radioactive substance, cytokine, interferon, targeting or reporter moiety, enzyme, peptide or protein, or an antigen-binding protein, such as an antibody or antigen-binding fragment, chemically or biologically linked to a therapeutic agent. The antigen-binding protein can be linked to a radioactive substance, cytokine, interferon, targeting or reporter moiety, enzyme, peptide or therapeutic agent at any position along the molecule as long as it can bind 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 agent can be a second different antibody that specifically binds to CoV-S. The type of therapeutic moiety that can be conjugated to the anti-CoV-S antigen-binding protein (e.g., antibody or fragment) will be one that takes into account the condition to be treated and the desired therapeutic effect to be achieved. 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(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 also olRev., 62: 119-58 (1982).

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

[0108] The antibodies in Table 1 each include CDR-H and CDR-L, V H , and V L , or HC and LC (including their variants described herein), and include multispecific molecules, such as antibodies or antigen-binding fragments.

[0109] In embodiments of the present invention, the antigen-binding domain that specifically binds to CoV-S and can be included in the multispecific molecule is (1) (i) A heavy chain variable domain sequence comprising the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences described in Table 1, and (ii) A light chain variable domain sequence comprising the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences described in Table 1, or (2) (i) A heavy chain variable domain sequence comprising the amino acid sequence described in Table 1, and (ii) A light chain variable domain sequence comprising the amino acid sequence described in Table 1, or (3) (i) A heavy chain immunoglobulin sequence comprising the amino acid sequence described in Table 1, and (ii) A light chain immunoglobulin sequence comprising the amino acid sequence described in Table 1.

[0110] In embodiments of the present 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 domains.

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

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

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

[0114] An effective amount or a therapeutically effective amount of an anti-CoV-S antigen-binding protein, such as an antibody or an antigen-binding fragment (e.g., those in Table 1), for treating or preventing viral infection refers to an amount of the antibody or fragment sufficient to reduce one or more signs and / or symptoms of infection in a subject to be treated, whether by inducing regression or elimination of such signs and / or symptoms or by inhibiting progression of such signs and / or symptoms. The dosage administered can vary depending on the age and size of the subject being administered, the target disease, condition, route of administration, etc. In embodiments of the present invention, for example, an effective amount or a therapeutically effective amount of an antibody of the present invention or an antigen-binding fragment thereof for treating or preventing viral infection in an adult subject is about 0.01 to about 200 mg / kg, for example, 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). Depending on the severity of the infection, the frequency and duration of treatment can be adjusted. In certain embodiments, the antigen-binding protein of the present invention can be administered as an initial dose, followed by one or more secondary doses. In certain embodiments, following the initial dose, subsequent doses of the antibody or its antigen-binding fragment can be administered in an amount that can be approximately the same as or less than the initial dose, and the subsequent doses can be separated by at least 1 day 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) in need of prevention and / or treatment of a disease or disorder such as a viral infection or cancer, preferably a human. The subject may be suffering from a viral infection, e.g., influenza infection, or may be predisposed to developing an infection. Subjects predisposed to developing an infection, or subjects at high risk of contracting an infection (e.g., of a 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 virus (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. Further, very young (e.g., 5 years old or younger) or elderly (e.g., 65 years old or older) subjects are at high risk. Further, a subject may be at risk of contracting a viral infection due to proximity to the occurrence of a disease, e.g., the subject lives in a highly populated city, or is a subject in whom a viral infection has been confirmed or suspected, or is in close proximity to an employment choice, e.g., a hospital worker, a pharmaceutical researcher, a traveler to an infected area, or a frequent visitor.

[0116] "Treat" or "treatment" refers to a subject having one or more signs or symptoms of a disease or infection, e.g., a viral infection, for which an 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 prophylactically administering to a subject at risk of viral infection an anti-CoV-S antigen-binding protein, such as an antibody of the invention or an antigen-binding fragment thereof (e.g., those of Table 1), to prevent such an infection. Passive antibody-based immunoprophylaxis has proven to be an effective strategy for preventing a subject from becoming infected with a virus. See, for example, 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 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, such as an antibody or antigen-binding fragment of the invention (e.g., those of Table 1), to inhibit the symptoms of a disease or infection (e.g., viral infection) in the subject's body, and for which the antigen-binding protein is effective (as discussed herein) when administered to the subject in an effective or therapeutically effective amount or dose.

[0118] In one embodiment of the present invention, signs or symptoms of viral infection in a subject are, for example, the survival or proliferation of a virus in the subject's body, 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 described above, in some embodiments, the subject can be a non-human animal, and the antigen-binding proteins (e.g., antibodies and antigen-binding fragments) discussed herein can be used to treat and / or prevent diseases in animals other than humans (e.g., cats, dogs, pigs, cows, horses, goats, rabbits, sheep, etc.) in an animal context.

[0120] The present invention provides a method for treating or preventing viral infection (e.g., coronavirus infection) or inducing regression or elimination or inhibiting progression of at least one sign or symptom (the sign or symptom being secondary to viral infection) of viral infection as follows, by administering to a subject in need thereof (e.g., a human) a therapeutically effective amount of an anti-CoV-S antigen-binding protein (e.g., those of Table 1), for example, by injection of the protein into the subject's body. · Fever or feeling of fever / chills, · Cough, · Sore throat, · Runny nose or nasal congestion, · Sneezing, · Muscle or body aches, · Headache, · Malaise (fatigue), · Vomiting, · Diarrhea, · Respiratory infection, · Chest discomfort, · Dyspnea, · Bronchitis, and / or · Pneumonia, and is carried out in a subject (e.g., a human) in need thereof.

[0121] Combinations and pharmaceutical compositions To prepare a pharmaceutical composition of an anti-CoV-S antigen-binding protein, such as an antibody and its antigen-binding fragment (e.g., those in Table 1), the antigen-binding protein is mixed with a pharmaceutically acceptable carrier or excipient. See, for example, Remington’s Pharmaceutical Sciences and U.S.Pharmacopeia:National Formulary,Mack Publishing Company,Easton,Pa.(1984), Hardman,et al.(2001)Goodman and Gilman’s The Pharmacological Basis of Therapeutics,McGraw-Hill,New York,N.Y., Gennaro(2000)Remington:The Science and Practice of Pharmacy,Lippincott,Williams,and Wilkins,New York,N.Y., 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,N.Y.In an embodiment of the invention,the pharmaceutical composition is sterile. Such compositions are part of the invention.

[0122] The scope of the present invention includes a dried, e.g., lyophilized, composition comprising an anti-CoV-S antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof (e.g., those of Table 1), or a pharmaceutical composition thereof (comprising a pharmaceutically acceptable carrier and substantially lacking water).

[0123] In a further embodiment of the invention, a further therapeutic agent administered to a subject related to the anti-CoV-S antigen-binding protein disclosed herein, e.g., an antibody or an antigen-binding fragment thereof (e.g., those of Table 1), is administered to the subject according to Physicians’ Desk Reference 2003 (Thomson Healthcare; 57 th edition (Nov. 1, 2002)).

[0124] The methods of administration can be various. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, cutaneous, transdermal, or intraarterial.

[0125] The present invention provides a method for administering an anti-CoV-S antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof ( e.g., those of Table 1), which includes introducing the protein into the body of a subject. For example, the method includes piercing the body of the subject with a syringe needle and injecting the antigen-binding protein into the body of the subject, e.g., into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0126] The present invention provides a container (e.g., a plastic or glass vial having a cap or chromatography column, hollow bore needle, or syringe cylinder) containing any of an anti-CoV-S antigen-binding protein, e.g., an antibody or an antigen-binding fragment thereof (e.g., those of Table 1), a polypeptide (e.g., HC, LC, V H , or V L ), or a polynucleotide (e.g., those of Table 2), or a vector described herein, or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier.

