Anti-SARS-COV-2-Spike Glycoprotein Antibodies and Antigen-Binding Fragments

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to a coronavirus spike protein, and methods of using such antibodies and fragments to treat or prevent a viral infection (e.g., a coronavirus infection).

Description

[Technical field]

[0001] Sequence Listing This application incorporates by reference a computer readable sequence listing in ST.26 XML format, entitled 11007WO01-Sequence, created on July 12, 2022, and containing 1,488,419 bytes.

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

[0003] Newly identified viruses, such as coronaviruses, can be difficult to treat because they are not well characterized. The emergence of these newly identified viruses highlights the need for the development of novel antiviral strategies. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a newly emerged coronavirus that causes the severe acute respiratory disease COVID-19. SARS-CoV-2 was first identified from an outbreak in Wuhan, China, and as of July 8, 2022, the World Health Organization has reported 551,296,228 confirmed cases resulting in 6,345,595 deaths. Clinical features of COVID-19 include fever, dry cough, and fatigue, and the disease can cause respiratory failure and death.

[0004] Given the ongoing threat to human health, especially the emergence of new variants of the SARS-CoV-2 virus, there remains an urgent need for preventive and therapeutic antiviral therapies for SARS-CoV-2 control. As this virus uses its spike glycoprotein for interaction with the cellular receptor ACE2 and the serine protease TMPRSS2 to enter target cells, this spike protein represents an attractive target for antibody therapeutics. 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 in the prevention and treatment of COVID-19. Summary of the Invention

[0005] There is a need for neutralizing therapeutic anti-SARS-CoV-2-spike protein (SARS-CoV-2-S) antibodies and their uses to treat or prevent viral infection. The present disclosure addresses this need, in part, by providing human anti-SARS-CoV-2-S antibodies, such as those in Table 4, and combinations thereof, including, for example, combinations with other therapeutic agents (e.g., anti-inflammatory agents, anti-malarial agents, anti-viral agents, or other antibodies or antigen-binding fragments), and methods of use thereof to treat viral infection.

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

[0007] In one aspect, the present disclosure provides an isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to a coronavirus spike protein (CoV-S), wherein the antibody has one or more of the following characteristics: (a) a specific binding affinity of about 10 -8 EC below M 50(b) exhibits extended survival in coronavirus-infected animals after administration to the coronavirus-infected animals compared to comparable coronavirus-infected animals that are not administered; and / or (c) comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy chain variable region (HCVR) of Table 4 that comprises an amino acid sequence having at least about 90% sequence identity with a HCVR, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light chain variable region (LCVR) of Table 4 that comprises an amino acid sequence having at least about 90% sequence identity with a LCVR.

[0008] In some cases, the antibody or antigen-binding fragment comprises (a) a heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of an antibody in Table 4 (e.g., an immunoglobulin HCVR), and / or (b) a light chain variable region comprising LCDR1, LCDR2, and LCDR3 of an antibody in Table 4 (e.g., an immunoglobulin LCVR).

[0009] In some cases, the antibody or antigen-binding fragment comprises (a) a heavy chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to an HCVR sequence in Table 4, and / or (b) a light chain immunoglobulin variable region comprising an amino acid sequence having at least 90% amino acid sequence identity to an LCVR sequence in Table 4.

[0010] In some embodiments, the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of a single antibody of Table 4. In some embodiments, the antibody or antigen-binding fragment comprises an immunoglobulin that comprises the HCVR and LCVR of a single antibody of Table 4.

[0011] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) comprising three complementarity determining regions (CDRs) comprised within the amino acid sequence of SEQ ID NO: 212; (b) a HCVR comprising an HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 214, 216, and 218, respectively; (c) a HCVR comprising the amino acid sequence of SEQ ID NO: 212; (d) a light chain variable region (LCVR) comprising three CDRs comprised within the amino acid sequence of SEQ ID NO: 220; (e) a LCVR comprising an LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 222, 126, and 224, respectively; (f) a LCVR comprising the amino acid sequence of SEQ ID NO: 220; (g) a light chain variable region (LCVR) comprising an LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 226, 228, and 229, respectively; (h) a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 228; (i) an HCVR / LCVR pair comprising CDRs contained within the amino acid sequence of SEQ ID NO: 212 / 222, respectively; (j) a HCVR comprising HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 214, 216, and 218, respectively, and a LCVR comprising LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 222, 126, and 224, respectively; (k) a HCVR comprising the amino acid sequence of SEQ ID NO: 212, and a LCVR comprising the amino acid sequence of SEQ ID NO: 220; or (l) a HC comprising the amino acid sequence of SEQ ID NO: 226, and a LC comprising the amino acid sequence of SEQ ID NO: 228.

