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

Description

[Technical Field]

[0001] Sequence List An official copy of the sequence listing will be submitted electronically via EFS-Web at the same time as the specification, as an ASCII-formatted sequence listing with the filename "10753WO01-Sequence.txt", created on June 25, 2020, and approximately 922,462 bytes in size. The sequence listing contained in this ASCII-formatted document is part of the specification and is incorporated herein by reference in its entirety.

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

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

[0004] To date, there have been no vaccines or treatments to prevent or treat SARS-CoV-2 infection. Given the ongoing threat to human health, there is an urgent need for prophylactic and therapeutic antiviral therapies to control SARS-CoV-2. This spike protein is an attractive target for antibody therapy because the virus uses its spike glycoprotein to interact with the cell receptor ACE2 and the serine protease TMPRSS2 to enter target cells. 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. [Overview of the Initiative]

[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. This disclosure addresses this need in part by providing human anti-SARS-CoV-2-S antibodies, such as those in Table 1, and combinations thereof, including combinations with other therapeutic agents (e.g., anti-inflammatory agents, antimalarial agents, antiviral agents, or other antibody or antigen-binding fragments), as well as methods for using them to treat viral infection.

[0006] This disclosure provides neutralizing human antigen-binding proteins that specifically bind to SARS-CoV-2-S, such as antibodies or antigen-binding fragments thereof.

[0007] In one embodiment, the disclosure relates to an isolated recombinant antibody or its antigen-binding fragment that specifically binds to coronavirus spike protein (CoV-S), wherein the antibody has the following characteristics: (a) about 10 -9 M-EC 50 (b) Coronavirus The present invention provides an isolated recombinant antibody or its antigen-binding fragment having one or more of the following characteristics: (c) showing increased survival in coronavirus-infected animals after administration compared to equivalent coronavirus-infected animals without such administration; and / or (c) comprising three heavy chain complementarity-determining regions (CDRs) (CDR-H1, CDR-H2, and CDR-H3) contained within a heavy chain variable region (HCVR) having 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) having an amino acid sequence having at least about 90% sequence identity with the LCVR of Table 1.

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

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

[0010] In some embodiments, the antibody or antigen-binding fragment comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 of the single antibody listed in Table 1. In some embodiments, the antibody or antigen-binding fragment comprises immunoglobulins containing HCVR and LCVR of the single antibody listed in Table 1.

[0011] In one embodiment, 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 discussed above or herein.

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

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

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

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

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

[0017] In one embodiment, this disclosure relates to the antibody or antigen-binding fragments discussed above or herein. A method for producing an antibody or antigen-binding fragment is provided, comprising: (a) introducing one or more polynucleotides encoding the antibody or antigen-binding fragment into host cells; (b) culturing the host cells under conditions favorable to the expression of the one or more polynucleotides; and (c) optionally isolating the antibody or antigen-binding fragment from the host cells and / or the culture medium in which the host cells grow. In some embodiments, the host cells are Chinese hamster ovary cells.

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

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

[0020] In one embodiment, the present disclosure provides a polynucleotide encoding the polypeptide discussed above.

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

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

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

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

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

[0027] In some embodiments of methods for treating or preventing coronavirus infection The patient is then administered one or more additional therapeutic agents. In some cases, one or more additional therapeutic agents are antiviral drugs or vaccines. In some cases, one or more additional therapeutic agents are selected from the group consisting of anti-inflammatory agents, antimalarial agents, antibodies or antigen-binding fragments that specifically bind to TMPRSS2, and antibodies or antigen-binding fragments that specifically bind to CoV-S. In some cases, the antimalarial agent is chloroquine or hydroxychloroquine. In some cases, the anti-inflammatory agent is an antibody such as sarilumab, tocilizumab, or gymcirumab. In some embodiments, the additional therapeutic agent is a secondary antibody or antigen-binding fragment containing 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 antibodies disclosed herein for use in the context of the methods of this 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 embodiment, the present disclosure provides a method for administering an antibody or antigen-binding fragment, as discussed above or herein, into the body of a subject, comprising injecting the antibody or antigen-binding fragment into the body of the subject. In some embodiments, the antibody or antigen-binding fragment is injected into the body of the subject subcutaneously, intravenously, or intramuscularly.

[0029] In any of the 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 embodiment, the Disclosure provides an isolated antibody or antigen-binding fragment that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence described 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 described 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 described in SEQ ID NO: 210.

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

[0032] In one embodiment, the Disclosure relates to an isolated antibody that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence described in SEQ ID NO: 832, wherein the isolated antibody comprises an immunoglobulin constant region and three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR1) contained within a heavy chain variable region (HCVR) comprising the amino acid sequence described in SEQ ID NO: 202. The present invention provides an isolated antibody comprising (2, and HCDR3) and three light chain complementarity-determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) containing the amino acid sequence described in SEQ ID NO: 210.

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

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

[0035] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some cases, 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 having the amino acid sequence described 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 antigen-binding fragment thereof that binds to the SARS-CoV-2 spike protein containing the amino acid sequence described in SEQ ID NO: 832. In some cases, the secondary antibody or antigen-binding fragment comprises three heavy-chain CDRs (HCDR1, HCDR2, and HCDR3) contained within an HCVR containing the amino acid sequence described in SEQ ID NO: 640, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR containing the amino acid sequence described in SEQ ID NO: 646. In some cases, the secondary antibody or antigen-binding fragment comprises HCDR1 containing the amino acid sequence described in SEQ ID NO: 642, HCDR2 containing the amino acid sequence described in SEQ ID NO: 499, HCDR3 containing the amino acid sequence described in SEQ ID NO: 644, LCDR1 containing the amino acid sequence described in SEQ ID NO: 648, LCDR2 containing the amino acid sequence described in SEQ ID NO: 650, and LCDR3 containing the amino acid sequence described in SEQ ID NO: 652. In some cases, the secondary antibody or antigen-binding fragment comprises an HCVR containing the amino acid sequence described in SEQ ID NO: 640 and an LCVR containing the amino acid sequence described in SEQ ID NO: 646. In some cases, the secondary antibody or its antigen-binding fragment comprises a heavy chain containing the amino acid sequence described in SEQ ID NO: 654 and a light chain containing the amino acid sequence described in SEQ ID NO: 656.

[0037] In one embodiment, the Disclosure provides an isolated antibody or antigen-binding fragment that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence described 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 described 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 described in SEQ ID NO: 646.

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

[0039] In one embodiment, the present disclosure provides an isolated antibody that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence described 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 described 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 described in SEQ ID NO: 646.

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

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

[0042] A pharmaceutical composition further comprising, in some embodiments, a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of a secondary antibody or its antigen-binding fragment that binds to the SARS-CoV-2 spike protein having the amino acid sequence described in SEQ ID NO: 832, an anti-inflammatory agent, an antimalarial agent, and an antibody or its antigen-binding fragment that binds to TMPRSS2.

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

[0044] In various embodiments, any combination of features or components of the embodiments discussed above or herein may be used, and such combinations are included within the scope of this disclosure. Any particular value discussed above or herein may be combined with other relevant values ​​discussed above or herein to enumerate ranges in which those values ​​represent upper and lower limits of a range, and such ranges are included within the scope of this disclosure. [Brief explanation of the drawing]

[0045] [Figure 1] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 2] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 3] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 4] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 5] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 6] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 7] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 8] This shows ELISA blockade data for selected anti-SARS-CoV-2-S antibodies against the SARS-CoV-2 spike protein, which prevents the spike protein from binding to its receptor, ACE2. [Figure 9A]Figure 9B shows the frequency of V gene pairs of heavy chain (X axis) and light chain (Y axis) of isolated neutralizing antibodies against SARS-CoV-2 from VelocImmune® mice (Figure 9A, N=185) and convalescent human donors (Figure 9B, N=68). The color and size of the circles correspond to the number of heavy chain and light chain pairs present in the repertoire of isolated neutralizing antibodies. Neutralization is defined as >70% by diluting the antibody 1:4 (approximately 2 μg / ml) in a VSV pseudoparticle neutralization assay. [Figure 9B] Figure 9B shows the frequency of V gene pairs of heavy chain (X axis) and light chain (Y axis) of isolated neutralizing antibodies against SARS-CoV-2 from VelocImmune® mice (Figure 9A, N=185) and convalescent human donors (Figure 9B, N=68). The color and size of the circles correspond to the number of heavy chain and light chain pairs present in the repertoire of isolated neutralizing antibodies. Neutralization is defined as >70% by diluting the antibody 1:4 (approximately 2 μg / ml) in a VSV pseudoparticle neutralization assay. [Figure 10A] The neutralizing efficacy is shown. Figure 10A shows the neutralizing efficacy of anti-SARS-CoV-2 spike mAbs. Serial dilutions of anti-spike mAbs, IgG1 isotype controls, and recombinant dimer ACE2 (hACE2.hFc) were added to Vero cells along with pVSV-SARS-CoV-2-S-mNeon, and mNeon expression was measured as a readout of viral infectivity 24 hours post-infection. The data are graphed as neutralization rates compared to a control infected with the virus alone. [Figure 10B] The neutralizing efficacy is shown. Figure 10B shows the neutralizing efficacy of individual antispike mAbs and mAb combinations against SARS-CoV-2-S virus in VeroE6 cells. [Figure 11]This graph displays epitope bin analysis from the matrix of various anti-SARS-CoV-2 mAb premix binding assays. Epitope binning was performed for nine anti-SARS-CoV-2 mAbs as described. Each graph had three phases (I, II, III). In Phase I, anti-SARS-CoV-2 mAb (20 ug / ml) was loaded onto an anti-human Fc probe. In Phase II, a human IgG1 blocking mAb solution (100 ug / ml) was used. In Phase III, a solution of 100 nM SARS-CoV-2 RBD-MMH premix complex with 600 nM anti-SARS-CoV-2 mAb binding sites flowed over the mAb capture probe. [Figure 12] A 3D surface model of the spike protein RBD domain structure is displayed, along with the results of the ACE2 interface and HDX-MS epitope mapping. RBD residues protected by the anti-SARS-CoV-2 spike antibody are indicated by shading representing the degree of protection determined by HDX-MS experiments. The RBD structure is reproduced from PDB6M17. [Figure 13A] Figure 13A shows the complexes of mAb10933 and mAb10987 with SARS-CoV-2 RBD. It displays a 3.9 ÅcryoEM map of the mAb10933+RBD+mAb10987 complex, shaded according to the chain of the refined model in Figure 13B. The RBD, the heavy and light chains of mAb10933, and the heavy and light chains of mAb10987 are identified. [Figure 13B] Figure 13A shows the complexes of mAb10933 and mAb10987 with SARS-CoV-2 RBD. It displays a 3.9 ÅcryoEM map of the mAb10933+RBD+mAb10987 complex, shaded according to the chain of the refined model in Figure 13B. The RBD, the heavy and light chains of mAb10933, and the heavy and light chains of mAb10987 are identified. [Figure 14]The cryoEM data statistics are displayed. Data collection and detailed statistics for the mAb10987+mAb10933+SARS-CoV-2 RBD complex structure shown in Figures 13A and 13B are reported. [Modes for carrying out the invention]

[0046] Before describing the methods 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 limit the scope of the present invention, as the scope is limited only by the appended claims.

[0047] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which this invention belongs. Any methods and materials similar to or equivalent to those described herein may be used in carrying out or testing the invention, but preferred methods and materials are described herein. All publications referenced herein are incorporated herein by reference in their entirety.

[0048] The terms "coronavirus" or "CoV" refer to any virus in the coronavirus family, including but not limited to SARS-CoV-2, MERS-CoV, and SARS-CoV. SARS-CoV-2 refers to a newly emerging coronavirus that was identified as the cause of a serious outbreak that originated in Wuhan, China, and is rapidly spreading to other parts of 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 a viral spike protein. The spike protein also binds to TMPRSS2, which activates the spike protein for viral membrane fusion, and is thereby cleaved.

[0049] The term "CoV-S," also called "S" or "S protein," refers to the coronavirus spike protein 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 1273-amino acid type I membrane glycoprotein that assembles into trimers that form a spike or peplomer on the surface of the enclosed 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 homologous receptors via a receptor-binding domain (RBD) present in the S1 subunit. The amino acid sequence of the full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence provided in Sequence ID No. 832. The term "CoV-S" includes protein variants of the CoV spike protein isolated from different CoV isolates, as well as recombinant CoV spike proteins or fragments thereof. This term also encompasses 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 caused by coronaviruses such as SARS-CoV-2, MERS-CoV, or SARS-CoV. This term includes coronavirus respiratory infections, which most often affect the lower respiratory tract. Symptoms may include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (renal failure and impaired renal function), septic shock, and, in severe cases, death.

[0051] virus The present invention includes methods 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 subject’s body is treatable or preventable by administration of an anti-CoV-S antibody or its antigen-binding fragment. In one embodiment of the present invention, “virus” is any virus that expresses a spike protein (e.g., CoV-S). The term “virus” also includes CoV-S-dependent respiratory viruses, which are viruses that infect 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 its antigen-binding fragment. For example, in one embodiment of the present invention, the virus includes 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 may include the genera alpha-coronavirus, beta-coronavirus, gamma-coronavirus, and delta-coronavirus. In some embodiments, the antibody 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 is specific to the coronavirus. It may be specific to a genus or a particular subgroup of a genus. "Viral infection" refers to the entry and multiplication of a virus into the body of an individual.