[0127] In one embodiment of the present disclosure, an anti-CoV-S antigen-binding protein, such as an antibody of the present invention or an antigen-binding fragment thereof (e.g., those in Table 1), is administered in association with one or more additional therapeutic agents. Additional therapeutic agents include, but are not limited to, anti-inflammatory agents, anti-malarial agents, secondary antibodies or antigen-binding fragments thereof that specifically bind to TMPRSS2, and secondary antibodies or antigen-binding fragments thereof that specifically bind 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 gemtuzumab. In some embodiments, the additional therapeutic agent is a secondary antibody or antigen-binding fragment disclosed herein, e.g., those in Table 1. In certain embodiments, one, two, three, four, or more antibodies of Table 1, or antigen-binding fragments thereof, can be administered in combination (e.g., simultaneously or sequentially). Specific combinations of the antibodies of Table 1 are described in the following exemplary table of antibody combinations (e.g., each number representing a specific combination is such that mAb10989 and mAb10987 are combination 1, mAb10989 and mAb10934 are combination 2, etc.). In some embodiments, the combination of antibodies can be selected from among those that bind to different epitope clusters. For example, specific 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, a combination of two antibodies can be selected, for example, from 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 a human subject, wherein 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 the variable domains cloned from such B cells are combined with constant regions that are not from those B cells to generate hybrid antibodies. Sources (donors) of such antibody variable regions are shown in the following exemplary table of human-derived antibody variable regions. In some embodiments, the composition can include a combination of an antibody or antigen-binding fragment thereof having a variable domain derived from Donor 1 and an antibody or antigen-binding fragment thereof having a variable domain derived from Donor 2. In some embodiments, the composition can include a combination of an antibody or antigen-binding fragment thereof having a variable domain derived from Donor 1 and an antibody or antigen-binding fragment thereof having a variable domain derived from Donor 3. In some embodiments, the composition can include a combination of an antibody or antigen-binding fragment thereof having a variable domain derived from Donor 2 and an antibody or antigen-binding fragment thereof having a variable domain derived from Donor 3. In some embodiments, the composition can include a combination of mAb10987 from Donor 1 (e.g., an antibody comprising the CDRs, variable region, or heavy and light chain sequences shown in Table 1) and mAb10989 from Donor 3 (e.g., an antibody comprising the CDRs, variable region, 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. A method for treating or preventing viral (e.g., coronavirus) infection in a subject in need thereof by administering an antibody or antigen-binding fragment of Table 1 in combination with an additional therapeutic agent is part of the present invention.

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

[0131] For example, in one embodiment of the present invention, the additional therapeutic agent is:

Chemical formula

Chemical formula

[0132] In one embodiment of the present invention, the antiviral agent is an antibody or antigen-binding fragment that specifically binds to a coronavirus, such as SARS-CoV-2, SARS-CoV, or MERS-CoV. Exemplary anti-CoV-S antibodies include, as described in International Patent Application Publication No. WO / 2015 / 179535, H4sH15188P, H1H15188P, H1H15211P, H1H15177P, H4sH15211P, H1H15260P2, H1H15259P2, H1H15203P, H4sH15260P2, H4sH15231P2, H1H15237P2, H1H15208P, H1H15228P2, H1H15233P2, H1H15264P2, H1H15231P2, H1H15253P2, H1H15215P, and H1H15249P2, or antigen-binding fragments thereof, but are not limited thereto. For example, the antibody or fragment may include a light chain immunoglobulin (e.g., V L or its light chain) containing CDR-L1, CDR-L2, and CDR-L3 of any of the aforementioned anti-CoV-S antibodies, and a heavy chain (e.g., V or its heavy chain) containing CDR-H1, CDR-H2, and H CDR-H3.

[0133] In certain embodiments of the present invention, the additional therapeutic agent is not aprotinin, leupeptin, cationic steroid antibacterial agent, influenza vaccine (e.g., inactivated, live, attenuated whole virus or subunit vaccine), or an antibody against influenza virus (e.g., anti-hemagglutinin antibody).

[0134] The term "in relation to" refers to a component, an anti-CoV-S antigen-binding protein, e.g., It is shown that the antibody of the present invention or an antigen-binding fragment thereof can be formulated together with another agent, for example, in a single composition for co-delivery, or separately in two or more compositions (e.g., a kit). Each component can be administered to a subject at a different time than when the other components are administered. For example, each administration can be given non-simultaneously at intervals (e.g., separately or continuously) over a given period. Further, the separate components can be administered by the same route or different routes (e.g., an anti-CoV-S antibody or an antigen-binding fragment thereof can be administered to a subject).

[0135] Kit There is further provided a kit comprising one or more components including, but not limited to, an anti-CoV-S antigen-binding protein, such as 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, further therapeutic agents as discussed herein. The antigen-binding protein and / or the further therapeutic agent can be formulated in a pharmaceutical composition as a single composition or separately in two or more compositions, for example, together with a pharmaceutically acceptable carrier.

[0136] In one embodiment of the invention, the kit comprises an anti-CoV-S antigen-binding protein, such as an antibody of the invention or an antigen-binding fragment thereof (e.g., of Table 1), or a pharmaceutical composition thereof, in one container (e.g., in a sterile glass or plastic vial), and a further 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, such as an antibody of the invention or an antigen-binding fragment thereof (e.g., of Table 1), or a pharmaceutical composition thereof, in combination with one or more further therapeutic agents optionally formulated together in a pharmaceutical composition within a single common container.

[0138] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit can include an instrument (e.g., an injection instrument) for performing such administration. For example, the kit can include one or more subcutaneous injection needles or other injection instruments, including an anti-CoV-S antigen-binding protein, such as an antibody of the invention or an antigen-binding fragment thereof (e.g., those in Table 1).

[0139] The kit can include a package insert containing information regarding the pharmaceutical composition and dosage form within the kit. Generally, such information serves to assist the patient and physician in using the enclosed pharmaceutical composition and dosage form effectively and safely. For example, the following information regarding the combinations of the invention can be provided in an insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, side effects, overdose, appropriate dosage and administration, provided methods, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0140] Diagnostic Use of Antibodies An anti-CoV-S antigen-binding protein, such as an antibody of the invention or an antigen-binding fragment thereof (e.g., those in Table 1), can be used to detect and / or measure CoV-S in a sample. Exemplary assays for CoV-S can include, for example, contacting the 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 is used as a capture ligand for selectively isolating 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. Detectable labels or reporter molecules are 3 H,[[]] 14 C,[[]] 32 P,[[]] 35 S, or[[]] 125 radioisotopes such as I, fluoro It can 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 a method for detecting the presence of a spike protein polypeptide in a sample, comprising contacting the sample with an anti-CoV-S antigen-binding protein and detecting the presence of CoV-S / anti-CoV-S antigen-binding protein complex, the presence of the complex indicating the presence of CoV-S.

[0141] The anti-CoV-S antigen-binding proteins of the present invention (e.g., those of Table 1) can be used in Western blot or immunoprotein blot procedures for detecting the presence of CoV-S or a fragment thereof 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 the sample to be tested for the presence of CoV-S, e.g., optionally transferring the proteins from the sample to be tested for the presence of CoV-S (e.g., from PAGE or SDS-PAGE electrophoretic separation of the 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 the anti-CoV-S antigen-binding protein of the present invention.

[0142] Such membranes can take the form of, for example, nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membranes, on which proteins to be tested for the presence of CoV-S are transferred (e.g., after electrophoretic separation in a gel), such as in non-denaturing PAGE (polyacrylamide gel electrophoresis) gels or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels. Before contacting the membrane with the anti-CoV-S antigen-binding protein, the membrane is optionally blocked, e.g., with skim milk powder, to bind to non-specific protein-binding sites on the membrane. (2) Wash the membrane one or more times to remove unbound anti-CoV-S antigen-binding protein and other unbound substances, and (3) Detect the bound anti-CoV-S antigen-binding protein.

[0143] Detection of the bound antigen-binding protein indicates the presence of the CoV-S protein on the membrane or substrate and in the sample. Detection of the bound antigen-binding protein can be by binding the antigen-binding protein to 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., those of Table 1)) disclosed herein can also be used for immunohistochemistry. Such methods form part of the present invention and include, for example: (1) Contacting a tissue to be tested for the presence of CoV-S protein with the anti-CoV-S antigen-binding protein of the present invention, and (2) Detecting the antigen-binding protein on or in the tissue.

[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 then the label is detected.

Examples

[0146] The following examples are presented to provide those skilled 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 that the inventors regard as the present invention. Efforts have been made to ensure accuracy with respect to the numerical values used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are 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 the human immunoglobulin heavy chain and kappa or 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 either the SARS-CoV-2-S vector or the SARS-CoV-2-S protein. The antibody immune response was monitored by a SARS-CoV-2-S specific immunoassay. 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. The hybridoma cell lines were screened and selected to identify cell lines that produce SARS-CoV-2-S specific antibodies. Anti-SARS-CoV-2-S antibodies were isolated directly from antigen-positive mouse B cells without fusing with myeloma cells as described in U.S. Patent No. 7,582,298, which is specifically incorporated herein by reference in its entirety. Using this method, fully human anti-SARS-CoV-2-S antibodies (i.e., antibodies having human variable and human constant domains) were obtained.

[0148] Antibody variable regions were also isolated from human blood samples. Whole blood was collected from patients 3 to 4 weeks after a PCR-positive test confirmed in the laboratory 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 to the SARS-CoV-2 spike protein were isolated by fluorescence-activated cell sorting (FACS). The isolated B cells were plated in single wells and mixed with antibody light and heavy variable region-specific PCR primers. cDNA of each single B cell was synthesized via a reverse transcriptase (RT) reaction. Next, each obtained RT product was divided and transferred to two corresponding wells for subsequent antibody heavy and light chain PCR. One set of the obtained 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. Next, the amplicons were amplified again by PCR using a 5' degenerate primer specific to the antibody heavy variable region framework 1 or a 5' degenerate primer specific to the antibody light chain variable region framework 1 and a 3' primer specific to the antibody constant region to generate amplicons for cloning. PCR products derived from the heavy and light chains of the antibody were cloned into expression vectors containing the heavy constant region and the light constant region, respectively, thereby creating expression vectors for hybrid antibodies. Expression vectors expressing pairs of full-length heavy and light chains were transfected into CHO cells to produce test antibody proteins.