[0012] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) comprising three complementarity determining regions (CDRs) comprised within the amino acid sequence of SEQ ID NO: 362; (b) a HCVR comprising an HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 364, 366, and 368, respectively; (c) a HCVR comprising the amino acid sequence of SEQ ID NO: 362; (d) a light chain variable region (LCVR) comprising three CDRs comprised within the amino acid sequence of SEQ ID NO: 370; (e) a LCVR comprising an LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 372, 106, and 374, respectively; (f) a LCVR comprising the amino acid sequence of SEQ ID NO: 370; (g) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 376; (h) a H chain variable region comprising the amino acid sequence of SEQ ID NO: 1077; C, (i) a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 378; (j) an HCVR / LCVR pair comprising CDRs comprised within the amino acid sequence of SEQ ID NO: 362 / 370, respectively; (k) an HCVR comprising HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 364, 366, and 368, respectively, and an LCVR comprising LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 372, 106, and 374, respectively; (l) an HCVR comprising the amino acid sequence of SEQ ID NO: 362, and an LCVR comprising the amino acid sequence of SEQ ID NO: 370; (m) an HC comprising the amino acid sequence of SEQ ID NO: 376, and an LC comprising the amino acid sequence of SEQ ID NO: 378; or (n) an HC comprising the amino acid sequence of SEQ ID NO: 1077, and an LC comprising the amino acid sequence of SEQ ID NO: 378.

[0013] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) comprising three complementarity determining regions (CDRs) comprised within the amino acid sequence of SEQ ID NO: 493; (b) a HCVR comprising an HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 495, 497, and 499, respectively; (c) a HCVR comprising the amino acid sequence of SEQ ID NO: 493; (d) a light chain variable region (LCVR) comprising three CDRs comprised within the amino acid sequence of SEQ ID NO: 501; (e) a LCVR comprising an LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 503, 505, and 507, respectively; (f) a LCVR comprising the amino acid sequence of SEQ ID NO: 501; (g) a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 509; (h) a H chain variable region comprising the amino acid sequence of SEQ ID NO: 1075; C, (i) a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 511; (j) an HCVR / LCVR pair comprising CDRs contained within the amino acid sequence of SEQ ID NO: 493 / 501, respectively; (k) an HCVR comprising HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 495, 497, and 3499, respectively, and an LCVR comprising LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 503, 505, and 507, respectively; (l) an HCVR comprising the amino acid sequence of SEQ ID NO: 493, and an LCVR comprising the amino acid sequence of SEQ ID NO: 501; (m) an HC comprising the amino acid sequence of SEQ ID NO: 509, and an LC comprising the amino acid sequence of SEQ ID NO: 511; or (n) an HC comprising the amino acid sequence of SEQ ID NO: 1075, and an LC comprising the amino acid sequence of SEQ ID NO: 511.

[0014] In some embodiments, the antibody or antigen-binding fragment comprises: (a) a heavy chain variable region (HCVR) comprising three complementarity determining regions (CDRs) comprised within the amino acid sequence of SEQ ID NO: 887; (b) a HCVR comprising an HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NOs: 889, 891, and 893, respectively; (c) a HCVR comprising the amino acid sequence of SEQ ID NO: 887; (d) a light chain variable region (LCVR) comprising three CDRs comprised within the amino acid sequence of SEQ ID NO: 895; (e) a LCVR comprising an LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 897, 164, and 899, respectively; (f) a LCVR comprising the amino acid sequence of SEQ ID NO: 895; (g) a light chain variable region (LCVR) comprising an LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NOs: 901, 902, and 903; (h) a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 903; (i) an HCVR / LCVR pair comprising CDRs contained within the amino acid sequences of SEQ ID NO: 887 / 895, respectively; (j) a HCVR comprising HCDR1, HCDR2, and HCDR3 comprising the amino acid sequences of SEQ ID NO: 889, 891, and 893, respectively, and a LCVR comprising LCDR1, LCDR2, and LCDR3 comprising the amino acid sequences of SEQ ID NO: 897, 164, and 899, respectively; (k) a HCVR comprising the amino acid sequence of SEQ ID NO: 887, and a LCVR comprising the amino acid sequence of SEQ ID NO: 895; or (l) a HC comprising the amino acid sequence of SEQ ID NO: 901, and a LC comprising the amino acid sequence of SEQ ID NO: 903.