[0052] Coronavirus virions are spherical, with a diameter of approximately 125 nm. The most striking feature of coronaviruses is the club-shaped spike protrusions emanating from the surface of the virion. These spikes characterize the virion, giving it the appearance of the solar corona and prompting the name coronavirus. Within the virion's envelope is a nucleocapsid. Coronaviruses have a helically symmetrical nucleocapsid, which is uncommon in positive-sense RNA viruses but far more common in negative-sense RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the coronavirus family. The initial attachment of a virion to a host cell is initiated by the interaction between the S protein and its receptor. The location of the receptor-binding domain (RBD) within the S1 region of the coronavirus S protein varies among viruses, with some having the RBD at the C-terminus of S1. The S protein / receptor interaction is a major determinant for coronavirus infection of a host species and also governs the virus's histotropy. Many coronaviruses utilize peptidases as cell receptors. Following receptor binding, the virus must then access the cytosol 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 an antigen-binding protein, such as an antibody that specifically binds to a CoV spike protein or its antigenic fragment, and the antigen-binding 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), as well as their polymers (e.g., IgM). Exemplary antibodies include, for example, those listed in Table 1. Each heavy chain has a heavy chain variable region ("HCVR" or "V").H "), and a heavy chain constant region (C H 1 domain, C H 2 domain, and C H 3 domain). Each light chain includes 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 (FRs). Each V H and V LThis includes three CDRs and four FRs arranged from the amino terminus to the carboxyl terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain CDRs are sometimes called HCDRs or CDR-H and are numbered as above (e.g., HCDR1, HCDR2, and HCDR3 or CDR-H1, CDR-H2, and CDR-H3). Similarly, the light chain CDRs are called LCDRs or CDR-L and are numbered LCDR1, LCDR2, and LCDR3 or CDR-L1, CDR-L2, and CDR-L3. In certain embodiments of the present invention, the FRs of the antibody (or its antigen-binding fragment) are identical to human germline sequences or are naturally or artificially modified. Exemplary human germline sequences include, but are not limited to, VH3-66 and Vk1-33. Accordingly, this disclosure provides an anti-CoV-S antibody or its antigen-binding fragment (e.g., an anti-SARS-CoV-2-S antibody or its antigen-binding fragment) containing the HCDR and LCDR sequences of Table 1 within the VH3-66 or Vk1-33 variable heavy chain or light chain region. This disclosure further relates to IgKV4-1, IgKV1-5, IgKV1-9, IgKV1-12, IgKV3-15, IgKV1-16, IgKV1-17, IgKV3-20, IgLV3-21, IgKV2-24, IgKV1-33, IgKV1-39, IgLV1-40, IgLV1-44, IgLV1-51, IgLV3-1, IgKV1-6, IgLV2-8, IgKV3-11, IgLV2-11, IgLV2-14, A light chain selected from IgLV2-23 or IgLV6-57, and IgHV1-69, IgHV3-64, IgHV4-59, IgHV3-53, IgHV3-48, IgHV4-34, IgHV3-33, IgHV3-30, IgHV3-23, IgHV3-20, IgHV1-18, IgHV3-15, IgHV3-11, IgHV3-9, IgHV1-8, IgHV3-7, IgHV2-5, IgHV1 The present invention provides an anti-CoV-S antibody or its antigen-binding fragment (e.g., an anti-SARS-CoV-2-S antibody or its antigen-binding fragment) containing the HCDR and LCDR sequences of Table 1 within a combination with a heavy chain selected from -2, IgHV2-70, IgHV3-66, IgHV5-51, IgHV1-46, IgHV4-39, IgHV4-31, IgHV3-30-3, IgHV2-26, or IgHV7-4-1. This 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, A light chain selected from IgLV3-1, IgKV1-6, IgLV2-8, IgKV3-11, IgLV2-11, IgLV2-14, IgLV2-23, or IgLV6-57, and IgHV1-69, IgHV3-64, IgHV4-59, IgHV3-53, IgHV3-48, IgHV4-34, IgHV The present invention provides an anti-CoV-S antibody or its antigen-binding fragment (e.g., an anti-SARS-CoV-2-S antibody or its antigen-binding fragment) containing the HCVR and LCVR sequences of Table 1 in combination with a heavy chain selected from 3-33, 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 the heavy and light chains of immunoglobulins contain three hypervariable regions, also called complementarity-determining regions (CDRs), located within a relatively conserved framework region (FR). 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 assignment to each domain is as follows: Sequences of Proteins of Immunological Interest, Kabat, et al.; National Institutes of Health, Bethesda, Md.; 5 th ed.;NIH Publ.No.91-3242(1991);Kabat(1978)Adv.Prot.Chem.32:1-75, Kabat,et al.,(1977)J.Biol.Chem.252:6609-6616,Chothia,et al.,(1987)J Mol.Biol.196:901-917, or according to the definition of Chothia, et al., (1989) Nature 342:878-883.

[0056] The present invention comprises monoclonal anti-CoV-S antigen-binding proteins, e.g., antibodies and their antigen-binding fragments, 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 constituting the population have identical amino acid sequences, except for possible spontaneous mutations that may be present in small amounts. “Pluried” such monoclonal antibodies and fragments in a composition refer to concentrations of identical (i.e., except for possible spontaneous mutations in the amino acid sequence, as discussed above) antibodies and fragments that are higher than those normally occurring in nature, for example, in the blood of a host organism such as a mouse or a human.

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

[0058] As used herein, the term “human” antigen-binding protein, such as an antibody, includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences, whether transplanted in human cells or 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 random or site-directed mutagenesis in vitro, or by somatic metamutation in vivo), such as in the CDR, particularly CDR3. However, as used herein, the term “human antibody” is not intended to include mAbs in which a CDR sequence derived from the germline of another mammalian species (e.g., mouse) is transplanted onto a human FR sequence. The term includes antibodies recombinantly produced in or in non-human mammals or in cells of non-human mammals. The term is not intended to include antibodies isolated from or produced in human subjects. See below.

[0059] The present invention comprises anti-CoV-S chimeric antigen-binding proteins, such as antibodies and their antigen-binding fragments, and methods of using them. As used herein, “chimeric 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 originate 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 their antigen-binding fragments, and methods of using them. As used herein, “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 derived from different animals, or the variable domain (not the constant region) is derived from the first animal. For example, the variable domain may be obtained from an antibody isolated from a human and expressed in a fixed constant region that has not been isolated from that antibody. Exemplary hybrid antibodies are described in Example 1, which refer to PCR products derived from the antibody heavy chain variable region and light chain variable region cloned into an expression vector containing the heavy chain constant region and light chain constant region, respectively. Hybrid antibodies are synthetic and not naturally occurring because the variable and constant regions they contain have not been isolated from a single natural source.

[0061] The term “recombinant” antigen-binding protein, such as an antibody or its antigen-binding fragment, refers to such molecules created, expressed, isolated, or obtained by techniques or methods known in the art, such as recombinant DNA technologies, including DNA splicing and transgenic expression. This term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice), or in cell (e.g., CHO cells) expression systems, or in non-human cell expression systems, or antibodies isolated from recombinant combinatorial human antibody libraries. In some embodiments, recombinant antibodies share sequences with antibodies isolated from organisms (e.g., mice or humans) but have been expressed via recombinant DNA technologies. Such antibodies may have different post-translational modifications (e.g., glycosylation) than antibodies isolated from organisms.

[0062] The recombinant anti-CoV-S antigen-binding proteins disclosed herein, such as antibodies and antigen-binding fragments, can also be generated in an E. coli / T7 expression system. In this embodiment, the nucleic acids encoding the anti-CoV-S antibody immunoglobulin molecules of the present invention (e.g., found in Table 1) can be inserted into a pET-based plasmid and expressed in an E. coli / T7 system. For example, the present invention includes 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 contains a polynucleotide encoding an immunoglobulin chain operably linked to a T7 promoter. For example, in an embodiment of the present invention, a bacterial host cell such as E. coli is expressed in a T7 RNA polymerase operably linked to a lac promoter. The RNA polymerase gene contains a polynucleotide, and the expression of the polymerase and its chain is induced by incubation of host cells 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 known methods for generating recombinant antibodies in the art. One example of a method for generating recombinant antibodies is disclosed in US4816567.

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

[0065] Accordingly, the present invention provides a recombination method for producing an anti-CoV-S antigen-binding protein, such as an antibody or its antigen-binding fragment or its immunoglobulin chain, comprising: (i) introducing an antigen-binding protein, for example, one or more polynucleotides encoding the light and / or heavy chains of immunoglobulins in Table 1, or a CDR (for example, including one or more nucleotide sequences from any of the sequences in Table 2) (the polynucleotide is in a vector and / or integrated into a host cell chromosome and / or operably ligated to a promoter); (ii) culturing host cells (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable for the expression of the polynucleotide; and (iii) optionally isolating the antigen-binding protein (e.g., an antibody or fragment) or chain from the host cells and / or the culture medium in which the host cells grow. For example, the polynucleotide may be used in gene editing systems (e.g. For example, after cutting a chromosome using CRISPR (e.g., CRISPR-Cas9, TALEN, megaTAL, zinc finger, or Argonaute), it can be incorporated into a host cell chromosome by targeting and inserting a vector such as adeno-associated virus (AAV). Targeted insertion can occur at host cell loci, such as albumin or immunoglobulin genomic loci. Alternatively, insertion can be performed at random loci using a vector, such as a lentivirus. When producing an antigen-binding protein (e.g., an antibody or antigen-binding fragment) containing more than one immunoglobulin chain, for example, an antibody containing two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of the chains in a single host cell leads to the association of the chains intracellularly, on the cell surface, or extracellularly, if such chains are secreted to form an antigen-binding protein (e.g., an antibody or antigen-binding fragment). The present invention includes methods in which only immunoglobulin heavy chains or only immunoglobulin light chains (e.g., any of those discussed herein, including mature fragments and / or their variable domains) are expressed. Such chains are useful, for example, as intermediates in the expression of antibodies or antigen-binding fragments containing such chains. For example, the present invention also includes anti-CoV-S antigen-binding proteins such as antibodies and their antigen-binding fragments, comprising a heavy chain immunoglobulin (including its variable domain or its CDR) encoded by a polynucleotide comprising the nucleotide sequence described in Table 2, and a light chain immunoglobulin (including its variable domain or its CDR) (which is the product of such a production method), and optionally, the purification method described herein. For example, in some embodiments, the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising an HCVR comprising the amino acid sequence described in Table 1 and an LCVR comprising the amino acid sequence described in Table 1, where 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, which is an antibody or fragment comprising HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences listed in Table 1, and LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences listed in Table 1, where 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, which is an antibody or fragment comprising a heavy chain comprising the HC amino acid sequences listed in Table 1 and a light chain comprising the LC amino acid sequences listed in Table 1.

[0066] Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for the expression of anti-CoV-S antigen-binding proteins. Such host cells are well known in the art and many 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, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cattle, horse, and hamster cells. 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 The present invention includes yeast and filamentous fungal cells, 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 K when measured by real-time label-free biolayer interferometry assays, e.g., at 25°C or 37°C, e.g., by Octet® HTX biosensor, or by surface plasmon resonance, e.g., by BIACORE®, or by lysis affinity ELISA. D CoV- The binding affinity to antigens such as the S protein (e.g., SARS-CoV-2-S) is at least about 10 -8 This refers to those antigen-binding proteins (e.g., mAbs) that are M. The present invention includes antigen-binding proteins that specifically bind to the CoV-S protein.

[0068] As used herein, the terms “antigen-binding portion” or “antigen-binding fragment” of an antibody, or “antigen-binding protein,” and similar terms include any naturally occurring, enzymatically available, synthetic, or genetically engineered polypeptides or glycoproteins that specifically bind to an antigen to form a complex. Non-limiting examples of antibody-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, and (iv) Fv fragments. Examples include (v) a single-chain Fv(scFv) molecule, (vi) a dAb fragment, and (vii) a minimal recognition unit consisting of amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity-determining region (CDR) such as the CDR3 peptide), or a restricted FR3-CDR3-FR4 peptide. Domain-specific antibodies, single-domain antibodies, domain deletion antibodies, chimeric antibodies, CDR-implanted antibodies, diabodies, triabodies, tetrabodies, minibodies, nanobodies (e.g., as defined in WO08 / 020079 or WO09 / 138519) (e.g., monovalent nanobodies, bivalent nanobodies, etc.), small modular immunotherapy drugs (SMIPs), and other manipulated 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 antibodies listed in Table 1 (e.g., CDR-H1, CDR-H2, and CDR-H3, or CDR-L1, CDR-L2, and CDR-L3).

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

[0070] In certain embodiments, the antigen-binding fragment of the antibody may include at least one variable domain covalently bound to at least one constant domain. Non-limiting exemplary stereochemistry of the variable and constant domains that may be found within the antigen-binding fragment of the antibody of the present invention includes (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 LExamples include: In any configuration of the variable domain and the constant domain, including any of the exemplary configurations listed above, the variable domain and the constant domain may be directly linked to each other, or they may be linked by a complete or partial hinge region or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60 or more) amino acids that result in a flexible or semi-flexible bond between adjacent variable domains and / or constant domains in a single polypeptide molecule. Furthermore, the antigen-binding fragment of the antibody of the present invention may bond with each other and / or one or more monomers V H Or V L In non-covalent bonds with the domain (e.g., via disulfide bonds), the domain may contain a homodimer or heterodimer (or other polymer) among the variable domain configurations and constant domain configurations 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 discussed further herein.

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

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

[0074] "Isolated" antigen-binding proteins, antibodies or their antigen-binding fragments, polypeptides, polynucleotides, and vectors do not contain, at least partially, other biological molecules from the cells or cell cultures from 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. Isolated antibodies or antigen-binding fragments may also not contain, at least partially, expression system components or their growth media, such as biological molecules from host cells. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or components of a pharmaceutical formulation containing antibodies or fragments.