[0149] The biological properties of exemplary antibodies generated according to the method of this example are described in detail in the examples described below.

[0150] Example 2: Amino Acid and Nucleotide Sequences of Heavy and Light Chain Variable Regions Table 1 shows the amino acid sequence identifiers of the heavy and light chain variable regions and CDRs of exemplary anti-SARS-CoV-2-S antibodies, as well as the heavy and light chain sequences. The corresponding nucleic acid sequence identifiers are set forth 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 murine constant regions (e.g., murine IgG1 Fc or murine 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, antibodies having a particular Fc isotype can be converted to antibodies having a different Fc isotype (e.g., an antibody having murine IgG1 Fc can be converted to an antibody having human IgG4, etc.), but in any event, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Tables 1 and 2 remain the same, and the binding properties to the antigen are expected to be the same or substantially similar regardless of the nature of the constant domain.

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

[0153] As described above, antibodies were obtained from hybridomas generated from VELOCIMMUNE® mice, directly isolated 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 bind to the SARS-CoV-2-spike protein receptor binding domain (RBD). A protein consisting of the RBD (amino acids 319-541) of SARS-CoV-2 expressed with a 6X histidine tag and two myc epitope tags at the C-terminus (SARS-CoV-2-S-RBD-mmH, see NCBI accession number MN908947.3) was coated onto 96-well plates in PBS buffer at 1 μg / ml at 4 °C overnight. Subsequently, non-specific binding sites were blocked using a 0.5 (weight / volume)% solution of BSA in PBS. Antibody supernatants or medium alone were diluted 1:40 or 1:50 with PSA + 0.5% BSA blocking buffer and transferred to the washed microtiter plates. After incubation at room temperature for 1 hour, the wells were washed and the supernatant bound to the plate 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). Next, the plates were developed using a TMB substrate solution (BD Biosciences) according to the manufacturer's recommendations, and the absorbance at 450 nm was measured with 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 evaluated as described above using a binding ELISA with the SARS-CoV-2-S-RBD-mmH protein coated onto microplates. Single-point antibody supernatants that bound to SARS-COV-2-S-RBD-mmH coated onto 96-well microtiter plates were detected with HRP-conjugated anti-hFc or anti-mFc antibodies.

[0156] The combined results of the three tests are summarized in Table 4. The SARS-CoV-2 binding signal (absorbance at 450 nm) is shown, and for each experiment, the background of medium only is provided as a negative reference. Samples marked IC (indeterminate) are reported without values because there were experimental anomalies on the plate. As shown compared to the medium-only control, the tested supernatants showed substantial binding to SARS-CoV-2-S-RBD.

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 using 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 the 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 conducted according to the following procedure. The above two types of VLPs 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 adhere the VLPs. Nonspecific binding sites were blocked with 2 (weight / volume)% BSA in PBS for 1 hour at room temperature. Supernatants containing antibodies generated from SARS CoV-2 immunized mice or infected human sera were added to the plate-bound particles along with a medium-only control diluted 1:10 or 1:20 with 1×PBS + 0.5% BSA buffer. Next, the plate was incubated for 1 hour at room temperature with shaking, then the plate was washed with 1×PBS, and unbound antibodies were removed using an AquaMax2000 plate washer (MDS Analytical Technologies). Plate-bound antibodies were detected at room temperature for 1 hour using a SULFO-TAGTM conjugated anti-human IgG antibody (Jackson Immunoresearch) or a SULFO-TAGTM conjugated anti-mouse IgG antibody (Jackson Immunoresearch). After washing, the plate was developed with read buffer (MSD) according to the manufacturer's recommended procedure, and the luminescence signal was recorded using a SECTOR Imager 600 (Meso Scale Development) instrument. The direct binding signal (in RLU) was captured, and the ratio of VLPs expressing SARS-CoV-2-S to VLPs unrelated to it was calculated.

[0160] The ability of anti-SARS-CoV-2-S monoclonal antibodies to bind to VLPs expressing SARS-CoV-2-S was evaluated using the immunobinding assay as described above, compared to binding to irrelevant VSV-expressing VLPs. Single-point binding to VLPs immobilized on a 96-well High Bind plate (MSD) was performed with antibody supernatant dilutions of 1:10 or 1:20 It was performed for 1 hour, combined, and detected using SULFO-TAGTM-conjugated anti-human IgG or anti-mouse IgG antibodies. The binding signal from electrochemiluminescence was recorded with a Sector Imager 600 (MSD). The RLU values of the antibodies binding to the VLPs were determined. The ratio was calculated by comparing the binding signal of the VLPs expressing SARS-CoV-2-S with that of the control VLPs.

[0161] The binding results from three experiments are summarized in Table 5. The signal observed from the VLPs expressing SARS-COV-2-S indicated binding, and the comparison with the negative VLPs provided a relative background. Samples of medium alone provided a baseline signal for the secondary antibody binding to samples without supernatant. Forty-six antibodies specifically bound more than 4-fold higher than the medium-alone samples (20 - 35 RLU) of the VLPs expressing SARS-CoV-2-S and showed binding signals in the range of 85 - 13,600 RLU. The ratio of VSV expressing SARS-CoV-2-S:VSV-VLP (negative control) was in the range of 1.1 - 22.7, and for many, the background of VSV-VLP was high. The ratio of mAb11002 being 0.9 might be due to the low concentration of the monoclonal antibody in the supernatant sample.

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 was developed using VSV-SARS-CoV-2-S pseudovirus.

[0163] As described above, the VSV pseudotype virus was generated by transiently transfecting 293T cells with a plasmid encoding the SARS-CoV-2 spike protein. The cells were plated in DMEM complete medium at 1.2×10 7 cells per plate in 15 cm plates one day prior to 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. Twenty-four hours after transfection, the cells were washed with 10 mL of PBS and infected with 0.1 VSVΔ G:mNeon virus at an MOI. The virus was incubated on the cells for 1 hour and gently rocked every 10 minutes. The cells were then overlaid with 20 mL of infection medium and washed three times with 10 mL of PBS 2 before incubating at 37 °C, 5% CO for 24 hours. The supernatant was collected in 250 mL centrifuge tubes on ice, centrifuged at 3000 rpm for 5 minutes to pellet any cell debris, aliquoted on ice, and frozen at -80 °C. The infectivity was tested in Vero cells prior to use in the neutralization assay. This material is called VSV-SARS-CoV-2-S.

[0164] Neutralization assay with VSV-SARS-CoV-2-S On day 1, Vero cells were seeded in T225 flasks at 80% confluence. 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 the cells 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 in 100 μL of pre-warmed complete DMEM per well of a 96-well black polystyrene microplate (Corning: 3904).

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

[0166] In a V-bottom 96-well plate, the dilution of each supernatant was made with 60 μl of infection medium. For the medium (negative) control, 60 μl of diluted acclimation 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 added to those wells. Next, the pseudovirus was incubated with the supernatant dilution at room temperature for 30 minutes. The medium was removed from the Vero cell plate, and 100 μL of the supernatant / pseudovirus mixture was transferred to the cells, and the plate was incubated at 37 °C and incubated for 24 hours in 5% CO 2 and incubated for 24 hours. The neutralization of VSV-SARS-CoV-2-S pseudovirus was evaluated using the final supernatant dilutions of 1:4 and 1:20, and in some samples 1:100.

[0167] On the third day, after 24 hours of incubation, the supernatant was removed from the cell wells and replaced with 100 μL of PBS. Next, the plate was read on a SpectraMax i3 equipped with a MiniMax imaging cytometer.

[0168] The ability of anti-SARS-CoV-2-S antibodies to neutralize VSV-based SARS-CoV-2-S-expressing pseudotype virus was evaluated using a neutralization fluorescence focus assay. The binding results of the three assays are summarized below. The neutralization efficacy of the antibody at each dilution is expressed as a percentage compared to the mock supernatant control. All antibodies showed neutralizing ability, and in particular, in the set of antibodies evaluated at 1:100, antibodies showing higher neutralization may represent a more potent neutralizing ability.