[0015] In one aspect, the disclosure provides an antigen-binding protein that competes with an antibody or antigen-binding fragment discussed above or herein for binding to CoV-S.

[0016] In one aspect, the present disclosure binds to the same epitope, or an overlapping epitope, on CoV-S as an antibody or antigen-binding fragment described above or discussed herein.

[0017] In some embodiments, the antibodies or antigen-binding fragments described above or discussed herein are multispecific.

[0018] In some embodiments, the antibody or antigen-binding fragment comprises one or more of the following properties: (a) inhibits coronavirus growth; (b) binds to the surface of coronavirus; (c) limits the spread of coronavirus infection of cells in vitro; and (d) protects mice engineered to express human ACE2 or TMPRSS2 protein from death and / or weight loss caused by coronavirus infection.

[0019] In any of the various embodiments described above or discussed herein, the CoV-S may be SARS-CoV-2-S.

[0020] In one aspect, the disclosure provides a conjugate comprising an antibody or antigen-binding fragment as described above or discussed herein bound to a CoV-S polypeptide. In some cases, the CoV-S is SARS-CoV-2-S.

[0021] In one aspect, the disclosure provides a method for making an antibody or antigen-binding fragment as described above or discussed herein, comprising: (a) introducing into a host cell one or more polynucleotides (e.g., a polynucleotide or pair of polynucleotides as discussed below) encoding the antibody or antigen-binding fragment, (b) culturing the host cell under conditions favorable for expression of the one or more polynucleotides, and (c) optionally isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown. Optionally, the host cell is a Chinese hamster ovary cell.

[0022] In one aspect, the disclosure provides an antibody or antigen-binding fragment that is the product of the methods discussed above.

[0023] In one aspect, the disclosure provides a polypeptide comprising: (a) an HCDR1, HCDR2, and HCDR3 of an HCVR domain of an antibody or antigen-binding fragment comprising an HCVR amino acid sequence set forth in Table 4; or (b) an LCDR1, LCDR2, and LCDR3 of an LCVR domain of an immunoglobulin chain comprising an LCVR amino acid sequence set forth in Table 4.

[0024] In various embodiments, the disclosure provides a polynucleotide encoding a polypeptide as discussed above, a vector comprising a polynucleotide, and / or a host cell comprising an antibody or antigen-binding fragment or polypeptide or a polynucleotide or vector. In various embodiments, the disclosure provides a polynucleotide encoding the HCVR, LCVR, or both the HCVR and LCVR of an antibody or antigen-binding fragment thereof as discussed above or herein. The HCVR and / or LCVR may be defined by the CDRs contained within the HCVR sequence, the LCVR sequence, or both the HCVR sequence and the LCVR sequence, respectively, set forth in Table 4. The HCVR and / or LCVR may also be defined by the heavy chain CDR sequence, the light chain CDR sequence, or both the heavy and light chain CDR sequences, respectively, set forth in Table 4. The HCVR and / or LCVR may be defined by the HCVR sequence, the LCVR sequence, or both the HCVR sequence and the LCVR sequence, respectively, set forth in Table 4. In various embodiments, the disclosure provides a polynucleotide encoding the heavy chain, the light chain, or both the heavy and light chains of an antibody as discussed above or herein. The heavy and / or light chains may be defined by the heavy and light chain sequences, respectively, set forth in Table 4. In various embodiments, the polynucleotide comprises a nucleic acid sequence set forth in Table 5. In various embodiments, the disclosure provides one or more vectors comprising a polynucleotide as discussed above, and / or a host cell comprising the polynucleotide or vector, or HCVR or LCVR, or assembled antibody or antigen-binding fragment thereof as discussed above or herein.In various embodiments, the disclosure provides a pair of polynucleotides, where (a) a first polynucleotide encodes (i) a HCVR comprising a CDR sequence included in the HCVR of an antibody of Table 4, (ii) a HCVR comprising HCDR1, HCDR2, and HCDR3 sequences described for an antibody of Table 4, (iii) a HCVR comprising a HCVR sequence of an antibody of Table 4, or (iv) a heavy chain (HC) comprising a HC sequence of an antibody of Table 4, and (b) a second polynucleotide encodes (i) a LCVR comprising a CDR sequence included in the LCVR of an antibody of Table 4, (ii) a LCVR comprising LCDR1, LCDR2, and LCDR3 sequences described for an antibody of Table 4, (iii) a LCVR comprising a LCVR sequence of an antibody of Table 4, or (iv) a light chain (LC) comprising a LC sequence of an antibody of Table 4. In various embodiments, the disclosure provides a pair of vectors comprising the first and second polynucleotides, respectively, as discussed above, and / or a host cell comprising the pair of vectors.