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

[0076] Methods for determining the epitopes of antigen-binding proteins, such as antibodies, fragments, or polypeptides, 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 antigens can be utilized (Tomer (2000) Prot. Sci. 9:487-496). Another method (e.g., coversin) that can be used to identify amino acids within polypeptides that interact with antigen-binding proteins (e.g., antibodies, fragments, or polypeptides) is hydrogen / deuterium exchange detected by mass spectrometry. Generally speaking, hydrogen / deuterium exchange methods involve deuterizing the protein of interest and then conjugating the antigen-binding protein, such as antibodies, fragments, or polypeptides, to the deuterium-labeled protein. Next, the CoV-S protein / antigen-binding protein complex is transferred to water. The exchangeable protons within amino acids protected by the antibody complex undergo a slower deuterium-to-hydrogen reverse exchange than the exchangeable protons within amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antigen-binding protein interface can retain deuterium and therefore exhibit a relatively larger mass compared to amino acids not included in the interface. After dissociation of the antigen-binding protein (e.g., antibody, fragment, or polypeptide), the target protein is subjected to protease cleavage and mass spectrometry to identify the deuterium-labeled residues corresponding to the specific amino acids that the antigen-binding protein interacts with. 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 its antigen-binding fragment) 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 its antigen-binding fragment) to the antigen. The term also includes competition between two antigen-binding proteins in both orientations, e.g., antibodies, i.e., a primary antibody that binds and blocks the binding of a secondary antibody, and vice versa. In certain embodiments, a first antigen-binding protein (e.g., an antibody) and a second antigen-binding protein (e.g., an antibody) may bind to the same epitope. Alternatively, the first and second antigen-binding proteins (e.g., antibodies) may bind to different but overlapping epitopes, such that the binding of one inhibits or blocks the binding of the 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 real-time label-free biolayer interferometry assays. Epitope mapping (e.g., via alanine scanning or hydrogen-deuterium exchange (HDX)) can be used to determine whether two or more antibodies are not competing (e.g., on spike protein receptor-binding domain (RBD) monomers), are competing for the same epitope, or are competing but for diverse microepitopes (e.g., identified via HDX). In embodiments of the present invention, competition between the first and second anti-CoV-S antigen-binding proteins (e.g., antibodies) is determined by the soluble CoV-S protein complexed with the second anti-CoV-S antigen-binding protein (e.g., antibody). This is determined by measuring the ability of a first immobilized anti-CoV-S antigen-binding protein (e.g., an antibody) to bind to the protein (which does not initially complex with the CoV-S protein). A decrease in the ability of the first anti-CoV-S antigen-binding protein (e.g., an antibody) to bind to the complexed CoV-S protein compared to the uncomplexed CoV-S protein indicates competition between the first and second anti-CoV-S antigen-binding proteins (e.g., antibodies). The degree of competition can be expressed as a percentage decrease in binding. Such competition can be measured using real-time label-free biolayer interferometry assays, such as the Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assay), or SPR (surface plasmon resonance).

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

[0079] For example, in embodiments of the present invention, competitive assays were performed at 25°C and a pH of about 7, for example, 7.4, in the presence of, for example, a buffer, salt, surfactant, and a nonspecific protein (e.g., bovine serum albumin).

[0080] Typically, the antibody or antigen-binding fragment of the present invention, modified in some way, retains the ability to specifically bind to CoV-S, for example, retaining at least 10% of its CoV-S binding activity (compared to the parent antibody) when its activity is expressed in molar terms. Preferably, the antibody or antigen-binding fragment of the present invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the CoV-S binding affinity of the parent antibody. The antibody or antigen-binding fragment of the present invention is also intended to contain conserved or non-conserved amino acid substitutions (referred to as “conserved variants” or “function-conserving variants” of the antibody) that do not substantially alter its biological activity.

[0081] "Variants" of polypeptides such as immunoglobulin chains (e.g., mAb8021 V H , V L HC, or LC, mAb8028 V H , V L HC, or LC, or mAb 8029 V H , V L A polypeptide (HC, or LC) is defined as a polypeptide containing at least approximately 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 amino acid sequences to the reference amino acid sequences described herein (e.g., SEQ ID NOs: 2, 10, 18, 20, 22, 30, 38, 40, 42, 50, 58, or 60) when the comparison is performed by the BLAST algorithm, and the parameters of the algorithm are each over the full length of the respective reference sequences. The sequence is selected to obtain the greatest match among these sequences (e.g., expected threshold: 10, word size: 3, maximum match in query range: 0, BLOSUM 62 matrix, gap cost: present 11, extended 1, conditional composition score matrix adjustment).

[0082] A "variant" of a polynucleotide is a polynucleotide that, when comparison is performed by the BLAST algorithm, contains nucleotide sequences that are at least approximately 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 NOs: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, or 59), and the algorithm parameters are selected to obtain the greatest possible match between each sequence over the full length of each reference sequence (e.g., expected threshold: 10, word size: 28, maximum match in query range: 0, match / mismatch score: 1, -2, gap cost: linear).

[0083] In embodiments of the present invention, the anti-CoV-S antigen-binding protein of the present invention, for example, an antibody and its antigen-binding fragment, includes 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 with the HCVR amino acid sequence listed 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 with the LCVR amino acid sequence listed in Table 1.

[0084] In addition, mutant anti-CoV-S antigen-binding proteins may include polypeptides comprising the amino acid sequences described herein, excluding one or more mutations (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) such as missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. For example, the present invention includes antigen-binding proteins comprising immunoglobulin light chain mutants comprising the LCVR amino acid sequence described in Table 1 but having one or more such mutations, and / or immunoglobulin heavy chain mutants comprising the HCVR amino acid sequence described in Table 1 but having one or more such mutations. In embodiments of the present invention, the mutant anti-CoV-S antigen-binding protein comprises immunoglobulin light chain mutants including CDR-L1, CDR-L2, and CDR-L3 (one or more of these CDRs (e.g., 1, 2, or 3) having one or more of these mutations (e.g., conservative substitutions)), and / or immunoglobulin heavy chain mutants including CDR-H1, CDR-H2, and CDR-H3 (one or more of these CDRs (e.g., 1, 2, or 3) having one or more of these mutations (e.g., conservative substitutions)). The substitutions may be included in the CDR, framework, or constant region.

[0085] The present invention further provides a mutant anti-CoV-S antigen-binding protein, such as an antibody or its antigen-binding fragment, comprising, for example, the heavy chain and light chain CDRs of Table 1 and one or more mutant CDRs described herein (e.g., one or more of CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and / or CDR-H3) having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity or similarity.

[0086] Embodiments of the present invention are also the corresponding V described herein. H , V L,HC, orLC amino acid sequence and more than 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) of the whole Immunoglobulin V containing an amino acid sequence having identical or similar amino acid sequence H and V L This also includes mutant antigen-binding proteins, such as anti-CoV-S antibodies and their antigen-binding fragments, which include HC and LC, but the CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3 of such immunoglobulins are not mutants and contain the CDR amino acid sequences listed in Table 1. Therefore, in such embodiments, the CDR within the mutant antigen-binding protein is not a mutant in itself.

[0087] Conservatively modified mutant anti-CoV-S antibodies and their antigen-binding fragments are also part of the present invention. “Conservatively modified mutant” or “conservative substitution” refers to a mutant in which one or more amino acids in a polypeptide are substituted by other amino acids having similar characteristics (e.g., charge, side chain size, hydrophobic / hydrophilicity, skeletal structure, and rigidity). 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 alter its biological activity (e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub.Co., p.224(4) th (See ED.) In addition, substitutions of structurally or functionally similar amino acids are 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 conserved amino acid substituents are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative permutation is any change that has a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256:1443 45.

[0089] Function-conserving variants of anti-CoV-S antibodies and their antigen-binding fragments are also part of the present invention. Any of the variants of anti-CoV-S antibodies and their antigen-binding fragments (discussed herein) may be “function-conserving variants.” Such function-conserving variants may, in some cases, be characterized as conservatively modified variants. As used herein, “function-conserving variant” refers to a variant of an anti-CoV-S antibody or its antigen-binding fragment in which one or more amino acid residues are changed without significantly altering one or more functional properties of the antibody or fragment. In embodiments of the present invention, the function-conserving variant anti-CoV-S antibody or its antigen-binding fragment comprises a variant amino acid sequence and exhibits one or more of the following functional properties. • Inhibiting the proliferation of coronaviruses (e.g., SARS-CoV-2, SARS-CoV, and / or MERS-CoV) in ACE2 and / or TMPRSS2 expressing cells (e.g., Calu-3 cells). • Does not significantly bind to MDCK / Tet-on cells that do not express ACE2 and / or TMPRSS2. • Limiting the spread of coronavirus infection in cells in vitro, e.g., Calu-3 (e.g., by SARS-CoV-2, SARS-CoV, and / or MERS-CoV), and / or Mice engineered to express human TMPRSS2 and / or ACE2 proteins are subjected to coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, if To protect against death caused by MERS-CoV (for example, when optionally combined with a second therapeutic agent, mice would otherwise be infected with a lethal dose of the virus). To protect mice engineered to express human TMPRSS2 and / or ACE2 proteins from weight loss caused by coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV) (e.g., mice are infected with a dose of the virus that would otherwise cause weight loss, when selectively combined with a second therapeutic agent).

[0090] "Neutralizing" or "antagonist" anti-CoV-S antigen-binding proteins, such as antibodies or antigen-binding fragments, refer to molecules that inhibit the activity of CoV-S to any detectable degree, such as being cleaved by proteases like TMPRSS2, or inhibiting CoV-S's ability to bind to receptors like ACE2, which mediate viral entry into or replication in host cells.

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

[0092] The antibodies described herein are also V H This is wild-type IgG4 (for example, residue 108 is S). This includes embodiments in which the IgG4 variant is fused to (for example, residue 108 is P).

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

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

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

[0096] The present invention provides a container (for example, a plastic or glass vial having a cap or chromatography column, a hollow bore needle, or a syringe cylinder) containing the anti-CoV-S antigen-binding protein of the present invention, for example, those listed 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) or a pharmaceutical composition thereof that specifically bind to CoV-S, for example, those listed in Table 1. The injection device may be packaged in a kit. An injection device is a device for introducing a substance into a subject's body via a parenteral route, for example, intramuscular, subcutaneous, or intravenous. For example, an injection device may be a syringe (e.g., pre-filled with the pharmaceutical composition, such as an autoinjector) comprising, for example, a cylinder or barrel for holding the fluid to be injected (e.g., an antibody or fragment thereof or a pharmaceutical composition thereof), a needle for suturing the skin and / or blood vessels for injecting the fluid, and a plunger for pushing the fluid out of the cylinder and through the needle hole. In one embodiment of the present invention, an injection device comprising an antigen-binding protein, for example, an antibody or an antigen-binding fragment thereof, or a pharmaceutical composition thereof, from the combination of the present invention is an intravenous (IV) injection device. Such an instrument may include an antigen-binding protein or its pharmaceutical composition in a cannula or trocar / needle, which can be attached to a tube that can be attached to a bag or reservoir for holding a fluid (e.g., saline solution) introduced into the subject's body through the cannula or trocar / needle. In one embodiment of the present invention, an antibody or a fragment thereof or its pharmaceutical composition may be introduced into the instrument when the trocar and cannula are inserted into the subject's vein and the trocar is removed from the inserted cannula. The IV instrument can be inserted, for example, into a peripheral vein (e.g., hand or arm), a superior or inferior vena cava, or into the right atrium of the heart (e.g., central IV), or into the subclavian vein, internal jugular vein, or femoral vein, and is advanced toward the heart until it reaches, for example, the superior vena cava or right atrium (e.g., a central venous line). In one embodiment of the present invention, the injection instrument is an auto-syringe, a jet injector, or an external infusion pump. The jet injector uses a narrow, high-pressure liquid jet that penetrates the epidermis. The antibody or fragment or its pharmaceutical composition is then introduced into the target body. An external infusion pump is a medical device that delivers an antibody or fragment or their pharmaceutical composition into the target body in a controlled amount. External infusion pumps 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 fluid supply; an elastomer pump holds fluid in an expandable balloon reservoir, and the pressure from the elastic wall of the balloon facilitates the fluid supply; in a peristaltic pump, a set of rollers grips the length of a flexible tube, pushing the liquid forward. In a multi-channel pump, fluid can be supplied from multiple reservoirs at multiple speeds.

[0098] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known method may 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 comprising any one of the following may be used: In a particular embodiment of the present invention, the antibody of the present invention is obtained from mice immunized with full-length natural CoV-S, or with attenuated live or inactivated virus, or with DNA encoding a protein or a fragment thereof. Alternatively, a CoV-S protein or a fragment thereof may be produced 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 may be a CoV-S polypeptide vaccine. In a particular embodiment, one or more additional immunization injections may be administered. In a particular embodiment, the immunogen may be a recombinant CoV-S polypeptide expressed in E. coli, or in any other eukaryotic or mammalian cell such as Chinese hamster ovary (CHO) cells.

[0099] High-affinity chimeric antibodies against CoV-S having human variable regions and mouse constant regions can first be isolated using VELOCIMMUNE® technology (see, e.g., US6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method to generate monoclonal antibodies. VELOCIMMUNE® technology involves generating transgenic mice in which the genome containing human heavy chain variable regions and human light chain variable regions is operably ligated to endogenous mouse constant region loci, so that the mouse produces antibodies containing human variable regions and mouse constant regions in response to antigen stimulation. The DNA encoding the variable regions of the antibody's heavy and light chains is isolated and operably ligated to the DNA encoding the human heavy chain constant region and 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 an antigen of interest, and lymphocytes (such as B cells) are collected from mice that express antibodies. Immortalized hybridoma cell lines can be prepared by fusing these lymphocytes with myeloma cell lines, and such hybridoma cell lines are screened and selected to identify hybridoma cell lines that produce antibodies specific to 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 directly isolated from antigen-specific lymphocytes.