Table 9-1

Table 9-2

Table 9-3

[0169] Example 6: Characterization of Antibodies in an Antibody-Dependent Cellular Cytotoxicity Surrogate Assay The ability of antibodies targeting the spike protein of SARS-CoV-2 to interact with FcγR3a, a Fc receptor significantly expressed on natural killer (NK) cells that induce antibody-dependent cellular cytotoxicity (ADCC), was measured in a surrogate bioassay using reporter cells and target cells conjugated to the antibody. This assay used Jurkat T cells engineered to express the reporter gene luciferase under the control of the transcription factor NFAT (NFAT-Luc) together with the high-affinity human FcγR3a 176 Val allotype receptor (Jurkat / NFAT-Luc / hFcγR3a 176 Val). Target cells were engineered Jurkat T cells that expressed human CD20 (used as a positive control for human IgG1 antibodies targeting CD20) and full-length SARS-CoV-2 spike protein controlled by a doxycycline-inducible promoter. Reporter cells were incubated with target cells, and the involvement of FcγR3a via the Fc domain of human IgG1 antibody bound to the target cells activated the transcription factor NFAT in the reporter cells, promoting the expression of luciferase, which was then measured via a luminescence 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), Tet3G transactivation protein (cloned using the Takara pEF1α-Tet3G vector, catalog number 631167), and 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 expression of the spike protein 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 were engineered to stably express a luciferase reporter construct for nuclear factor of activated T cells (NFAT) together with the high-affinity human FcγR3a 176 Val allotype receptor (amino acids M1-K254 of NCBI accession number P08637 VAR_003960). Engineered reporter cells were maintained in RPMI1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 500 μg / ml G418 growth medium.

[0172] Thirty-six hours prior to the start of the surrogate ADCC assay, 5 × 10 5 target cells / ml were induced in RPMI + 10% Tet-free FBS + P / S / G cell culture medium containing 1 μg / ml doxycycline (Sigma). One day prior to the experiment, reporter cells were split to a density of 7.5 × 10 5 cells / ml in RPMI1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 500 μg / ml G418 growth medium.

[0173] ​Briefly, on the day of the experiment, target cells and reporter cells were transferred to assay medium (RPMI + 10% Tet - free FBS + P / S / G), and added to a 384 - well white microtiter plate at a ratio of 3:2 (3×10 4 / wells of target cells and 2×10 4 / wells of reporter cells). Subsequently, anti - SARS - CoV - 2 - S antibody supernatants at various concentrations were added. To normalize the detected ADCC activity of the anti - SARS - CoV - 2 - S antibody supernatants, positive control (CD20 antibody containing human IgG1) samples and antibody - free negative control samples were included in each plate. The plates were incubated at 37°C / 5% CO for 5 hours, then an equal volume of ONE - Glo™ (Promega) reagent was added to lyse the cells and detect luciferase activity. The emitted light was captured as relative light units (RLU) on a multi - label plate reader Envision (PerkinElmer), and the data were analyzed and normalized using the following equation. 2 ated After that, an equal volume of ONE - Glo(™)(Promega) reagent was added to lyse the cells and detect luciferase activity. The emitted light was captured as relative light units (RLU) on a multi - label plate reader Envision (PerkinElmer), and the data were analyzed and normalized using the following equation.

Equation

[0174] The ability of the anti - SARS - CoV - 2 - S antibody to activate the FcγR3a receptor was evaluated in a surrogate ADCC assay using Jurkat / N FAT - Luc / FcγR3a 176 Val) as reporter cells and 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 the calculated % of the positive control. A range of %ADCC activity indicating activation of FcγR3a by the antibody supernatants was observed. All samples showed some measure of surrogate ADCC activity, and 10 of the antibody supernatants showed superior surrogate ADCC activity compared to that observed in 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 on Luminex microspheres by streptavidin as follows: approximately 1,000 ten thousand MagPlex microspheres (Luminex Corp., MagPlex Microspheres, catalog numbers MC10000 and MC12000) were vortexed in 500 μL of 0.1 M NaPO 4 , pH 6.2 (activation buffer) and It was resuspended and centrifuged to remove the supernatant. Since the microspheres are sensitive to light, they were protected from light. The microspheres were resuspended in 160 μL of activation buffer, followed by 20 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog number 24525), and then 20 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog number 22980) was added at 25 °C to activate the carboxylic acid groups (-COOH). After 10 minutes, 600 μL of 50 mM MES, pH 5 (coupling buffer) was added to lower the pH of the reaction solution to 5.0. The microspheres were vortexed and centrifuged to remove the supernatant. The activated microspheres were immediately mixed with 500 μL of 25 μg / mL protein antigen or streptavidin in the coupling buffer and incubated at 25 °C for 2 hours. 50 μL of 1 M Tris-HCl, pH 8.0 was added to stop the coupling reaction. The microspheres were vortexed, centrifuged, and washed three times with 800 μL of 0.005% PBS (0.05% Tween 20) to remove uncoupled proteins and other reaction components. The microspheres were resuspended in 1 mL of 2% PBS BSA 0.05% sodium azide at 10 million microspheres / mL. For antigen capture by streptavidin, 500 μL of 12.5 μg / mL biotinylated protein in PBS was added to the streptavidin-conjugated microspheres and incubated at 25 °C for 1 hour. The microspheres were vortexed, centrifuged, and washed three times with 800 μL of PBS, and then blocked using 500 μL of 30 mM biotin (Millipore-Sigma, catalog number B4501) in 0.15 M Tris pH 8.0. After incubating the microspheres for 30 minutes, they were vortexed, centrifuged, and washed three times with 800 μL of PBS. The microspheres were resuspended in 1 mL of 2% PBS 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 individual anti-SARS-CoV-2 supernatants containing antibodies. After incubating the samples and microspheres at 25 °C for 2 hours, they were washed twice with 200 μL of DPBS containing 0.05% Tween 20. To detect the level of bound antibody to individual microspheres, 100 μL of 2.5 μg / mL R-phycoerythrin-conjugated goat F(ab’)2 anti-human kappa (Southern Biotech, catalog number 2063-09) in blocking buffer (for antibodies with mouse Fc region) or 100 μL of 1.25 μg / mL R-phycoerythrin affinity-purified F(ab’) 2 fragment goat anti-mouse IgG, F(ab’) 2 fragment specific (Jackson Immunoresearch, catalog number 115-116-072) was added and incubated at 25 °C for 30 minutes. After 30 minutes, the samples were washed twice with 200 μl of wash buffer and resuspended in 150 μL of wash buffer. The plates were read on a 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] The results of the Luminex binding are shown in Tables 8 and 9 as the median fluorescence intensity (MFI) signal intensity. The results indicate that 46 anti-SARS-CoV-2-S antibody supernatants specifically bound to the SARS-CoV-2-S RBD protein. These results also show that 5 of these antibodies cross-react with the SARS coronavirus spike RBD protein and exhibit binding signals exceeding 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, the antigen was 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 vortexed and resuspended in 500 μL of 0.1 M NaPO 4 , pH 6.2, and centrifuged to remove the supernatant. The microspheres were resuspended in 160 μL of activation buffer and centrifuged again to remove the supernatant. The microspheres were resuspended in 160 μL of activation buffer 20 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog number 24525) was added, followed by 20 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog number 22980) at 25 °C to activate the carboxylic acid group (-COOH). After 10 minutes, 600 μL of 50 mM MES, pH 5 (coupling buffer) was added to lower the pH of the reaction solution to 5.0. 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 the coupling buffer and incubated at 25 °C for 2 hours. The coupling reaction solution was quenched by adding 50 μL of 1 M Tris-HCl, pH 8.0. 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 out of the 16 microsphere regions containing amine-binding proteins were modified for the binding assay as follows: The microspheres were washed twice with 5% DMSO in PBS, 500 μl of a chemical or enzyme was dissolved according to the manufacturer's recommendations, and added to the above amine-binding microspheres at 10 nM. Subsequently, this was 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 protein-unmodified (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. After incubating the samples and microspheres at 25 °C for 2 hours, they were washed twice with 200 μL of DPBS containing 0.05% Tween 20. To detect the level of bound antibody to individual microspheres, 100 μL of 2.5 μg / mL R-phycoerythrin-conjugated goat F(ab’)2 anti-human kappa (Southern Biotech, catalog number 2063-09) in blocking buffer (for antibodies with hFc) or 100 μL of 1.25 μg / mL R-phycoerythrin affinity-purified F(ab’) 2 fragment goat anti-mouse IgG, F(ab’) 2 fragment specific (Jackson Immunoresearch, catalog number 115-116-072), or 100 μL of 1.25 μg / mL R-phycoerythrin anti-His (Biolegend, catalog number 362603) in blocking buffer (ACE-2 control, R&D, catalog number 933-ZN) was added and incubated at 25 °C for 30 minutes. After 30 minutes, the samples were washed twice with 200 μl of wash buffer and resuspended in 150 μL of wash buffer. The plates were read on a FlexMap 3D® (Luminex Corp.) and Luminex xPonent® software version 4.3 (Luminex Corp.).