[0025] In some embodiments, a pair of polynucleotides encode components of an antibody or antigen-binding fragment thereof, for example, (a) a first polynucleotide encodes an HCVR that includes a CDR contained in an HCVR that includes the amino acid sequence of SEQ ID NO: 212, and a second polynucleotide encodes an LCVR that includes a CDR contained in an LCVR that includes the amino acid sequence of SEQ ID NO: 220, or (b) a first polynucleotide encodes an HCVR that includes HCDR1, HCDR2, and HCDR2 that include the amino acid sequences of SEQ ID NOs: 214, 216, and 218, respectively; (c) the first polynucleotide encodes an HCVR comprising an amino acid sequence of SEQ ID NO:212 and the second polynucleotide encodes an LCVR comprising an amino acid sequence of SEQ ID NO:220; or (d) the first polynucleotide encodes an HC comprising an amino acid sequence of SEQ ID NO:226 and the second polynucleotide encodes an LC comprising an amino acid sequence of SEQ ID NO:228.

[0026] In some embodiments, a pair of polynucleotides encode components of an antibody or antigen-binding fragment thereof, for example, (a) a first polynucleotide encodes an HCVR that includes CDRs contained in an HCVR that includes the amino acid sequence of SEQ ID NO: 362, and a second polynucleotide encodes an LCVR that includes CDRs contained in an LCVR that includes the amino acid sequence of SEQ ID NO: 370, or (b) a first polynucleotide encodes an HCVR that includes HCDR1, HCDR2, and HCDR2 that include the amino acid sequences of SEQ ID NOs: 364, 366, and 368, respectively, and a second polynucleotide encodes an HCVR that includes CDRs contained in an LCVR that includes the amino acid sequence of SEQ ID NO: 372, 106, and 374, respectively. (c) the first polynucleotide encodes an HCVR comprising the amino acid sequence of SEQ ID NO: 362 and the second polynucleotide encodes an LCVR comprising the amino acid sequence of SEQ ID NO: 370; (d) the first polynucleotide encodes an HC comprising the amino acid sequence of SEQ ID NO: 376 and the second polynucleotide encodes an LC comprising the amino acid sequence of SEQ ID NO: 378; or (e) the first polynucleotide encodes an HC comprising the amino acid sequence of SEQ ID NO: 1077 and the second polynucleotide encodes an LC comprising the amino acid sequence of SEQ ID NO: 378.

[0027] In some embodiments, a pair of polynucleotides encode components of an antibody or antigen-binding fragment thereof, for example, (a) a first polynucleotide encodes an HCVR that includes CDRs contained in an HCVR that includes the amino acid sequence of SEQ ID NO: 493, and a second polynucleotide encodes an LCVR that includes CDRs contained in an LCVR that includes the amino acid sequence of SEQ ID NO: 501, or (b) one polynucleotide encodes an HCVR that includes HCDR1, HCDR2, and HCDR2 that include the amino acid sequences of SEQ ID NOs: 495, 497, and 499, respectively, and a second polynucleotide encodes an HCVR that includes CDRs contained in an LCVR that includes the amino acid sequence of SEQ ID NO: 503, 505, and 507, respectively. (c) the first polynucleotide encodes an HCVR comprising the amino acid sequence of SEQ ID NO:493 and the second polynucleotide encodes an LCVR comprising the amino acid sequence of SEQ ID NO:501; (d) the first polynucleotide encodes an HC comprising the amino acid sequence of SEQ ID NO:509 and the second polynucleotide encodes an LC comprising the amino acid sequence of SEQ ID NO:511; or (e) the first polynucleotide encodes an HC comprising the amino acid sequence of SEQ ID NO:1075 and the second polynucleotide encodes an LC comprising the amino acid sequence of SEQ ID NO:511.