[0101] First, a high-affinity chimeric antibody possessing a human variable region and a mouse constant region is isolated. As in the experimental sections below, the antibody is subjected to desired characteristics including affinity, selectivity, and epitopes. The mouse constant region is characterized and selected. The mouse constant region is replaced with a desired human constant region to produce the fully human antibody of the present invention, e.g., wild-type or modified IgG1 or IgG4. The selected constant region may vary depending on the specific application, but high affinity antigen-binding features and target specificity features reside in the variable region.

[0102] Anticoronavirus spike protein antibodies containing Fc variants According to certain embodiments of the present invention, an anti-CoV-S antigen-binding protein, such as an antibody or antigen-binding fragment, is provided, which comprises an Fc domain containing one or more mutations that enhance or decrease antibody binding to, for example, an FcRn receptor at an acidic pH compared to a neutral pH. For example, the present invention provides an Fc domain with C H 2 or C HThe anti-CoV-S antibody contains mutations in three regions, and these mutations increase the affinity of the Fc domain to FcRn in an acidic environment (e.g., in endosomes with a pH in the range of approximately 5.5 to 6.0). Such mutations may result in an increased serum half-life of the antibody when administered to animals. Non-restrictive 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 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 the modifications 428L (e.g., M428L) and 434S (e.g., N434S), the modifications 428L, 259I (e.g., V259I), and 308F (e.g., V308F), the modifications 433K (e.g., H433K) and 434 (e.g., 434Y), the modifications 252, 254, and 256 (e.g., 252Y, 254T, and 256E), the modifications 250Q and 428L (e.g., T250Q and M428L), and the modifications 307 and / or 308 (e.g., 308F or 308P). In yet another embodiment, the modifications include the modifications 265A (e.g., D265A) and / or 297A (e.g., N297A).

[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 containing an Fc domain, such as an antibody or an antigen-binding fragment.

[0104] Any possible combination of the aforementioned Fc domain mutations, the V described herein H and / or V L containing 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] <000058​​​​​​​​​​​​​​​​​​​​​​The region may include. According to certain embodiments, the antibody of the present invention includes a chimeric hinge region. Chimera C H Includes regions. For example, the chimeric hinge is the human IgG1 hinge region, human The chimeric hinge region may include an "upper hinge" amino acid sequence (amino acid residues at EU numbering positions 216-227) derived from the human IgG1 hinge region, human IgG2 hinge region, or human IgG4 hinge region, combined with a "lower hinge" sequence (amino acid residues at EU numbering positions 228-236) derived from the IgG2 hinge region or the human IgG4 hinge region. According to certain embodiments, the chimeric hinge region includes amino acid residues derived from the human IgG1 or human IgG4 upper hinge and amino acid residues derived from the human IgG2 lower hinge. Chimeric C as described herein H Antibodies containing a region are, in certain embodiments In this context, it may exhibit modified Fc effector function without adversely affecting the therapeutic or pharmacokinetic properties of the antibody. (See, for example, 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 part, such as a toxoid, for example, a therapeutic part ("immunoconjugate"), or to an antiviral drug for treating influenza virus infection. In embodiments of the present invention, an anti-CoV-S antibody or fragment is conjugated to one of the further therapeutic agents described herein. As used herein, the term "immunoconjugate" refers to an antigen-binding protein, such as an antibody or antigen-binding fragment, chemically or biologically linked to a radioactive substance, cytokine, interferon, target or reporter part, enzyme, peptide or protein, or therapeutic agent. An antigen-binding protein can be conjugated to a radioactive substance, cytokine, interferon, target or reporter part, 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 drug may be a second distinct antibody that specifically binds to CoV-S. The type of therapeutic portion that can be conjugated to an anti-CoV-S antigen-binding protein (e.g., antibody or fragment) should take 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 olRev.,62:119-58(1982).

[0107] multispecific antibodies The present invention includes anti-CoV-S antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, and 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, including 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 multispecific 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 include, for each of those antibodies, 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 domains that specifically bind to CoV-S that may be included in the multispecific molecule are (1) (i) Heavy chain variable domain sequences including the CDR-H1, CDR-H2, and CDR-H3 amino acid sequences listed in Table 1, and (ii) Light chain variable domain sequences including the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences described in Table 1, or (2) (i) Heavy chain variable domain sequences including the amino acid sequences listed in Table 1, and (ii) Light chain variable domain sequences containing the amino acid sequences listed in Table 1, or (3) (i) Heavy chain immunoglobulin sequences containing the amino acid sequences listed in Table 1, and (ii) Contains a light chain immunoglobulin sequence that includes the amino acid sequence listed in Table 1.

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

[0111] In one embodiment of the present invention, the bispecific antigen-binding fragment is a first scFv (e.g., V in Table 1) having binding specificity to a first epitope (e.g., CoV-S). H and V L (including the sequence) and a second sc having binding specificity to a second different epitope Includes Fv. For example, in embodiments of the present invention, the first and second scFv are linkers, e.g., peptide linkers (e.g., (GGGGS) n G (Sequence ID 834) The sequences are linked by an S-linker, where n is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Other bispecific antigen-binding fragments include F(ab)2 of the bispecific IgG antibody containing the heavy and light chain CDRs in Table 1, and another antibody that binds to a different epitope. This includes F(ab)2.

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

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

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

[0115] As used herein, the term “Subject” refers to mammals (e.g., rats, mice, cats, dogs, cattle, sheep, horses, goats, rabbits), preferably humans, that require prevention and / or treatment of a disease or disorder such as a viral infection or cancer. Subjects may have a viral infection, such as influenza, or may be predisposed to developing an infection. Subjects predisposed to developing an infection, or at high risk of developing an infection (e.g., coronavirus or influenza virus), include subjects with a weakened immune system due to an autoimmune disease, subjects receiving immunosuppressive therapy (e.g., after organ transplantation), subjects suffering from human immunodeficiency syndrome (HIV) or acquired immunodeficiency syndrome (AIDS), subjects with a form of anemia that depletes or destroys white blood cells, subjects receiving radiation or chemotherapy, or subjects suffering from inflammatory disorders. Furthermore, subjects who are very young (e.g., under 5 years of age) or elderly (e.g., over 65 years of age) are at higher risk. Furthermore, the subjects may be at risk of contracting the virus due to their proximity to the disease outbreak, for example, by living in a densely populated city, or in close proximity to individuals with confirmed or suspected viral infections, or by their employment choices, such as living in close proximity to hospital workers, pharmaceutical researchers, travelers to or frequent visitors to infected areas.

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

[0117] The present invention also encompasses the prophylactic administration of an anti-CoV-S antigen-binding protein, such as the antibody of the present invention or its antigen-binding fragment (e.g., Table 1), to subjects at risk of viral infection in order to prevent such infection. Passive antibody-based immunoprophylaxis has proven to be an effective strategy for preventing subjects from becoming infected with the virus. 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 See Control of influenza A infections, Immunotherapy. 2012 February;4(2):175-186, Prabhu et al., Antivir Ther. 2009;14(7):911-21, Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 hemagglutinin against lethal H5N1 influenza. "Preventing" or "preventing" means administering an anti-CoV-S antigen-binding protein, such as the antibody or antigen-binding fragment of the present invention (e.g., Table 1), to a subject to inhibit signs of disease or infection (e.g., viral infection) in the subject's body, and the antigen-binding protein is effective when administered to the subject in an effective or therapeutically effective amount or dose for that purpose (as discussed herein).

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

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

[0120] The present invention provides for the treatment or prevention of a viral infection (e.g., coronavirus infection), or for the induction or elimination or inhibition of the progression of at least one sign or symptom of a viral infection (the sign or symptom is secondary to the viral infection), by administering a therapeutically effective amount of anti-CoV-S antigen-binding protein (e.g., as shown in Table 1) to a target, for example by injection of the protein into the target's body. • Fever or feeling of fever / chills ·cough, ·sore throat, • Runny nose or nasal congestion, ·sneeze, • Muscle or body pain, ·headache, • Fatigue (tiredness) ·vomiting, ·diarrhea, • Respiratory tract infections, • Chest discomfort, ·Difficulty breathing, • Bronchitis, and / or ·pneumonia, This provides a method for performing this action on subjects that require it (e.g., humans).

[0121] Combinations and pharmaceutical compositions To prepare pharmaceutical compositions of anti-CoV-S antigen-binding proteins, such as antibodies and their antigen-binding fragments (e.g., Table 1), the antigen-binding proteins are mixed with pharmaceutically acceptable carriers or excipients. (e.g., Remington's Pharmaceutical Sciences and US 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, NY, Gennaro (2000) Remington: The Science and See Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993); Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990); Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990); Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000); Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. In an embodiment of the invention, the pharmaceutical composition is sterile. Such a composition is part of the present invention.

[0122] The scope of the present invention includes dried, for example, lyophilized compositions or pharmaceutical compositions thereof (containing a pharmaceutically acceptable carrier but substantially lacking water) comprising an anti-CoV-S antigen-binding protein, such as an antibody or an antigen-binding fragment thereof (for example, those in Table 1).

[0123] In further embodiments of the present invention, further therapeutic agents administered to a subject, relating to anti-CoV-S antigen-binding proteins disclosed herein, such as antibodies or their antigen-binding fragments (e.g., Table 1), are described in Physicians' Desk Reference 2003 (Thomson Healthcare; 57 th It will be administered to the subjects according to the edition (Nov. 1, 2002)

[0124] The method of administration may vary. Routes of administration include oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, inhalation, topical, cutaneous, percutaneous, or intra-arterial.

[0125] The present invention relates to an anti-CoV-S antigen-binding protein, for example, an antibody or its antigen-binding fragment. For example, the present invention provides a method for administering (as shown in Table 1), which involves introducing the protein into the body of a subject. For example, the method involves puncturing the body of the subject with a syringe needle and injecting the antigen-binding protein into the body of the subject, for example, into the subject's vein, artery, tumor, muscle tissue, or subcutaneous tissue.

[0126] The present invention relates to anti-CoV-S antigen-binding proteins, such as antibodies or their antigen-binding fragments (e.g., Table 1), polypeptides (e.g., HC, LC, V in Table 1). H , or V L 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 any of the following: a ), a polynucleotide (e.g., those listed in Table 2), a vector as described herein, or a pharmaceutically acceptable carrier comprising the pharmaceutically acceptable carrier.

[0127] In one embodiment of this disclosure, an anti-CoV-S antigen-binding protein, such as an antibody of the present invention or its antigen-binding fragment (e.g., of Table 1), is administered in association with one or more further therapeutic agents. These further therapeutic agents include, but are not limited to, anti-inflammatory agents, antimalarial agents, secondary antibodies or their antigen-binding fragments that specifically bind to TMPRSS2, and secondary antibodies or their antigen-binding fragments that specifically bind to CoV-S. In some embodiments, the antimalarial agent is chloroquine or hydroxychloroquine. In some embodiments, the anti-inflammatory agent is an antibody such as sarilumab, tocilizumab, or gymcirumab. In some embodiments, the further therapeutic agents are secondary antibodies or antigen-binding fragments disclosed herein, such as those of Table 1. In certain embodiments, one, two, three, four, or more antibodies or their antigen-binding fragments from Table 1 may be administered in combination (e.g., simultaneously or sequentially). Specific antibody combinations in Table 1 are listed in the following table of exemplary antibody combinations (for example, each number representing a specific combination is such that mAb10989 and mAb10987 is combination 1, mAb10989 and mAb10934 is combination 2, and so on). In some embodiments, antibody combinations may be selected from those that bind to different epitope clusters. For example, the 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. Therefore, the combination of two antibodies can be selected from, for example, cluster 1 and cluster 2, cluster 1 and cluster 3, cluster 1 and cluster 4, cluster 1 and cluster 5, cluster 2 and cluster 3, cluster 2 and cluster 4, cluster 2 and cluster 5, cluster 3 and cluster 4, cluster 3 and cluster 5, and cluster 4 and cluster 5.In some embodiments, the antibody that specifically binds to TMPRSS2 is H1H7017N, as described in International Patent Publication 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 comprises compositions comprising two (or more) anti-SARS-CoV-2-S antibodies or antigen-binding fragments containing variable domains from human subjects, wherein the two (or more) antibodies or antigen-binding fragments originate from different subjects (e.g., two different human subjects). Antibody variable regions derived from human B cells are discussed, for example, in Examples 1 and 2 (Table 3), and variable domains cloned from such B cells are combined with constant regions not from those B cells to produce hybrid antibodies. Sources (donors) of such antibody variable regions are shown in the following table of exemplary human-derived antibody variable regions. In some embodiments, the composition may comprise a combination of an antibody or antigen-binding fragment having a variable domain derived from donor 1 and an antibody or antigen-binding fragment having a variable domain derived from donor 2. In some embodiments, the composition may comprise a combination of an antibody or antigen-binding fragment having a variable domain derived from donor 1 and an antibody or antigen-binding fragment having a variable domain derived from donor 3. In some embodiments, the composition may include a combination of an antibody or antigen-binding fragment having a variable domain derived from donor 2 and an antibody or antigen-binding fragment having a variable domain derived from donor 3. In some embodiments, the composition may include a combination of mAb10987 from donor 1 (e.g., an antibody containing a CDR, variable region, or heavy and light chain sequences shown in Table 1) and mAb10989 from donor 3 (e.g., an antibody containing a CDR, variable region, or heavy and light chain sequences shown in Table 1). [Table 3]

[0129] In some embodiments, further therapeutic agents are antiviral drugs and / or vaccines. As used herein, the term “antiviral agent” refers to any anti-infective drug or therapy used to treat, prevent, or improve 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 a viral (e.g., coronavirus) infection in a subject requiring such treatment or prevention by administering the antibodies or antigen-binding fragments of Table 1 in conjunction with further therapeutic agents is part of the present invention.