[0182] The results of the Luminex binning are shown in Table 10 as the median fluorescence intensity (MFI) signal intensity. To determine the clusters, the data were normalized to intact protein (unmodified microspheres) and clustered. Forty-six anti-SARS-CoV-2 antibodies were classified into nine clusters with two or more antibodies, and 11 antibodies were classified as single nodes. The 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 The equilibrium dissociation constants (K D ) of different SARS-CoV-2-S antibodies from primary supernatants of CHOt cells or hybridomas were determined by real-time surface plasmon resonance-based Biacor Determined using an e T200 / Biacore8K biosensor. All binding studies were performed at 25 °C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05 v / v% Surfactant Tween-20, pH 7.4 (HBS-ET) running buffer. 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 number BR-1008-38) to capture anti-SARS-CoV-2 antibodies. Binding studies were performed with the human SARS-CoV-2 RBD extracellular domain expressed with a C-terminal myc-myc-hexahistidine tag (SARS-COV-2 RBD-MMH), the SARS-CoV-2 RBD extracellular domain expressed with a C-terminal mouse IgG2a (SARS-COV-2 RBD-mFc), or the SARS-CoV-2 RBD extracellular domain expressed with a C-terminal human IgG1 (SARS-COV-2 RBD-hFc). Single concentrations 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 were injected at a flow rate of 30 μL / min for 1.5 min, and the dissociation of the various SARS-CoV-2 RBD reagents bound to the antibody was monitored for 2 min 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 s injection of 20 mM phosphoric acid for the mouse anti-human Fc specific monoclonal antibody surface or a 40 s injection of 10 mM glycine, HCl, pH 1.5 for the rabbit anti-mouse Fcγ specific polyclonal antibody. Association rate (K a ) and dissociation rate (K d) was determined by fitting the real-time binding sensorgram 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. The binding dissociation equilibrium constant (K D ) and the dissociation half-life (t1 / 2) were calculated from the kinetic rate as follows:

Number

[0184] The binding rate parameters of different SARS-CoV-2 monoclonal antibodies binding to different anti-SARS-COV-2 RBD reagents of the present 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 The 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 was expressed with the Fc portion of human IgG1 at the C-terminus (refer to SARS-CoV-2 RBD-hFc, NCBI accession number MN908947.3) and consisted of the receptor-binding domain (RBD) portion (amino acids Arg319 - Phe541) of the SARS-CoV-2 spike protein. The human ACE2 protein used in the experiment was purchased from R&D Systems and consisted of amino acids Glutamine 18 - Serine 740 with a C-terminal - 10X histidine tag (hACE2-His, NCBI accession number Q9BYF1).

[0187] The experiment was conducted according to the following procedure. Monoclonal anti-Penta-His antibody (Qiagen) was coated overnight at 4°C at 1 μg / ml in PBS on a 96-well microtiter plate. The hACE2-His receptor was added at 0.2 μg / ml in PBS and allowed to bind for 2 hours at room temperature. Subsequently, non-specific binding sites were blocked using a 0.5% (weight / volume) solution of BSA in PBS. On other microtiter plates, a fixed amount of 10 pM or 15 pM (shown in Table 13) of SARS-CoV-2 RBD-hFc protein was bound to the antibody diluted 1:10 or 1:20 with PBS + 0.5% BSA. These antibody-protein complexes were transferred to the microtiter plate coated with hACE2-His after 1 hour of incubation. After incubating for 1.5 hours at room temperature, the wells were washed, and the SARS-CoV-2 RBD-hFc protein bound to the plate was detected with a goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson). Next, the plate was developed using a TMB substrate solution (BD Biosciences, catalog number 555214) according to the manufacturer's recommendations, and the absorbance at 450 nm was measured with a VictorX5 plate reader.

[0188] Data analysis was performed by calculating the rate of decrease in signal of the fixed SARS-CoV-2-S RBD-hFc concentration in the presence and absence of the antibody. In the calculation, the binding signal of a sample of a certain SARS-CoV-2-S RBD-hFc in the absence of the antibody on each plate was referred to as 100% binding or 0% blockade, and the SARS-CoV-2 baseline signal of the medium sample only in the absence of RBD-hFc was referred to as 0% binding or 100% blockade.

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

[0190] The blocking results of the three assays are summarized in Table 13. The SARS-CoV-2-S binding signal (450 nm) and % blocking calculated with G are shown. Various blockings were observed in the test samples. For samples where NA is shown in columns 6 and 7, plate correction values are included in columns 4 and 5 as the data was consistent with a single plate switch occurring in these samples. Of the 46 antibody supernatants, 43 blocked more than 50% of the SARS-CoV-2-S RBD-hFc binding to plate-coated human ACE2, and 16 of them blocked more than 90% 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 to 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] The HDX-MS experiments were performed on an integrated HDX / MS platform, which consists 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 gradient, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0193] The labeling solution was prepared as PBS buffer in D 2 O at pD 7.0 (10 mM phosphate buffer, 140 mM NaCl, and 3 mM KCl, equivalent to pH 7.4 at 25 °C). For deuterium labeling, 10 μL of RBD protein or RBD protein pre-mixed with each of the 12 antibodies above was incubated with 90 μL of D O labeling solution at 20 °C for 2 samples Incubated twice at various time points. For mAb10989, mAb10987, mAb10934, and mAb10933, the time points were 0 minutes (non-deuterated control), 5 minutes, and 10 minutes. For mAb10920, mAb10922, mAb10936, mAb10954, mAb10964, mAb10977, mAb10984, and mAb10986, the time points were 0 minutes (non-deuterated control) and 10 minutes. The deuteration reaction was quenched by adding 90 μL of pre-cooled 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. Next, the quenched samples were injected into the Leaptec HDX PAL system for on-line pepsin / protease XIII digestion . The digested peptides were trapped on a C18 column (2.1 mm × 5 mm, Waters) and separated on another C18 column (2.1 mm × 50 mm, Waters) with a gradient of -5 °C for 20 minutes (for mAb10989, mAb10987, mAb10934, and mAb10933) or a 10-minute gradient from 0% to 90% mobile phase B solution (mobile phase A solution: 0.5% formic acid and 4.5% aqueous acetonitrile solution, mobile phase B solution: 0.5% formic acid in acetonitrile) (for mAb10920, mAb10922, mAb10936, mAb10954, mAb10956, mAb10964, mAb10977, and mAb10984). The eluted peptides were analyzed by LC-MS / MS or LC-MS mode using a Thermo Q Exactive HF mass spectrometer.

[0194] LC-MS / MS data from the non-deuterated RBD protein sample was searched against a database containing the amino acid sequences of the RBD protein, pepsin, protease XIII, and their reverse sequences using the Byonic search engine (Protein Metrics). The search parameters were set as default using non-specific enzymatic digestion and human glycosylation as common variable modifications. Next, the list of identified peptides was 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 incorporation (ΔD) = D-incorporation (RBD-mAb) - D-incorporation (RBD only)

Number

[0195] A total of 190 peptides from RBD were identified from both RBD only and RBD complexed with the mAb10989 sample, representing 86.06% sequence coverage of RBD. Peptides showing a decrease in deuterium incorporation of more than 5% upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as significantly protected. The peptide corresponding to amino acids 467 - 513 of 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 the mAb10987 sample, representing 86.06% sequence coverage of the RBD. Peptides showing a decrease in deuterium incorporation of more than 5% upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being significantly protected. The peptide corresponding to amino acids 432 - 452 (CVIAWNSNNLDSKVGGNYNYL) (SEQ ID NO: 836) of the RBD 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 the mAb10934 sample, representing 86.06% sequence coverage of the RBD. Peptides showing a decrease in deuterium incorporation of more than 5% upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being significantly protected. The 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 the mAb10933 sample, representing 86.06% sequence coverage of the RBD. Peptides showing a decrease in deuterium incorporation of more than 5% upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being significantly protected. The peptide corresponding to amino acids 467 - 510 (DISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRV) (SEQ ID NO: 839) of the RBD 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 the mAb10920 sample, representing 83.27% sequence coverage of the RBD. Peptides showing a decrease in deuterium incorporation of more than 5% upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being 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 the mAb10922 sample, representing 87.25% sequence coverage of the RBD. Peptides showing a decrease in deuterium incorporation of more than 5% upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being significantly protected. The peptide corresponding to amino acids 432 - 452 (CVIAWNSNNLDSKVGGNYNYL) (SEQ ID NO: 836) of the RBD 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 the mAb10936 sample, representing 82.07% sequence coverage of the RBD. Peptides showing a decrease in deuterium incorporation of more than 5% upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being significantly protected. Peptides RBD 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) of the RBD 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 the mAb10954 sample, representing 87.25% sequence coverage of the RBD. Peptides showing a decrease of more than 5% in deuterium incorporation upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) 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 the mAb10964 sample, representing 83.67% sequence coverage of the RBD. Peptides showing a decrease of more than 5% in deuterium incorporation upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) 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 the mAb10977 sample, representing 87.25% sequence coverage of the RBD. Peptides showing a decrease of more than 5% in deuterium incorporation upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being 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 the mAb10984 sample, representing 87.25% sequence coverage of the RBD. Peptides showing a decrease of more than 5% in deuterium incorporation upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being 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 the mAb10986 sample, representing 87.25% sequence coverage of the RBD. Peptides showing a decrease of more than 5% in deuterium incorporation upon mAb binding (i.e., Δ%D values less than -5% such as -6%, -10%, etc.) were defined as being 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, most of the neutralizing antibodies tested contact the RBD in a way 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 pattern 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 can neutralize SARS-CoV-2 variants, these antibodies were screened against a panel of VSV pseudotype viruses expressing wild-type and mutant spike proteins. VSV pseudotype 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. One day before transfection with 15 μg / plate of spike DNA, cells were seeded at 1.2×10 7 cells per plate in 15 cm plates in DMEM complete medium (1000 mL DMEM, Gibco, 100 ml FBS, Gibco, 10 mL PSG, Gibco) using 125 μL of Lipofectamine LTX, 30 μL of PLUS reagent, and up to 3 mL of Opti-Mem. Trans After 24 hours of fection, the cells were washed with 10 mL of PBS and infected with 0.1 VSVΔ G:mNeon virus at an MOI. The virus was incubated on the cells for 1 hour with gentle rocking every 10 minutes. After washing the cells three times with 10 mL of PBS, the cells were overlaid with 20 mL of infection medium (1000 mL DMEM, Gibco, 10 mL sodium pyruvate, Gibco, 7 mL BSA, Sigma, 5 mL gentamicin, Gibco) and then incubated at 37 °C, 5% CO 2 for 24 hours . The supernatant of the pseudovirus was collected in 250 mL centrifuge tubes on ice, centrifuged at 3000 rpm for 5 minutes to pellet any cell debris, aliquoted on ice, and frozen at -80 °C. Before use in the neutralization assay, the infectivity was tested in Vero cells. This material is VSVΔ G:mNeon / spike pseudovirus, or VSVΔ G:mNeon / spike_(mutant amino acid mutation) (e.g., VSVΔ G:mNeon / It is called Spike_H49Y).