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

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

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

[0133] In certain embodiments of the present invention, further therapeutic agents are not aprotinin, leupeptin, cationic steroid antibacterial agents, influenza vaccines (e.g., dead, live, attenuated whole virus or subunit vaccines), or antibodies against influenza viruses (e.g., antihemagglutinin antibodies).

[0134] The term "in relation to" refers to constituent components, anti-CoV-S antigen-binding proteins, for example, The present invention demonstrates that the antibody or its antigen-binding fragment can be formulated together with another drug, 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 may be given asynchronously at intervals over a given period (e.g., separately or sequentially). Furthermore, the separate components can be administered to a subject via the same or different routes (e.g., by an anti-CoV-S antibody or its antibody-binding fragment).

[0135] kit Further kits are provided comprising one or more components, including but not limited to anti-CoV-S antigen-binding proteins, such as antibodies or antigen-binding fragments (e.g., Table 1) as discussed herein, in association with one or more additional components, including but not limited to further therapeutic agents as discussed herein. Antigen-binding proteins and / or further therapeutic agents can be formulated in pharmaceutical compositions 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 present invention, the kit comprises an anti-CoV-S antigen-binding protein, for example, the antibody of the present invention or its antigen-binding fragment (for example, from Table 1), or a pharmaceutical composition thereof in one container (for example, in a sterile glass or plastic vial), and a further therapeutic agent in another container (for example, in a sterile glass or plastic vial).

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

[0138] If the kit contains a pharmaceutical composition for parenteral administration to a subject, the kit may include equipment for carrying out such administration (e.g., an injection device). For example, the kit may include one or more subcutaneous needles or other injection devices containing an anti-CoV-S antigen-binding protein, such as the antibody of the present invention or its antigen-binding fragment (e.g., from Table 1).

[0139] The kit may include an insert containing information about the pharmaceutical compositions and dosage forms contained within the kit. Generally, such information helps patients and physicians to use the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information relating to the combination of the present invention may be provided in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and uses, contraindications, warnings, precautions, side effects, overdose, appropriate dosage and administration, methods provided, appropriate storage conditions, references, manufacturer / distributor information, and patent information.

[0140] Diagnostic use of antibodies Anti-CoV-S antigen-binding proteins, such as the antibodies of the present invention or their antigen-binding fragments (e.g., those in Table 1), can be used to detect and / or measure CoV-S in a sample. An exemplary assay for CoV-S may include, for example, contacting a sample with the anti-CoV-S antigen-binding protein of the present invention, where the anti-CoV-S antigen-binding protein is labeled with a detectable label or reporter molecule, or used as a capture ligand for selectively isolating CoV-S from the sample. The presence of an anti-CoV-S antigen-binding protein complexed with CoV-S indicates the presence of CoV-S in the sample. Alternatively, an unlabeled anti-CoV-S antibody can be used in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule is: 3 H, 14 C, 32 P, 35 S, or 125 Radioactive isotopes such as I, The fluorescent or chemiluminescent moiety may be ruolecein 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 (ELISA), radioimmunoassays (RIA), 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 the CoV-S / anti-CoV-S antigen-binding protein, the presence of which indicates the presence of CoV-S.

[0141] The anti-CoV-S antigen-binding proteins of the present invention (e.g., those in Table 1) can be used in Western blotting or immunoprotein blotting procedures to detect the presence of CoV-S or fragments 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 a sample to be tested for the presence of CoV-S, for example, comprising the steps of optionally transferring a protein from the sample to be tested for the presence of CoV-S (e.g., from PAGE or SDS-PAGE electrophoresis separation of proteins in the sample) to a membrane or other solid substrate using a method 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 fragments 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., polynylidene fluoride (PVDF)) membranes, to which proteins to be tested for the presence of CoV-S in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel have been transferred (e.g., after electrophoretic separation in the gel). Before contacting the membrane with the anti-CoV-S antigen-binding protein, the membrane is selectively blocked, for example, with skim milk powder, to bind to nonspecific protein binding sites on the membrane. (2) Wash the membrane once or more to remove unbound anti-CoV-S antigen-binding proteins and other unbound substances, (3) Detect the bound anti-CoV-S antigen-binding protein.

[0143] The detection of bound antigen-binding proteins indicates the presence of CoV-S proteins on the membrane or substrate and in the sample. Detection of bound antigen-binding proteins may be achieved 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 disclosed herein (e.g., antibodies and antigen-binding fragments (e.g., Table 1)) may also be used for immunohistochemistry. Such methods form part of the present invention and include, for example, the following: (1) Contacting the 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) To detect antigen-binding proteins on or within tissues.

[0145] If the antigen-binding protein itself is detectably labeled, it can be detected directly. Alternatively, the antigen-binding protein can be bound by a detectably labeled secondary antibody, after which the label can be detected. [Examples]

[0146] The following examples are provided to give a complete disclosure and explanation of how the methods and compositions of the present invention are prepared and used, and are not intended to limit the scope of what the inventors consider to be the present invention. Although efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantity, temperature, etc.), some experimental errors and deviations should be taken into consideration. Unless otherwise indicated, parts are parts by weight, molecular weight is the average molecular weight, temperature is in degrees Celsius, room temperature is approximately 25°C, and pressure is atmospheric pressure or near-atmospheric pressure.

[0147] Example 1: Generation of human antibodies against 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 either the human immunoglobulin heavy chain and kappa light chain variable region or the human immunoglobulin heavy chain and λ light chain variable region. Each mouse was immunized with a vector expressing the SARS-CoV-2-S receptor-binding domain (RBD) (NCBI accession number (MN908947.3), amino acids 1-1273, SEQ ID NO: 832), followed by SARS-CoV-2-S vector or 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. Hybridoma cell lines were screened and selected to identify cell lines that produce SARS-CoV-2-S specific antibodies. As described in U.S. Patent No. 7,582,298 (which is incorporated herein by reference in its entirety), anti-SARS-CoV-2-S antibodies were directly isolated from antigen-positive mouse B cells without fusing to myeloma cells. Using this method, fully human anti-SARS-CoV-2-S antibodies (i.e., antibodies possessing both a human variable domain and a human constant domain) were obtained.

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

[0149] The biological properties of exemplary antibodies produced according to the method of this embodiment are described in detail in the following examples.

[0150] Example 2: Amino acid and nucleotide sequences of the heavy chain variable region and light chain variable region Table 1 shows the amino acid sequence identifiers for 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 listed in Table 2. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

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

[0152] The variable regions of antibodies derived 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 main strains of VI antibodies utilized VH3-53 paired with VK1-9, VK1-33, or VK1-39, while the human-derived antibodies utilized VH3-66 paired with VK1-33 or VH2-70 paired with VK1-39. Further analysis of the overlaid sequences showed strong overlap in the isolated kappa chain repertoire 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 mean CDR length of the heavy chains was similar between VI and human-derived antibodies, with mean lengths of 13 amino acids and 14.5 amino acids, respectively. The average kappa CDR length was the same for VI and human-derived antibodies, with 9 amino acids, and the lambda chains were similar in average length, at 11.1 amino acids and 10.6 amino acids, respectively. The availability of humanized mouse and human-derived antibodies has increased the diversity of the V gene, allowing for the later identification of non-competitive 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 supernatant by conjugated ELISA An ELISA binding assay was performed to identify antibody supernatant bound to the SARS-CoV-2 spike protein receptor-binding domain (RBD). A protein consisting of SARS-CoV-2 RBD (amino acids 319-541) 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 overnight at 4°C with 1 μg / ml in PBS buffer in a 96-well plate. Nonspecific binding sites were then blocked using a 0.5% (wt / vol) solution of BSA in PBS. Only the antibody supernatant or medium was diluted 1:40 or 1:50 with PSA + 0.5% BSA blocking buffer and transferred to a washed microtiter plate. After incubation at room temperature for 1 hour, the wells were washed, and the supernatant bound to the plate was detected with either goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson Immunoresearch) or anti-mouse IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson Immunoresearch). The plates were then developed using TMB substrate solution (BD Biosciences) according to the manufacturer's recommendations, and the absorbance at 450 nm was measured using a VictorX5 plate reader.

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

[0156] Table 4 summarizes the binding results of the three tests. The SARS-CoV-2 binding signal (absorbance 450 nm) is shown, with a medium-only background provided as a negative reference for each experiment. Samples marked IC (indeterminate) are reported without a value due to experimental anomalies in the plate. The tested supernatant showed substantial binding to SARS-CoV-2-S-RBD, as shown in comparison to the medium-only control. [Table 7-1] [Table 7-2]

[0157] Example 4: Antibody that binds 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 an electrochemiluminescence-based detection platform (MSD) with virus-like particles (VLPs) expressing the SARS-CoV-2 spike protein.

[0158] To transiently express the SARS-CoV-2 spike protein (NCBI accession number MN908947.3, amino acids 16-1211, SEQ ID NO: 833), vesicular stomatitis virus (VSV) lacking glycoprotein G (VSV delta G) was pseudotyped with the SARS-CoV-2 spike protein (VSV-SARS-CoV-2-S) and generated 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 using the following procedure. The two types of VLPs described above were diluted in PBS and seeded onto a 96-well carbon electrode plate (MULTI-ARRAY high-bind plate, MSD). The VLPs were incubated overnight at 4°C to adhere to the plate. Nonspecific binding sites were blocked at room temperature for 1 hour with 2% (weight / volume) BSA in PBS. The supernatant containing antibodies generated from SARS-CoV-2 immunized mice or infected human serum was added to the plate-bound particles along with a control of medium alone diluted 1:10 or 1:20 in 1×PBS + 0.5% BSA buffer. The plates were then incubated at room temperature for 1 hour with shaking, after which the plates were 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 SULFO-TAG™-conjugated anti-human IgG antibody (Jackson Immunoresearch) or SULFO-TAG™-conjugated anti-mouse IgG antibody (Jackson Immunoresearch). After washing, the plates were developed with read buffer (MSD) according to the manufacturer's recommended procedure, and the luminescence signals were recorded using a SECTOR Imager 600 (Meso Scale Development) instrument. Direct binding signals (at RLUs) were captured, and the ratio of VLPs expressing SARS-CoV-2-S to VLPs unrelated to SARS-CoV-2-S was calculated.

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

[0161] Table 5 summarizes the binding results from the three experiments. Signals observed from VLPs expressing SARS-CoV-2-S indicate binding, and comparison with negative VLPs provides relative background. Medium-only samples provide baseline signals for secondary antibodies that bind to samples without supernatant. Forty-six antibodies bound specifically to SARS-CoV-2-S expressing VLPs more than four times higher than to medium-only samples (20–35 RLU), showing binding signals in the range of 85–13,600 RLU. The ratio of SARS-CoV-2-S expressing VSV to VSV-VLP (negative control) ranged from 1.1 to 22.7, with many showing higher background for VSV-VLP. The ratio of 0.9 for mAb11002 may be due to the low concentration of 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 infection 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 using the VSV-SARS-CoV-2-S pseudovirus was developed.

[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 on 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 a multiplicity of infection (MOI). The virus was incubated on the cells for 1 hour with gentle rocking every 10 minutes. Then, 20 mL of infection medium was overlaid and the cells were washed three times with 10 mL of PBS before incubating for 24 hours at 37 °C and 5% CO2. The supernatant was collected into 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 before use in the neutralization assay. This material is called VSV-SARS-CoV-2-S.

[0164] [[ID=十三]] 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 they 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 per well in 100 μL of pre-warmed complete DMEM in 96-well black polystyrene microplates (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 ul 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, 100 μL of the supernatant / pseudovirus mixture was transferred to the cells, and the plate was incubated at 37 °C and 5% CO2 for 24 hours. Using the final supernatant dilutions of 1:4 and 1:20, and in some samples 1:100, the neutralization of VSV-SARS-CoV-2-S pseudovirus was evaluated.

[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 neutralization ability, and in particular, in the set of antibodies evaluated at 1:100, antibodies showing higher neutralization may represent a more potent neutralization ability.

Table 9-1

Table 9-2

Table 9-3

[0169] Example 6: Characterization of antibodies in antibody-dependent cell-mediated toxicity surrogate assays The ability of antibodies targeting the SARS-CoV-2 spike protein to interact with FcγR3a, an Fc receptor prominently expressed in natural killer (NK) cells that induce antibody-dependent cell-mediated cytotoxicity (ADCC), was measured using a surrogate bioassay with antibody-bound reporter and target cells. This assay targeted high-affinity human FcγR3a 176 Val allotype receptor (Jurkat / NFAT-Luc / hFcγR3a 176 Along with Val, Jurkat T cells engineered to express the reporter gene luciferase under the control of the transcription factor NFAT (NFAT-Luc) were used. Target cells were engineered Jurkat T cells expressing the full-length SARS-CoV-2 spike protein regulated by human CD20 (used as a positive control for a human IgG1 antibody targeting CD20) and a doxycycline-inducible promoter. Reporter cells were incubated with target cells, and the transcription factor NFAT in the reporter cells was activated via the Fc domain of the human IgG1 antibody bound to the target cells, promoting luciferase expression, which was then measured via 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 transactivating protein (cloned using 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-CoV-2 spike protein expressing cells were sorted for high spike protein expression and subsequently 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 have high affinity for human FcγR3a 176 Val allotype receptor co The cells were engineered to stably express a luciferase reporter construct in nuclear factor-activated T cells (NFAT) (amino acids M1-K254 of NCBI accession number P08637 VAR_003960). The engineered reporter cells were maintained in RPMI1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 500 μg / ml G418 growth medium.

[0172] 36 hours until the start of the surrogate ADCC assay, 5 × 10 5 1 μg / ml of target cells RPMI + 10% Tet-free FBS containing ml of doxycycline (Sigma) Reporter cells were induced in P / S / G cell culture medium. The day before the experiment, reporter cells were cultured in RPMI1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 500 μg / ml G418 growth medium for 7.5 × 10⁶ cells. 5 The cells were divided into densities of cells / ml.

[0173] To put it simply, on the day of the experiment, target cells and reporter cells are transferred to assay medium (RPMI + 10% Tet-free FBS + P / S / G) in a 3:2 ratio (3 × 10⁻¹⁰). 4 / Well target cells and 2 × 10 4 (Wel reporter cells) 384 wells white micro In addition to the titer plates, various concentrations of anti-SARS-CoV-2-S antibody supernatant were subsequently added. To normalize the detected ADCC activity of the anti-SARS-CoV-2-S antibody supernatant, a positive control sample (CD20 antibody containing human IgG1) and a negative control sample without antibody were included in each plate. The plates were incubated at 37°C / 5% CO2 for 5 hours. After batting, cells were lysed with an equal volume of ONE-Glo® (Promega) reagent, and luciferase activity was detected. The emitted light was captured in relative light units (RLU) on a multi-label plate reader Envision (PerkinElmer), and the data were analyzed and normalized using the following equation.

number

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

[0175] Table 7 summarizes the results, showing the calculated percentages of raw luciferase activity and positive control. A range of %ADCC activity indicating FcγR3a activation by antibody supernatant was observed. All samples showed some degree of surrogate ADCC activity, and 10% of the antibody supernatant showed better surrogate ADCC activity than 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, the 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 NaPO4, pH 6.2 (activation buffer) to The microspheres were resuspended and centrifuged to remove the supernatant. Since the microspheres are light-sensitive, they were protected from light. The microspheres were resuspended in 160 μL of activation buffer, and the carboxylic acid group (-COOH) was activated by adding 20 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog no. 24525) followed by 20 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog no. 22980) at 25°C. After 10 minutes, the pH of the reaction mixture was lowered to 5.0 by adding 600 μL of 50 mM MES, pH 5 (coupling buffer), the microspheres were vortexed, and the supernatant was removed by centrifugation. Activated microspheres were immediately mixed with 500 μL of 25 μg / mL protein antigen or streptavidin in coupling buffer and incubated at 25°C for 2 hours. The coupling reaction was stopped by adding 50 μL of 1 M Tris-HCl, pH 8.0, the microspheres were vortexed, centrifuged, and washed three times with 800 μL of PBS 0.005% (Tween20 0.05%) to remove uncoupling proteins and other reaction components. The microspheres were resuspended at 10 million microspheres / mL in 1 mL of PBS 2% BSA 0.05% sodium azide. For 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. Microspheres were vortexed, centrifuged, and washed three times with 800 μL of PBS, then blocked with 500 μL of 30 mM biotin (Millipore-Sigma, catalog number B4501) in 0.15 M Tris pH 8.0. After incubation for 30 minutes, the microspheres were vortexed, centrifuged, and washed three times with 800 μL of PBS. The microspheres were resuspended at 1 mL of PBS 2% BSA 0.05% sodium azide at a concentration of 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 no. EPX-44444-000). These were then mixed with 25 μL of individual anti-SARS-CoV-2 supernatant containing antibody. After incubation at 25°C for 2 hours, the samples and microspheres were washed twice with 200 μL of DPBS containing 0.05% Tween20. To detect the level of antibody binding to individual microspheres, 100 μL of 2.5 μg / mL- R-phycoerythrin-conjugated goat F(ab')2 anti-human kappa (Southern Biotech, catalog no. 2063-09) or 100 μL of 1.25 μg / mL R-phycoerythrin Affinipure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment specific (Jackson Immunoresearch, catalog no. 115-116-072) was added to the blockage buffer (for antibodies with human Fc region) 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 using Luminex FlexMap 3D (Luminex Corp.) and Luminex xPonent (Luminex Corp.) software version 4.3. The SARS-CoV-2 proteins used in the assay were as follows: RBD_(R319-F541).mmh: Sequence ID 829 RBD_(R319-F541).mFc: Sequence ID 830 RBD_(R319-F541).hFc): Sequence ID 831

[0178] Luminex binding results are shown in Tables 8 and 9 as median fluorescence intensity (MFI) signal intensities. 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 five of these antibodies cross-reacted with the SARS coronavirus spike RBD protein, exhibiting 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 the anti-SARS-CoV-2-S antibody. In this assay, the antigen was amine-conjugated as described in the Luminex binding assay above. Briefly, approximately 9 million MagPlex microspheres (Luminex Corp., MagPLex Microspheres, catalog numbers MagPLex MC10000 and MC12000) were vortexed into 500 μL of 0.1 M NaPO4, pH 6.2 and then dispersed. The heart was separated and the supernatant was removed. The microspheres were resuspended in 160 μL of activation buffer. The carboxylic acid group (-COOH) was activated by adding 20 μL of 50 mg / mL N-hydroxysuccinimide (NHS, Thermo Scientific, catalog number 24525), followed by 20 μL of 50 mg / mL 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC, Thermo Scientific, catalog number 22980) at 25°C. After 10 minutes, the pH of the reaction mixture was lowered to 5.0 by adding 600 μL of 50 mM MES, pH 5 (coupling buffer), the microspheres were vortexed, and the supernatant was removed by centrifugation. The activated microspheres were immediately mixed with 500 μL of 20 μg / mL SARS-CoV-2 spike protein (RBD)(R319-F541)-mmH in coupling buffer and incubated at 25°C for 2 hours. The coupling reaction mixture 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 a concentration of 9 million microspheres / mL.

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

[0181] Protein-modified and protein-unmodified (intact) microspheres were mixed at 2700 beads / ml, and 75 μL of microspheres were seeded into a 96-well ProcartaPlex 96-well flat-bottom plate (ThermoFisher, catalog no. EPX-44444-000). These were then mixed with 25 μL of individual anti-SARS-CoV-2-S supernatant-containing antibodies. The samples and microspheres were incubated at 25°C for 2 hours and then washed twice with 200 μL of DPBS containing 0.05% Tween20. To detect the level of antibody binding to individual microspheres, 100 μL of 2.5 μg / mL- of R-phycoerythrin-conjugated goat F(ab')2 anti-human kappa (Southern Biotech, catalog no. 2063-09) in a blocking buffer (for antibodies with hFc), or 100 μL of 1.25 μg / mL of R-phycoerythrin Affinipure F(ab')2 fragment goat anti-mouse IgG, F(ab')2 fragment specific (Jackson Immunoresearch, catalog no. 115-116-072) in a blocking buffer (for antibodies with mFc), or 100 μL of 1.25 μg / mL of R-phycoerythrin anti-His (Biolegend, catalog no. 362603) in a blocking buffer (ACE-2 control, R&D, catalog no. 933-ZN) was added and incubated at 25°C for 30 minutes. After 30 minutes, the samples were washed twice with 200 μl of washing buffer and resuspended in 150 μl of washing buffer. The plates were read using FlexMap 3D (Luminex Corp.) and Luminex xPonent (Luminex Corp.) software version 4.3.

[0182] The results of Luminex binning are shown in Table 10 as median-first-effect (MFI) signal intensities. To determine clusters, the data were normalized to intact proteins (unmodified microspheres) and clustered. The 46 anti-SARS-CoV-2 antibodies were classified into nine clusters with two or more antibodies, and 11 antibodies were classified as single nodes. 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 characteristics and profiles, suggesting a collection of antibodies bound 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 rate of anti-SARS-CoV-2-S monoclonal antibody Equilibrium dissociation constant (K) of different SARS-CoV-2-S antibodies from the primary supernatant of CHOt cells or hybridomas D ) is a real-time surface plasmon resonance-based Biacor The determination was made using the e T200 / Biacore8K biosensor. All binding studies were performed at 25°C with 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 tip surface was first derivatized by amine coupling with a mouse anti-human Fc-specific mAb or a rabbit anti-mouse Fcγ monoclonal antibody (GE, catalog no. BR-1008-38) to capture the anti-SARS-CoV-2 antibody. Binding studies were conducted using 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 C-terminal mouse IgG2a (SARS-CoV-2 RBD-mFc), or the SARS-CoV-2 RBD extracellular domain expressed with 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 minutes, and the dissociation of various SARS-CoV-2 RBD reagents bound to the antibody was monitored in HBS-ET running buffer for 2 minutes. At the end of each cycle, the SARS-CoV-2 RBD antibody capture surface was regenerated using either a 10-second injection of 20 mM phosphate for the mouse anti-human Fc-specific monoclonal antibody surface, or a 40-second injection of 10 mM glycine, HCl, pH 1.5 for the rabbit anti-mouse Fcγ-specific polyclonal antibody. The association rate (K) was then measured. a ) and dissociation rate (K dThe bond-dissociation equilibrium constant (K) was determined by fitting the real-time bond sensorgram to a 1:1 bond model with a mass transport limitation using BiaEvaluation software v3.1, Biacore Insight Evaluation software v2.0, or curve fitting software. D ) and dissociation half-life (t1 The second ( / 2) was calculated from the velocity as follows:

number

[0184] Tables 11 and 12 show the binding rate parameters of different SARS-CoV-2 monoclonal antibodies that bind to different anti-SARS-CoV-2 RBD reagents of the present invention at 25°C. [Table 17-1] [Table 17-2] [Table 17-3] [Table 18-1] [Table 18-2] [Table 18-3]

[0185] Example 10: Characterization of anti-SARS-CoV-2-S monoclonal antibodies by blocking ELISA. An ELISA-based blockade assay was developed to determine the ability of anti-SARS-CoV-2-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 had a C-terminus (SARS-CoV-2 The receptor-binding domain (RBD) portion (amino acids Arg319~Phe541) of the SARS-CoV-2 spike protein was expressed in the Fc portion of human IgG1 using RBD-hFc (see NCBI accession number MN908947.3). The human ACE2 protein used in the experiment was purchased from the R&D system and consists 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 using the following procedure: Monoclonal anti-Penta-His antibody (Qiagen) was coated overnight at 4°C with 1 μg / ml of PBS on a 96-well microtiter plate. The hACE2-His receptor was added at 0.2 μg / ml in PBS and conjugated at room temperature for 2 hours. Subsequently, nonspecific binding sites were blocked using a 0.5 (wt / vol)% 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 conjugated with antibody diluted 1:10 or 1:20 in PBS + 0.5% BSA. After 1 hour of incubation, these antibody-protein complexes were transferred to microtiter plates coated with hACE2-His. After incubation at room temperature for 1.5 hours, the wells were washed, and the SARS-CoV-2 RBD-hFc protein bound to the plates was detected with goat anti-human IgG antibody conjugated to horseradish peroxidase (HRP) (Jackson). The plates were then developed using TMB substrate solution (BD Biosciences, catalog no. 555214) according to the manufacturer's recommendations, and the absorbance at 450 nm was measured using a VictorX5 plate reader.

[0188] Data analysis was performed by calculating the percentage decrease in signal for fixed SARS-CoV-2-S RBD-hFc concentrations in the presence and absence of antibodies. In the calculation, the binding signal of a constant SARS-CoV-2-S RBD-hFc sample in the absence of antibodies for each plate was referred to as 100% binding or 0% blockage. Baseline signals in culture samples where RBD-hFc was absent were referenced 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 RBDs to human ACE2 was evaluated using a blockade ELISA format. Single-point test antibody supernatant blockade of 10 pM or 15 pM SARS-CoV-2S RBD-hFc binding to hACE2-His, presented on anti-His antibodies coated on 96-well microtiter plates, was detected with HRP-conjugated anti-hFc antibodies.

[0190] Table 13 summarizes the blockade results of the three assays. The SARS-CoV-2-S binding signal (450 nm) and the % blockade calculated in G are shown. Various blockades were observed in the test samples. For samples where NA is indicated in columns 6 and 7, plate-corrected values ​​are included in columns 4 and 5, as the data were consistent with the single-plate switch occurring in these samples. Of the 46 antibody supernatants, 43 blocked more than 50% of SARS-CoV-2-S RBD-hFc binding to plate-coated human ACE2, and of those, 16 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 antibody against 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 given in Ehring (1999) Analytical Biochemistry 267(2):252-259, and Engen This is described in and Smith (201) Anal. Chem. 73:256A-265A.

[0192] HDX-MS experiments are 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 gradients, and a Thermo Q Exactive HF mass spectrometer for peptide mass measurement.

[0193] Labeled solution was prepared as PBS buffer in D2O at pD 7.0 (10 mM ri (Equivalent to pH 7.4 at 25°C with sodium acid buffer, 140 mM NaCl, and 3 mM KCl). For deuterium labeling, 10 μL of RBD protein or RBD protein pre-mixed with each of the 12 antibodies listed above is mixed with 90 μL of D2O-labeled solution and incubated at 20°C. The samples were incubated twice at different time points. For mAb10989, mAb10987, mAb10934, and mAb10933, the time points were 0 minutes (undeuterated control), 5 minutes, and 10 minutes. For mAb10920, mAb10922, mAb10936, mAb10954, mAb10964, mAb10977, mAb10984, and mAb10986, the time points were 0 minutes (undeuterated control) and 10 minutes. The deuteration reaction was performed 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. The quenched samples were then subjected to Leaptec HDX The PAL system was injected, and online pepsin / protease XIII digestion was performed. 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) by a gradient of 20 minutes at -5°C (for mAb10989, mAb10987, mAb10934, and mAb10933), or by 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 Thermo Q Exactive HF mass spectrometry in LC-MS / MS or LC-MS mode.