[0209] On the first day, Vero cells were seeded in a T225 flask to a confluence density of 80%, the cells were washed with PBS (Gibco: 20012-043), TrypLE was added to separate the cells from the flask, and complete DMEM was added to inactivate the trypsin. 20,000 Vero cells were seeded into 100 μL of pre-warmed complete DMEM per well in a 96-well black polystyrene microplate (Corning: 3904). On the second day, VSVΔ G:mNeon / Spike pseudovirus was thawed on ice and diluted with infection medium. Antibodies were diluted in a U-bottom 96-well plate, and 210 μl of infection medium was used to generate dilution solutions of each antibody at a two-fold assay concentration. 120 μL of the diluted antibody was transferred to a new U-bottom plate, and medium and IgG1 control antibody were added to each plate. 120 μl of the diluted pseudovirus was added to all wells except the medium control wells. 120 μL of infection medium was added to those wells. The pseudovirus containing the antibody was incubated at room temperature for 30 minutes, and then the medium was removed from the Vero cells. 100 μL of the antibody / pseudovirus mixture was added to the cells, and then incubated at 37 °C in 5% CO 2 for 24 hours. On the third day, the supernatant was removed from the cell wells and replaced with 100 μL of PBS. The plate was read on a SpectraMax i3 equipped with a MiniMax imaging cytometer.

[0210] In addition to testing the neutralization ability against the non-replicating VSV-SARS-CoV-2-S virus, the antibodies were also tested against the SARS-CoV-2 virus. Monoclonal antibodies and antibody combinations were serially diluted in DMEM (Quality Biological), 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 (final concentration 2 mM, 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 mixture was incubated at 37 °C for 60 minutes. After incubation, the virus titer of the mixture was determined by plaque assay. Finally, the 50% plaque reduction neutralization titer (PRNT50) value (serum dilution at which plaque formation was reduced by 50% compared to the untreated control) was calculated using a four-parameter logistic curve fitted to the percent neutralization data (GraphPad software, La Jolla, CA).

[0211] Half of the maximum inhibitory concentration (IC50) of individual monoclonal antibodies against the VSV-SARS-CoV-2 spike protein (S) expressing pseudovirus encoding the Wuhan-Hu-1 (NCBI accession number MN908947.3) sequence of the spike protein (S-wt) was measured in Vero cells (Table 26). Most of the antibodies showed neutralizing potency in the picomolar range (pM), and some showed neutralizing potency in the nanomolar (nM) range.

[0212] As previously reported, recombinant ACE2 mediates the neutralization of VSV spike pseudoparticles It was able to be done, but its efficacy was far inferior to that of monoclonal antibodies, with a decrease of more than 1 / 1000 of the efficacy compared to the best neutralizing mAb (Figure 10A). In addition, the potent neutralizing activities of mAb10987, mAb10989, mAb10933, and mAb10934 were confirmed in a neutralization assay including the neutralization of SARS-CoV-2 in VeroE6 cells (Figure 10B). All neutralization assays produced similar efficacies with the four mAbs (mAb10987, mAb10989, mAb10933, and mAb10934), and there was no combination showing synergistic neutralizing activity (Figure 10B).

Table 32-1

Table 32-2

[0213] Amino acid variants of the spike (S) protein were identified from more than 700 publicly available SARS-CoV-2 sequences representing isolates circulating worldwide and cloned into VSV pseudoparticles. To evaluate the effect of each variant on the neutralizing efficacy of monoclonal antibodies, a neutralization assay was performed using pseudoparticles encoding the variants. Table 27 exemplifies the relative neutralizing efficacy of monoclonal antibodies against variants encoding pseudoparticles compared to 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. Except for mAb10985 and the R408I variant, there was no antibody showing loss of neutralizing efficacy at a concentration of 5 μg / ml. These data indicate a broad functional neutralization range of monoclonal antibodies against SARS-CoV-2 spike variants circulating worldwide.

[0214] To further investigate the effect of S protein variants on the neutralizing potency of monoclonal antibodies, a full neutralization curve was run to determine the IC50 values of the most potent neutralizing antibodies against a subset of variants located within the receptor-binding domain (RBD) of the S protein. Table 28 shows the IC50 neutralization values for each variant pseudoparticle. A maximum three-fold intrinsic variation can be observed between pseudoparticle neutralization assays and does not indicate a change in neutralizing potency. These data show that the antibodies retain neutralizing 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 D ) of various SARS-CoV-2 RBD reagents binding to purified CHOt anti-SARS-COV-2 monoclonal antibodies (mAbs) were determined by real-time surface Determined using a plasmon resonance-based Biacore T200 / Biacore 8K biosensor. All binding studies were performed at 25 °C and 37 °C in 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05 v / v% surfactant Tween-20, pH 7.4 (HBS-ET) running buffer. The Biacore CM5 sensor chip surface was first derivatized by amine coupling with a mouse anti-human Fc-specific mAb (Regeneron, mAb2567) to capture anti-SARS-COV-2 bmAb. Binding studies were performed with the extracellular domain of the human SARS-COV-2 RBD expressed with a C-terminal myc-myc-hexahistidine (SARS-COV-2 RBD-MMH) and the extracellular domain of the SARS-COV-2 RBD expressed with a C-terminal mouse IgG2a (SARS-COV-2 RBD-mFc). By using these reagents, the ability of the antibody to bind to monomeric and dimeric RBD peptides could be tested respectively.

[0216] Different concentrations of hSARS-COV-2 RBD-MMH (90 nM - 3.33 nM, 3-fold dilution) and SARS-COV-2 RBD-mFc (3-fold dilution of 30 nM - 1.11 nM) prepared in HBS-ET running buffer were injected at a flow rate of 50 μL / min for 3 minutes, and the dissociation of various SARS-COV-2 RBD reagents bound to the mAb was monitored for 6 - 10 minutes in HBS-ET running buffer. At the end of each cycle, the SARS-COV-2 RBD mAb capture surface was regenerated by injecting 20 mM phosphoric acid onto the mouse anti-human Fc-specific mAb surface for 12 seconds. Association rate (K a ) and dissociation rate (K d ) were determined by fitting the real-time binding sensorgram 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. Binding dissociation equilibrium constant (KD ) and dissociation half-life (t1 / 2) were calculated from the migration speed as follows: as follows:

Number

[0217] The binding rate parameters of different SARS-CoV-2 mAbs that bind to different anti-SARS-CoV-2 RBD reagents of the present invention at 25 °C and 37 °C are shown in Tables 29 to 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: As determined by ELISA, the anti-SARS-CoV-2 antibody blocks the binding of RBD to hACE2 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 was composed of the receptor-binding domain (RBD) portion (amino acids Arg319 - Phe541) of the SARS-CoV-2 spike protein 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 consisted of amino acids Gln18 - Ser740 with a C-terminal-10X histidine tag (hACE2-His, NCBI accession number Q9BYF1).