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

number

[0195] A total of 190 peptides from RBD were identified from both RBD alone and RBD complexed with mAb10989 samples, representing 86.06% sequence coverage of RBD. Peptides showing a decrease of 5% or more in deuterium uptake upon mAb binding (i.e., Δ%D values ​​less than -5%, such as -6%, -10%) 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 RBD were identified from both RBD alone and RBD complexed with mAb10987 samples, representing 86.06% sequence coverage of RBD. Peptides showing a decrease of 5% or more in deuterium uptake upon mAb binding (i.e., Δ%D values ​​less than -5%, such as -6%, -10%) were defined as significantly protected. The peptide corresponding to amino acids 432-452 of RBD (CVIAWNSNNLDSKVGGNYNYL) (SEQ ID NO: 836) was significantly protected by mAb10987.

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

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

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

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

[0201] A total of 81 peptides from RBD were identified from both RBD alone and RBD complexed with mAb10936 samples, representing 82.07% sequence coverage of RBD. Peptides showing a decrease of 5% or more in deuterium uptake upon mAb binding (i.e., Δ%D values ​​less than -5%, such as -6%, -10%) were defined as 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) were significantly protected by mAb10936.

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

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

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

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

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

[0207] In summary, the majority of the neutralizing antibodies tested contact the RBDs in a manner that overlaps with the RBD residues that constitute 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 is 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 plasmids encoding the SARS-CoV-2 spike protein or the same plasmids containing nucleotide mutations encoding known variants of the SARS-CoV-2 spike protein amino acid sequence. All plasmids were confirmed by Sanger sequencing. Cells were transfected at a rate of 1.2 × 10⁶ cells per plate in DMEM complete medium (1000 mL DMEM, Gibco, 100 mL FBS, Gibco, 10 mL PSG, Gibco) one day before transfection with 15 μg / plate of spike DNA, using 125 μL lipofectamine LTX, 30 μL PLUS reagent, and up to 3 mL Opti-Mem. 7 Individual cells were seeded onto a 15 cm plate. 24 hours after infection, the cells were washed with 10 mL of PBS and 0.1 VSVΔ in 10 mL of Opti-Mem. G:mNeon The virus was infected at the MOI. The cells were incubated for 10 hours, gently agitated every 10 minutes. After washing the cells three times with 10 mL of PBS, they were covered with 20 mL of infection medium (1000 mL of DMEM, Gibco, 10 mL of sodium pyruvate, Gibco, 7 mL of BSA, Sigma, 5 mL of gentamicin, Gibco) and incubated at 37°C and 5% CO2 for 24 hours. The pseudovirus supernatant was collected in a 250 mL centrifuge tube on ice, centrifuged at 3000 rpm for 5 minutes to pellet any cell debris, dispensed on ice, and frozen at -80°C. Infectivity was tested in Vero cells before use in the neutralization assay. This material was used in the VSVΔ assay. G:mNeon / Spike pseudovirus, or VSVΔ G:mNeon / Spike_(mutant amino acid mutation) (e.g., VSVΔ) G:mNeonIt is called / Spike_H49Y).

[0209] On day 1, Vero cells were seeded in a T225 flask to a concentration of 80%, washed with PBS (Gibco:20012-043), separated from the flask with TrypLE, and inactivated trypsin with complete DMEM. 20,000 Vero cells were seeded in 100 μL of preheated complete DMEM per well of a 96-well black polystyrene microplate (Corning:3904). On day 2, VSVΔ G:mNeon The spike pseudovirus was thawed on ice and diluted in infection medium. Antibodies were used. Dilutions of each antibody were prepared in 96-well plates with a bottom base at 2-fold assay concentration using 210 μl of infection medium. 120 μl of diluted antibody was transferred to a new U-bottom plate, and the medium and IgG1 control antibody were added to each plate. 120 μl of diluted pseudovirus was added to all wells except the medium control well. 120 μl of infection medium was added to those wells. After incubating the antibody-containing pseudovirus at room temperature for 30 minutes, the medium was removed from the Vero cells. 100 μL of the antibody / pseudovirus mixture was added to the cells, and then incubated for 24 hours in 5% CO2 at 37°C. On day 3, the supernatant was removed from the cell wells. The plate was then replaced with 100 μL of PBS. The plate was read using a SpectraMax i3 equipped with a MiniMax imaging cytometer.

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

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

[0212] As previously reported, recombinant ACE2 mediates the neutralization of VSV spike pseudoparticles. While neutralization was possible, its efficacy was far inferior to that of monoclonal antibodies, showing a decrease of more than 1 / 1000th in potency compared to the best-performing neutralizing mAb (Figure 10A). In addition, the potent neutralizing activity of mAb10987, mAb10989, mAb10933, and mAb10934 was confirmed in neutralization assays, including the neutralization of SARS-CoV-2 in VeroE6 cells (Figure 10B). All neutralization assays produced similar potency with the four mAbs (mAb10987, mAb10989, mAb10933, and mAb10934), and no combination showed synergistic neutralizing activity (Figure 10B). [Table 32-1] [Table 32-2]

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

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

[0215] Example 13: Biacore binding rate of purified anti-SARS-CoV-2-S monoclonal antibody Equilibrium dissociation constant (K) of various SARS-CoV-2 RBD reagents that bind to purified CHOt anti-SARS-CoV-2 monoclonal antibody (mAb). D ) is real-time surface Binding was determined using plasmon resonance-based Biacore T200 / Biacore 8K biosensors. All binding studies were performed at 25°C and 37°C with 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 initially 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 human SARS-CoV-2 RBD extracellular domain expressed with C-terminal myc-myc-hexahistidine (SARS-CoV-2 RBD-MMH) and the SARS-CoV-2 RBD extracellular domain expressed with C-terminal mouse IgG2a (SARS-CoV-2 RBD-mFc). These reagents allowed us to test the ability of antibodies to bind to monomeric and dimeric RBD peptides, respectively.

[0216] Different concentrations of hSARS-COV-2 RBD-MMH (90 nM–3.33 nM, 3-fold dilution) and SARS-COV-2 RBD-mFc (30 nM–1.11 nM, 3-fold dilution), prepared with 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 mAbs was monitored with HBS-ET running buffer for 6–10 minutes. At the end of each cycle, the SARS-COV-2 RBD mAb capture surface was measured at 20 mM on the mouse anti-human Fc specific mAb surface. It was regenerated by injecting phosphoric acid for 12 seconds. Association rate (K a ) and dissociation rate (K d The bond-dissociation equilibrium constant (K) was determined by fitting the real-time bond sensorgram to a 1:1 bond model with a mass transport limitation using BiaEvaluation software v3.1, Biacore Insight Evaluation software v2.0, or curve fitting software.D ) and the dissociation half-life (t1 / 2) are determined from the motion velocity. I calculated it 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 RBD binding to hACE2. We used an ELISA-based blockade assay to determine the ability of anti-SARS-CoV-2 antibodies to block the binding of the SARS-CoV-2 spike protein receptor-binding domain (RBD) to its receptor, human angiotensin-converting enzyme 2 (hACE2).

[0219] The SARS-CoV-2 protein used in this assay consisted of the receptor-binding domain (RBD) portion (amino acids Arg319~Phe541) of the SARS-CoV-2 spike protein, expressed at 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 the R&D system and tagged with a C-terminal 10X histidine tag (hACE2-His, NCBI accession number). It is composed of amino acids Gln18 to Ser740, which have the symbol Q9BYF1.

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

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

number

[0222] Antibodies that blocked less than 50% of binding at the highest concentration tested are classified as non-blocking agents, and IC50 values ​​have not been reported for these 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 SARS-CoV-2 RBD-hFc was titrated with a wide range of concentrations of anti-SARS-CoV-2-S antibody, and the inhibition of RBD binding to hACE2-His by the presence of the antibody was evaluated. Plate-bound RBD-hFc was detected with HRP-conjugated anti-hFc antibody.

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

[0225] Example 15: Intercompetition between mAb10987, mAb10989, mAb10933, and mAb10934 mAb10987, mAb10989, mAb10933, and mAb10934 were investigated using cross-competitive binding assays (Figure 11) to determine the potential for combining them to form antibody cocktails. We identified several pairs of non-competitive mAbs with a certain picomolar neutralizing ability (e.g., mAb10987 and mAb0933).

[0226] Epitope binning of anti-SARS-CoV-2-S mAbs was performed using a ForteBio Octet HTX biolayer interferometry system (Molecular Devices ForteBio) containing a running buffer of 10 mM HEPES, 150 mM NaCl, 0.05% (v / v) Tween-20, pH 7.4, and 1 mg / mL BSA. Using a premix sandwich configuration containing mutually competing mAbs in a pairwise combinatorial manner to bind to the SARS-CoV-2 RBD-MMH protein, the assay was performed at 30°C with continuous agitation at 1000 rpm. After obtaining an initial baseline in running buffer, 20 μg / mL of anti-COVID19 mAb was captured on an anti-human Fc(AHC) biosensor chip for 300 seconds. To block any remaining free unsaturated binding sites on the AHC biosensor chip, all sensors were exposed to a blocking solution containing 100 μg / mL of unrelated IgG1 for 240 seconds. Following this process, the biosensors were immersed for 300 seconds in wells containing a premix solution of 100 nM of SARS-CoV-2 RBD-MMH protein and a 600 nM anti-COVID19 mAb binding site of the second mAb. Binding responses at each step were recorded, and specific signals were normalized by subtracting self-blocking mAb competitive controls from the dataset. Data analysis is handled by Epitope This was performed using Binning with Octet Data Analysis HT 10.0 software.

[0227] Comparing the cross-competition binding assay with the HDX-MS results described above provides structural insights into the mechanism by which non-competitive antibody pairs can simultaneously bind to RBD, thus potentially making them ideal partners in therapeutic antibody cocktails. mAb10987 and mAb10933 represent such an antibody pair. mAb10933 targets a spike-like loop region on one edge of the ACE2 interface. Within that region, the residues exhibiting the most significant HDX protection by mAb10933 are upward-facing, suggesting that the Fab region of mAb10933 binds to RBD from above, leading to a critical collision between mAb10933 and ACE2. To avoid competition with mAb10933, mAb10987 binds only to the HDX-defined protection region from the front or lower left (front view of mAb10987 in Figure 12). This is consistent with the neutralization data described above, as mAb10987 is oriented to a position where it is likely to interfere with ACE2.

[0228] Example 16: Structural determination of antibody-binding 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). Fab fragments of mAb10933 and mAb10987 were separated using the FabALACTICA kit (Genovis). 600 μg of mAb10933 Fab and 600 μg of mAb10987 Fab were mixed with 300 μg of SARS-CoV-2-S RBD and incubated on ice for approximately 1 hour. The mixture was then injected into a Superdex200 increasing gel filtration column equilibrated to 50 mM Tris pH 7.5 and 150 mM NaCl. Peak fractions containing the mAb10933 Fab-mAb10987 Fab-RBD complexes were collected and concentrated using a 10 kDa MWCO centrifuge filter. For the preparation of the cryoEM grid, the protein sample was diluted to 1.5 mg / mL and 0.15% PMAL-C8 amphipol was added. 3.5 μL of protein was deposited onto an UltrAufoil grid (1.2 / 1.3, 300 mesh) that had just been washed with plasma. Excess solution was absorbed using filter paper and plunge-frozen in liquid ethane using a Vitrobot Mark IV. The cryoEM grid was transferred to a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan). The film is The images were collected using an EPU (Thermo Fisher) at a magnification of 105,000x, corresponding to a pixel size of 0.85 Å. A dose rate of 15 electrons per second per pixel was used, and each movie is 2 seconds long, corresponding to a total dose of approximately 40 electrons per Ų.

[0229] All cryoEM data processing was performed using cryoSPARC v2.14.2. 2,821 films were adjusted using patch motion correction and patch CTF estimation. Based on the estimated focus blur values ​​and CTF-fit resolution, 2,197 aligned micrographs were selected for further processing. The initial set of particles picked using the blob picker was subjected to 2D classification to generate a template for template picking. The 989,553 particles picked by template picking were subjected to multiple rounds of 2D classification to remove particles containing unbound fabs 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 purification of this class of particles, followed by further heterogeneous purification, yielded a map with a resolution of 3.9 Å (FSC = 0.143) containing 48,140 particles used for model construction. Models for RBD (obtained from PDB code 6M17) and two Fabs (obtained from previous antibody structures of mAb10987 excluding the lambda light chain, obtained from PDB code 5U15) were manually placed on this map. These models were then manually reconstructed using Coot, and their real-space alignment with the map was adjusted using Phenix.

[0230] Reviewing the data above, single-particle cryoEMs of the SARS-CoV-2 spike RBD complex bound to the Fab fragments of mAb10933 and mAb10987 demonstrate that the two antibodies in this cocktail can simultaneously bind to different regions of the RBD (Figures 13A, 13B, and 14). 3D reconstruction maps of the complex at a nominal resolution of 3.9 Å show that both Fab fragments bind to different epitopes of the RBD, confirming that they are non-competitive antibodies. mAb10933 binds to the upper part of the RBD and extensively overlaps with the ACE2 binding site. On the other hand, the epitope of mAb10987 is located on the side of the RBD, considerably farther from the mAb10933 epitope, and has little to no overlap with the ACE2 binding site.

[0231] Example 17: Mutual competition among anti-SARS-CoV-2-S mAbs Binding competition between anti-SARS-CoV-S monoclonal antibodies (mAbs) was determined using real-time label-free biolayer interferometry (BLI) assays on the Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C with 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05 v / v% Tween-20 surfactant, 1 mg / mL BSA, pH 7.4 (HBS-EBT) buffer, while shaking the plate at a speed of 1000 rpm. To evaluate whether two mAbs could compete with each other for binding to their respective epitopes on the extracellular domain of SARS-CoV-2-S RBD expressed with C-terminal myc-myc-hexahistidine (SARS-CoV-2 RBD-MMH), approximately 0.51 nm of SARS-CoV-2-S RBD-MMH was initially captured on an Octet biosensor chip coated with an anti-penta-His antibody (Fortebio Inc, #18-5122) by immersing the biosensor chip in 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 a first anti-SARS-CoV-2-S monoclonal antibody (hereinafter referred to as mAb-1) by immersing it in a well containing a 50 μg / mL solution of mAb-1 for 5 minutes. Next, the biosensor chip was immersed for 5 minutes in a well containing a 50 μg / mL solution of the second anti-SARS-CoV-2-S monoclonal antibody (hereafter referred to as mAb-2). The biosensor chips were immersed. The biosensor chips were washed with HBS-ETB buffer between each step of the experiment. Real-time binding responses were monitored throughout the entire experiment, and binding responses at the end of each step were recorded. The responses of mAb-2 to SARS-CoV-2 RBD-MMH pre-complexed with mAb-1 were compared to determine the competitive / non-competitive behavior of different anti-SARS-CoV-2 monoclonal antibodies, as shown in Table 34. [Table 42-1] Table 42-2 Table 42-3 Table 42-4 Table 42-5 Table 42-6 Table 42-7 Table 42-8 Table 42-9 Table 42-10 Table 42-11 Table 42-12 Table 42-13 Table 42-14 Table 42-15 Table 42-16 Table 42-17 Table 42-18 Table 42-19 Table 42-20 Table 42-21 Table 42-22 Table 42-23 Table 42-24 Table 42-25 Table 42-26 Table 42-27 Table 42-28 Table 42-29 Table 42-30 Table 42-31 Table 42-32 Table 42-33 Table 42-34 Table 42-35

[0232] Example 18: pH sensitivity of anti-SARS-CoV-2-S monoclonal antibody bound to monomeric SARS-CoV-2-S RBD reagent, measured at 37°C. Dissociation rate constants (K) of various anti-SARS-CoV-2-S monoclonal antibodies in pH 7.4, pH 6.0, and pH 5.0 buffers. d ) is real-time surface plasmon Binding was determined using a 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 a mouse anti-human Fc-specific mAb (Regeneron) to capture an anti-SARS-CoV-2-S monoclonal antibody. Binding studies were conducted using 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 minutes. The bound SARS-COV-2-S RBD-MMH was then dissociated for 5 minutes in PBS-T-pH 7.4, PBS-T-pH 6.0, or PBS-T-pH 5.0 running buffer.

[0233] Using Scrubber 2.0c curve fitting software, the dissociation rate constants (k) of four pH running buffers were fitted to a 1:1 binding model by fitting the real-time binding sensorogram to a 1:1 binding model. d) was determined. Dissociation half-life (T1 / 2) and do K d It was calculated from the values.

number

[0234] Following dissociation at 37°C in PBS-T-pH7.4 and PBS-T-pH6.0, the K of SARS-CoV-2-S RBD-MMH that binds to different anti-SARS-CoV-2-S monoclonal antibodies in PBS-T-pH7.4 d The t1 / 2 values ​​are shown in Table 35. The K of SARS-CoV-2-S RBD-MMH, which binds to different anti-SARS-CoV-2-S monoclonal antibodies in PBS-T-pH7.4, follows dissociation at 37°C in PBS-T-pH7.4 and PBS-T-pH5.0. d And the t1 / 2 values ​​are shown in Table 36. This shows a comparison of the dissociative half-lives (t1 / 2) of SARS-CoV-2 RBD-MMH in buffers at pH 7.4, pH 6.0, and pH 5.0. [Table 43-1] [Table 43-2] [Table 43-3] [Table 44-1] [Table 44-2] [Table 44-3]

[0235] Example 19: Anti-SARS-CoV-2-S antibody that binds 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 SARS-CoV-2 spike protein on an electrochemiluminescence-based detection platform (MSD).

[0236] Pseudotyped bullous 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 negative binding controls.

[0237] The experiment was conducted using the following procedure. VLPs from the two sources mentioned above were diluted in PBS and seeded onto 96-well carbon electrode plates (MULTI-ARRAY high-bind plates, MSD). The VLPs were incubated overnight at 4°C to adhere to the cells. Nonspecific binding sites were blocked at room temperature for 1 hour with 2% (weight / volume) BSA in PBS. Anti-SARS-CoV-2 antibody and unbound human IgG1 control were added to the plate-bound cells in PBS + 0.5% BSA at concentrations ranging from 0.0008 nM to 50 nM. Two doses of antibody-free buffer were added, and the plates were incubated at room temperature for 1 hour with shaking. The plates were then washed with 1× PBS and washed in an AquaMax 2000 plate washer (MDS). Unbound antibodies were removed using Analytical Technologies. Plate-bound antibodies were detected at room temperature for 1 hour using SULFO-TAG™-conjugated anti-human IgG antibody (Jackson Immunoresearch). After washing, plates were developed with read buffer (MSD) according to the manufacturer's recommended procedure, and luminescence signals were recorded using a SECTOR Imager 600 (Meso Scale Development) instrument. Direct binding signals (at RLU) were captured for VLPs expressing SARS-CoV-2 and VLPs expressing only VSVs.

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

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

[0240] Example 20: Anti-SARS-CoV-2-S antibody that binds 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) utilizing SARS-CoV-2-S expressing cells via electrochemiluminescence was developed.

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

[0242] The experiment was conducted using the following procedure. Cells from the two cell lines described above were induced with 1 μg / ml doxycycline at 37°C for 36 hours, then harvested, spun down, washed with PBS, diluted with PBS, and seeded onto 96-well carbon electrode plates (using a MULTI-ARRAY high-bind plate (MSD)). Cells were incubated overnight at 4°C to adhere. Nonspecific binding sites were blocked at room temperature for 1 hour with 2 (weight / volume)% BSA in PBS. Anti-SARS-CoV-2 antibody and unbound human IgG1 control were added to the plate-bound cells in a concentration range of 0.0008 nM to 50 nM, diluted in PBS + 0.5% BSA. Antibody-free buffer was added twice, and the plates were incubated at room temperature for 1 hour with shaking. The plates were then washed with 1× PBS using an AquaMax2000 plate washer (MDS Analytical Technologies). Unbound antibodies were removed. Plate-bound antibodies were detected at room temperature for 1 hour using SULFO-TAG™-conjugated anti-human IgG antibody (Jackson Immunoresearch). After washing, the plates were developed with read buffer (MSD) according to the manufacturer's recommended procedure, and the luminescence signal was detected using a SECTOR Imager 600 (Meso Scale). The signals were recorded using a development instrument. Direct binding signals (RLUs) were 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 immunobinding assays. Binding to immobilized cells on 96-well high-binding plates (MSDs) was performed using a series of antibody dilutions, and bound antibodies were detected using SULFO-TAG™-conjugated anti-human IgG. Binding signals from electrochemiluminescence were recorded using a Sector Imager 600 (MSD). All antibodies showed concentration-dependent binding, and the ratio of binding to spike-expressing cells to parental cells was analyzed at concentrations of 5.5 nM and 0.20 nM.

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

[0245] All references cited herein are incorporated by reference to the same extent as each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent is specifically and individually indicated as being incorporated by reference. This incorporation by reference is intended by the applicant to relate to all individual publications, database entries (e.g., Genbank sequence or GeneID entry), patent applications, or patents, even if such citations are not directly adjacent to the specific incorporation by reference. This general incorporation by reference is not diminished by including, if present, specific incorporations within this specification. References cited herein are not intended as an acknowledgment that the references are relevant prior art, nor do they constitute any acknowledgment of the content or date of these publications or documents. The sequences excluded from the ST.26 format sequence list are shown in Table 39. [Table 47]

Claims

1. An isolated host cell comprising a first polynucleotide encoding the heavy chain variable region (HCVR) of an antibody or antigen-binding fragment that binds to the SARS-CoV-2 spike protein having the amino acid sequence described in SEQ ID NO: 832, and a second polynucleotide encoding the light chain variable region (LCVR) of the antibody or antigen-binding fragment, wherein the HCVR comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), and HCDR1 is the amino acid sequence described in SEQ ID NO: 642 The isolated host cell comprising an acid sequence, wherein HCDR2 comprises the amino acid sequence described in SEQ ID NO: 499, HCDR3 comprises the amino acid sequence described in SEQ ID NO: 644, and LCVR comprises three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3), wherein LCDR1 comprises the amino acid sequence described in SEQ ID NO: 648, LCDR2 comprises the amino acid sequence described in SEQ ID NO: 650, and LCDR3 comprises the amino acid sequence described in SEQ ID NO:

652.

2. The isolated host cell according to claim 1, wherein the first polynucleotide comprises the HCDR1 nucleic acid sequence described in SEQ ID NO: 641, the HCDR2 nucleic acid sequence described in SEQ ID NO: 498, and the HCDR3 nucleic acid sequence described in SEQ ID NO:

643.

3. The isolated host cell according to claim 1, wherein the second polynucleotide comprises the LCDR1 nucleic acid sequence described in SEQ ID NO: 647, the LCDR2 nucleic acid sequence described in SEQ ID NO: 649, and the LCDR3 nucleic acid sequence described in SEQ ID NO:

651.

4. The isolated host cell according to claim 1, wherein the first polynucleotide comprises the HCDR1 nucleic acid sequence described in SEQ ID NO: 641, the HCDR2 nucleic acid sequence described in SEQ ID NO: 498, and the HCDR3 nucleic acid sequence described in SEQ ID NO: 643, and the second polynucleotide comprises the LCDR1 nucleic acid sequence described in SEQ ID NO: 647, the LCDR2 nucleic acid sequence described in SEQ ID NO: 649, and the LCDR3 nucleic acid sequence described in SEQ ID NO:

651.

5. The first polynucleotide, the second polynucleotide, or the first and second An isolated host cell according to any one of claims 1 to 4, wherein both polypeptides are RNA polynucleotides.

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 lipid nanoparticles.

7. The isolated host cell according to claim 6, wherein the vector is a lentiviral vector or an adeno-associated virus vector.

8. An isolated host cell comprising: a first polynucleotide encoding the heavy chain variable region (HCVR) of an antibody or antigen-binding fragment that binds to the SARS-CoV-2 spike protein, comprising the amino acid sequence described in SEQ ID NO: 832, wherein the HCVR comprises the amino acid sequence described in SEQ ID NO: 640; and a second polynucleotide encoding the light chain variable region (LCVR) of the antibody or antigen-binding fragment, wherein the LCVR comprises the amino acid sequence described in SEQ ID NO:

646.

9. The first polynucleotide comprises the HCVR nucleic acid sequence described in Sequence ID No. 639; The antibody further comprises an immunoglobulin heavy chain constant region; The antibody further comprises an immunoglobulin heavy chain constant region which is the IgG1 constant region; The antibody contains the heavy chain amino acid sequence described in SEQ ID NO: 654; or, The isolated host cell according to claim 8, wherein the first polynucleotide comprises the heavy chain nucleic acid sequence described in SEQ ID NO:

653.

10. The second polynucleotide comprises the LCVR nucleic acid sequence described in Sequence ID No. 645; The antibody comprises the light chain amino acid sequence described in Sequence ID No. 656; The second polynucleotide comprises the light chain nucleic acid sequence described in Sequence ID No. 655; or, The isolated host cell according to claim 8 or 9, wherein the second polynucleotide is an RNA polynucleotide.

11. An isolated host cell according to any one of claims 8 to 10, wherein the first polynucleotide and / or the second polynucleotide are contained in a vector or lipid nanoparticles.

12. The isolated host cell according to claim 11, wherein the vector is a lentiviral vector or an adeno-associated virus vector.

13. A polynucleotide encoding a heavy chain variable region (HCVR) and a light chain variable region (LCVR) of an antibody or antigen-binding fragment that binds to the SARS-CoV-2 spike protein containing the amino acid sequence described in SEQ ID NO: 832, wherein the HCVR comprises three heavy chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3), where HCDR1 comprises the amino acid sequence described in SEQ ID NO: 642, HCDR2 comprises the amino acid sequence described in SEQ ID NO: 499, and HCDR3 comprises the amino acid sequence described in SEQ ID NO: 644, and the LCVR comprises three light chain complementarity-determining regions (CDRs) (LCDR1, LCDR2, and LCDR3), where LCDR1 comprises the amino acid sequence described in SEQ ID NO: 648, LCDR2 comprises the amino acid sequence described in SEQ ID NO: 650, and LCDR3 comprises the amino acid sequence described in SEQ ID NO:

652.

14. The HCVR comprises the amino acid sequence described in Sequence ID No. 640, and the LCVR comprises the sequence The polynucleotide according to claim 13, comprising the amino acid sequence described in number 646.

15. A vector or lipid nanoparticle comprising the polynucleotide described in claim 13 or 14.

16. An isolated host cell comprising the polynucleotide described in claim 13 or 14, or the vector or lipid nanoparticles described in claim 15.

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