[0220] The experiment was conducted according to the following procedure. Monoclonal anti-Penta-His antibody (Qiagen) was coated at 1 μg / ml in PBS on a 96-well microtiter plate at 4 °C overnight. The hACE2-His receptor was added at 0.2 μg / ml in PBS and allowed to bind for 2 hours at room temperature (RT). Subsequently, non-specific binding sites were blocked using a 0.5 (weight / volume)% solution of BSA in PBS. In other microtiter plates, a fixed amount of 100 pM of SARS-CoV-2 RBD-hFc protein was bound at dilution rates of 0.0008 nM - 50 nM with anti-SARS-COV-2 antibodies and isotype IgG1 antibody controls in PBS + 0.5% BSA. After a 1-hour incubation, the mixed solution was transferred to hACE2-His coated on the microtiter plate. After incubating for 1.5 hours at room temperature, the wells were washed and SARS-COV2 bound to the plate was detected with a goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson). Next, the plate was developed using a TMB substrate solution (BD Biosciences, #555214) according to the manufacturer's recommendations, and the absorbance at 450 nm was measured with a VictorX5 plate reader.

[0221] The binding data were analyzed using the sigmoid dose-response model within Prism (trademark) software (GraphPad). The calculated IC50 values, defined as the concentration of antibody required to block 50% of the SARS-CoV-2 RBD-hFc binding to plate-coated hACE2-His, were used as an indicator of blocking efficacy. The blocking rate 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 less than 50% of the 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 evaluated 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 the presence of antibodies against RBD binding to hACE2-His was evaluated. The RBD-hFc bound to the plate was detected with an HRP-conjugated anti-hFc antibody.

[0224] The blocking IC50 and maximum block at the highest test concentration 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 showed antibody concentration-dependent blocking of RBD.hFc binding to hACE-2. The IC50 values ranged from 41 pM to 4.5 nM, and the maximum block ranged from 55% to approximately 100% at the highest antibody concentration tested. Two of the 46 antibodies tested did not show blocking activity under the assay conditions. As expected, irrelevant isotype control antibodies did not show blocking activity.

Table 41-1

Table 41-2

[0225] Example 15: Cross-competition among mAb10987, mAb10989, mAb10933, and mAb10934 mAb10987, mAb10989, mAb10933, and mAb10934 were examined in a cross-competitive binding assay (Figure 11) to identify any picomolar neutralizing-capable non-competing mAb pairs that could potentially form an antibody cocktail (e.g., mAb10987 and mAb0933). Identified several pairs of non-competing mAbs with a picomolar neutralizing capacity (e.g., mAb10987 and mAb0933).

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

[0227] Comparing the cross-competition binding assay with the results of the above HDX-MS, structural insights into the mechanism by which non-competing antibody pairs can bind to the RBD simultaneously are obtained, and thus they can 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 showing the most significant HDX protection by mAb10933 are upward-facing, suggesting that the Fab region of mAb10933 binds to the RBD from above and that mAb10933 makes a significant collision with ACE2. To avoid competition with mAb10933, mAb10987 binds only to the protection regions defined by HDX from the front or lower left (front view of mAb10987 in Figure 12). This is consistent with the above neutralization data because mAb10987 is oriented at a position where it is likely to interfere with ACE2.

[0228] Example 16: Determination of the Structure of Antibody-Bound Spike Protein To better understand the binding of mAb10933 and mAb10987 to the spike protein RBD, structural analysis was performed via cryo-electron microscopy (cryoEM). 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, incubated on ice for approximately 1 hour, and then injected into a Superdex200 Increase gel filtration column equilibrated with 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 the preparation of cryoEM grids, the protein sample was diluted to 1.5 mg / mL and 0.15% PMAL-C8 amphipol was added. 3.5 μL of the protein was deposited onto an UltrAufoil grid (1.2 / 1.3, 300 mesh) freshly washed with plasma. Excess solution was blotted using filter paper and plunge-frozen into liquid ethane using a Vitrobot Mark IV. The cryoEM grid was transferred to a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan). Movies were collected using EPU (Thermo Fisher) at a magnification of 105,000 corresponding to a pixel size of 0.85 Å. A dose rate of 15 electrons per pixel per second was used, and each movie was 2 seconds, corresponding to a total dose of approximately 40 electrons per Å2.

[0229] All cryoEM data processing was performed using cryoSPARC v2.14.2. 2,821 movies were processed using patch motion correction and patch CTF estimation. Based on the estimated defocus values and CTF-corrected resolutions, 2,197 aligned micrographs were selected for further processing. An initial set of particles picked using 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 the particles in this class followed by homogeneous refinement yielded a map at 3.9 Å resolution (FSC = 0.143) containing 48,140 particles used for model building. 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) were manually placed into this map. These models were then manually rebuilt using Coot and real space was adjusted against the map using Phenix.

[0230] Examination of the above data shows that single particle cryoEM of the complex of SARS-CoV-2 spike RBD bound to the Fab fragments of mAb10933 and mAb10987 indicates that the two antibodies in this cocktail can simultaneously bind to different regions of the RBD (Figure 13A, Figure 13B, and Figure 14). The 3D reconstruction map of the complex at a nominal resolution of 3.9 Å shows that both Fab fragments bind to different epitopes of the RBD, confirming that they are non-competing antibodies. mAb10933 binds to the upper part of the RBD and extensively overlaps with the binding site of ACE2. On the other hand, the epitope of mAb10987 is on the side of the RBD, quite distant from the mAb10933 epitope and hardly or not overlapping with the ACE2 binding site at all.

[0231] Example 17: Mutual competition between anti-SARS-CoV-2-S mAbs The 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 carried out in a buffer of 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) at 25 °C, with the plate shaken at 1000 rpm. To evaluate whether two mAbs can compete with each other for binding to their respective epitopes on the extracellular domain of SARS-COV-2-S RBD expressed with the C-terminal myc-myc-hexahistidine (SARS-COV-2 RBD-MMH), approximately 0.51 nm of SARS-COV-2-S RBD-MMH was first captured on an anti-penta-His antibody-coated Octet biosensor chip (Fortebio Inc, #18-5122) by immersing the biosensor chip into a well containing a 10 μg / mL solution of SARS-COV-2-S RBD-MMH for 1 minute. Next, the biosensor chip capturing SARS-COV-2-S RBD-MMH was saturated with the first anti-SARS-CoV-2-S monoclonal antibody (hereinafter referred to as mAb-1) by immersing it into a well containing a 50 μg / mL mAb-1 solution for 5 minutes. Then, the biosensor chip was immersed into a well containing a 50 μg / mL solution of the second anti-SARS-CoV-2-S monoclonal antibody (hereinafter referred to as mAb-2) for 5 minutes. The biosensor chip was washed with HBS-ETB buffer between each step of the experiment. The real-time binding response was monitored throughout the experiment, and the binding response at the end of each step was recorded. The response of mAb-2 binding to SARS-COV-2 RBD-MMH pre-complexed with mAb-1 was compared, and the competition / non-competition behavior of different anti-SARS-CoV-2 monoclonal antibodies was determined 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 antibodies that bind to monomeric SARS-CoV-2-S RBD reagent measured at 37 °C Dissociation rate constants (K d ) of various anti-SARS-CoV-2-S monoclonal antibodies in pH 7.4, pH 6.0, and pH 5.0 buffers were determined using a real-time surface plasmon resonance (SPR)-based Biacore T200 biosensor. All binding studies were performed at 37 °C using three running buffers: (i) PBS, 0.05 v / v% surfactant Tween-20, pH 7.4 (PBS-T-pH 7.4); (ii) PBS, 0.05 v / v% surfactant Tween-20, pH 6.0 (PBS-T-pH 6.0); and (iii) PBS, 0.05 v / v% surfactant Tween-20, pH 5.0 (PBS-T-pH 5.0). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with mouse anti-human Fc-specific mAb (Regeneron) to capture anti-SARS-CoV-2-S monoclonal antibodies. Binding studies were performed with the extracellular domain of human SARS-CoV-2-S RBD expressed with 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 at a flow rate of 25 μL / min for 3 min, and then the bound SARS-CoV-2-S RBD-MMH was dissociated for 5 min in PBS-T-pH 7.4, PBS-T-pH 6.0, or PBS-T PBS-T-pH 5.0 running buffer.

[0233] The dissociation rate constants (k d) was determined. The dissociation half-life (T1 / 2) and and K d value were calculated.

Number

[0234] Following dissociation at 37 °C in PBS-T-pH7.4 and PBS-T-pH6.0, the K d and t1 / 2 values of SARS-COV-2-S RBD-MMH binding to different anti-SARS-CoV-2-S monoclonal antibodies in PBS-T-pH7.4 are shown in Table 35 . Following dissociation at 37 °C in PBS-T-pH7.4 and PBS-T-pH5.0, the K d and t1 / 2 values of SARS-COV-2-S RBD-MMH binding to different anti-SARS-CoV-2-S monoclonal antibodies in PBS-T-pH7.4 are shown in Table 36 . Comparison of the dissociation half-life (t1 / 2) of SARS-COV-2 RBD-MMH in buffers of pH7.4, pH6.0, and pH5.0.

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 binding 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 using 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 from HEK293T cells to transiently express the SARS-CoV-2 spike protein (accession number MN908947.3, amino acids 16 - 1211). VLPs expressing only VSV were also generated as a negative binding control.

[0237] The experiment was conducted according to the following procedure. VLPs from the above two sources were diluted with PBS and seeded onto a 96-well carbon electrode plate (MULTI-ARRAY high-binding plate, MSD), and incubated overnight at 4°C to adhere the VLPs. Nonspecific binding sites were blocked with 2% (weight / volume) BSA in PBS for 1 hour at room temperature. To the cells bound to the plate, anti-SARS-CoV-2 antibodies and non-binding human IgG1 controls were diluted in PBS + 0.5% BSA in the concentration range of 0.0008 nM to 50 nM, and buffer without antibody was added twice, and the plate was incubated for 1 hour at room temperature with shaking. Next, the plate was washed with 1×PBS, and unbound antibodies were removed using an AquaMax 2000 plate washer (MDS Analytical Technologies). Plate-bound antibodies were detected using a SULFO-TAGTM-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 luminescence signal was recorded with a SECTOR Imager 600 (Meso Scale Development) instrument. The direct binding signal (in RLU) was captured for VLPs expressing SARS-CoV-2 and VLPs of VSV only.

[0238] The ability of anti-SARS-CoV-2-S monoclonal antibodies to bind to SARS-CoV-2-S-expressing VLPs was evaluated using an immunobinding assay, compared to binding to irrelevant VSV-expressing VLPs. Binding to VLPs immobilized on 96-well High Bind plates (MSD) was performed with a series of antibody dilutions, and the bound antibodies were detected using SULFO-TAGTM-conjugated anti-human IgG. The binding signal from electrochemiluminescence was recorded with a Sector Imager 600 (MSD). The RLU values of the antibodies binding to VLPs were determined. All antibodies showed concentration-dependent binding, and the ratio of binding to VSV only in VLPs expressing SARS-COV-2-S 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, and the ratio to VSV was 3 or more 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, and the ratio to VSV VLPs was less than 3, but the signal at 5 nM was higher for VSV / spike than for VSV. As expected, the irrelevant IgG1 isotype antibody showed minimal binding.

Table 45-1

Table 45-2

[0240] Example 20: Anti-SARS-CoV-2-S Antibodies That 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) using electrochemiluminescence to utilize SARS-CoV-2-S-expressing cells was developed.

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

[0242] The experiment was conducted according to the following procedure. After inducing the above two lines of cells with 1 μg / ml of doxycycline at 37 °C for 36 hours, they were harvested, spun down, washed with PBS, diluted with PBS, and seeded onto a 96-well carbon electrode plate (using a MULTI-ARRAY high-binding plate (MSD)) and incubated overnight at 4 °C to allow the cells to adhere. Nonspecific binding sites were blocked with 2% (weight / volume) BSA in PBS for 1 hour at room temperature. To the cells bound to the plate, anti-SARS-CoV-2 antibodies and non-binding human IgG1 controls were diluted in PBS + 0.5% BSA in a concentration range of 0.0008 nM to 50 nM, and buffer without antibody was added twice, and the plate was incubated at room temperature for 1 hour with shaking. Next, the plate was washed with 1× PBS and Unbound antibodies were removed. Plate-bound antibodies were detected using SULFO-TAGTM-conjugated anti-human IgG antibody (Jackson Immunoresearch) for 1 hour at room temperature. After washing, the plates were developed with read buffer (MSD) according to the manufacturer's recommended procedure, and the luminescence signal was recorded with a SECTOR Imager 600 (Meso Scale Development) instrument. The direct binding signal (RLU) was captured in SARS-CoV-2-S-expressing cells and negative control cell lines.

[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 evaluated using an immunobinding assay. Binding to immobilized cells on 96-well high-binding plates (MSD) was performed with a series of antibody dilutions, and the bound antibodies were detected using SULFO-TAGTM-conjugated anti-human IgG. The binding signal from electrochemiluminescence was recorded with a Sector Imager 600 (MSD). All antibodies showed concentration-dependent binding, and the ratio of binding of spike-expressing cells to parental cells was analyzed at concentrations of 5.5 nM and 0.20 nM.

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

Table 46-1

Table 46-1

[0245] All references cited in this specification 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 reference to incorporation by reference is intended by the applicant to relate to each and every publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent, even if such citation is not directly adjacent to the specific recitation that it is incorporated by reference. Inclusion of a specific recitation of incorporation by reference, if present, within this specification in no way weakens this general recitation of incorporation by reference. Citation of a reference in this specification is not intended as an admission that the reference is prior art relevant thereto, nor does it constitute any admission as to the content or date of these publications or documents.

Claims

1. A first polynucleotide encoding the heavy chain variable region (HCVR) of an antibody or an 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, and a second polynucleotide encoding the light chain variable region (LCVR) of the antibody or an antigen-binding fragment thereof, wherein the HCVR comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 204, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 206, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 208, and the LCVR comprises three heavy chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3), the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 212, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 55, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 214, said isolated host cell.

2. The isolated host cell according to claim 1, wherein the first polynucleotide comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 203, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 205, and the HCDR3 nucleic acid sequence set forth in SEQ ID NO:

207.

3. The isolated host cell according to claim 1, wherein the second polynucleotide comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 211, the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 54, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO:

213.

4. The isolated host cell according to claim 1, wherein the first polynucleotide comprises the HCDR1 nucleic acid sequence set forth in SEQ ID NO: 203, the HCDR2 nucleic acid sequence set forth in SEQ ID NO: 205, and the HCDR3 nucleic acid sequence set forth in SEQ ID NO: 207, and the second polynucleotide comprises the LCDR1 nucleic acid sequence set forth in SEQ ID NO: 211, the LCDR2 nucleic acid sequence set forth in SEQ ID NO: 54, and the LCDR3 nucleic acid sequence set forth in SEQ ID NO:

213.

5. The isolated host cell according to any one of claims 1 to 4, wherein the first polynucleotide, the second polynucleotide, or both the first and second polynucleotides are RNA polynucleotides. ​ **Claim 6**: The isolated host cell according to any one of claims 1 to 5, wherein each of the first polynucleotide and the second polynucleotide is contained in a vector or a lipid nanoparticle. **Claim 7** The isolated host cell according to claim 6, wherein the vector is a lentiviral vector or an adeno-associated viral vector. **Claim 8** A first polynucleotide encoding a heavy chain variable region (HCVR) of an antibody or an 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, wherein the HCVR comprises the amino acid sequence set forth in SEQ ID NO: 202, and a second polynucleotide encoding a light chain variable region (LCVR) of the antibody or an antigen-binding fragment thereof, wherein the LCVR comprises the amino acid sequence set forth in SEQ ID NO: 210, the isolated host cell comprising the same. **Claim 9** The first polynucleotide comprises the HCVR nucleic acid sequence set forth in SEQ ID NO: 201; The antibody further comprises an immunoglobulin heavy chain constant region; The antibody further comprises an immunoglobulin heavy chain constant region that is an IgG1 constant region; The antibody comprises the heavy chain amino acid sequence set forth in SEQ ID NO: 216; or The first polynucleotide comprises the heavy chain nucleic acid sequence set forth in SEQ ID NO: 215, the isolated host cell according to claim 8. **Claim 10** The second polynucleotide comprises the LCVR nucleic acid sequence set forth in SEQ ID NO: 209; The antibody comprises the light chain amino acid sequence set forth in SEQ ID NO: 218; The second polynucleotide comprises the light chain nucleic acid sequence set forth in SEQ ID NO: 217; or The second polynucleotide is an RNA polynucleotide, the isolated host cell according to claim 8 or 9. **Claim 11** The isolated host cell according to any one of claims 8 to 10, wherein the first polynucleotide and / or the second polynucleotide is contained in a vector or a lipid nanoparticle. **Claim 12** The isolated host cell according to claim 11, wherein the vector is a lentiviral vector or an adeno-associated viral vector. **Claim 13** A polynucleotide encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or an antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in Sequence Listing No. 832, wherein the HCVR comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), the HCDR1 comprises the amino acid sequence set forth in Sequence Listing No. 204, the HCDR2 comprises the amino acid sequence set forth in Sequence Listing No. 206, and the HCDR3 comprises the amino acid sequence set forth in Sequence Listing No. 208, and the LCVR comprises three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3), the LCDR1 comprises the amino acid sequence set forth in Sequence Listing No. 212, the LCDR2 comprises the amino acid sequence set forth in Sequence Listing No. 55, and the LCDR3 comprises the amino acid sequence set forth in Sequence Listing No. 214, said polynucleotide. **Claim 14** The polynucleotide according to claim 13, wherein the HCVR comprises the amino acid sequence set forth in Sequence Listing No. 202 and the LCVR comprises the amino acid sequence set forth in Sequence Listing No.

210. **Claim 15** A vector or lipid nanoparticle comprising the polynucleotide according to claim 13 or 14. **Claim 16** An isolated host cell comprising the polynucleotide according to claim 13 or 14, or comprising the vector according to claim 15. ​

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