[0028] In some embodiments, a pair of polynucleotides encode components of an antibody or antigen-binding fragment thereof, for example, (a) a first polynucleotide encodes an HCVR that includes a CDR contained in an HCVR that includes the amino acid sequence of SEQ ID NO: 887, and a second polynucleotide encodes an LCVR that includes a CDR contained in an LCVR that includes the amino acid sequence of SEQ ID NO: 895, or (b) a first polynucleotide encodes an HCVR that includes HCDR1, HCDR2, and HCDR2 that include the amino acid sequences of SEQ ID NOs: 889, 891, and 893, respectively; (c) the first polynucleotide encodes an HCVR comprising an amino acid sequence of SEQ ID NO: 887 and the second polynucleotide encodes an LCVR comprising an amino acid sequence of SEQ ID NO: 895; or (d) the first polynucleotide encodes an HC comprising an amino acid sequence of SEQ ID NO: 901 and the second polynucleotide encodes an LC comprising an amino acid sequence of SEQ ID NO: 903.

[0029] In one aspect, the disclosure provides a composition or kit comprising an antibody or antigen-binding fragment as described above or discussed herein, in association with an additional therapeutic agent.

[0030] In one aspect, the disclosure provides a pharmaceutical composition comprising an antibody or antigen-binding fragment as described above or discussed herein, a pharma- ceutically acceptable carrier, and, optionally, an additional therapeutic agent. Optionally, the composition or kit is associated with an additional therapeutic agent that is an antiviral drug or a vaccine. In some embodiments, the additional therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an anti-malarial agent, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S. In some embodiments, the anti-malarial agent is chloroquine or hydroxychloroquine. In some embodiments, the anti-inflammatory agent is an antibody. Optionally, the antibody is sarilumab, tocilizumab, or gimsilumab. Optionally, the composition or kit comprises a second antibody or antigen-binding fragment comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of Table 4. Optionally, the antibody that binds to CoV-S is casirivimab or imdevimab, In one aspect, the disclosure provides a container or injection device comprising an antigen binding protein or composition as described above or discussed herein.

[0031] In one aspect, the disclosure provides a method for treating or preventing infection with a coronavirus in a subject in need thereof through administration of a therapeutically effective amount of an antigen binding protein as described above or discussed herein. In some cases, the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, and MERS-CoV.

[0032] In some embodiments, the subject is administered one or more additional therapeutic agents. Optionally, the one or more additional therapeutic agents are an antiviral agent or a vaccine. Optionally, the one or more additional therapeutic agents are selected from the group consisting of an anti-inflammatory agent, an anti-malarial agent, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S. In some embodiments, the anti-malarial agent is chloroquine or hydroxychloroquine. In some embodiments, the anti-inflammatory agent is an antibody. Optionally, the antibody is sarilumab, tocilizumab, or gimsilumab. Optionally, the subject is administered a second antibody or antigen-binding fragment comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of Table 4. In some cases, the subject is administered a second antibody or antigen-binding fragment that includes the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of an antibody described in U.S. Patent No. 10,787,501, e.g., mAb 10933, mAb 10987, or mAb 10985. In some cases, the antibody that binds to CoV-S is casirivimab or imdevimab.

[0033] In one aspect, the disclosure provides a method for administering an antibody or antigen-binding fragment as described above or discussed herein into the body of a subject, comprising injecting the antibody or antigen-binding fragment into the body of the subject. In some cases, the antibody or antigen-binding fragment is injected subcutaneously, intravenously, or intramuscularly into the body of the subject.

[0034] In any of the various embodiments of the antibodies or antigen-binding fragments, compositions, kits, complexes, polypeptides, polynucleotides, vectors, cells, or methods described above or discussed herein, the antibodies or antigen-binding fragments may comprise a VH3-66 or Vk1-33 variable domain sequence.

[0035] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, wherein the isolated antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 887, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 895.

[0036] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 889, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 891, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 893, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 897, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 164, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 899. Optionally, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 887. Optionally, the isolated antibody or antigen-binding fragment thereof comprises a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 895. Optionally, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 887, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 895.

[0037] In one aspect, the disclosure provides an isolated antibody that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, the isolated antibody comprising an immunoglobulin constant region, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 887, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 895.

[0038] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 889, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 891, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 893, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 897, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 164, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 899. Optionally, the isolated antibody comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 887, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 895. Optionally, the isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 901, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 903. In some embodiments, the isolated antibody comprises an immunoglobulin constant region that is an IgG1 constant region. In some embodiments, the isolated antibody is a recombinant antibody. Optionally, the isolated antibody is multispecific.

[0039] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody as discussed above and a pharma- ceutically acceptable carrier or diluent. Optionally, the pharmaceutical composition further comprises a second therapeutic agent. Optionally, the second therapeutic agent is selected from the group consisting of a second antibody or antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, an anti-inflammatory agent, an anti-malarial agent, and an antibody or antigen-binding fragment thereof that binds to TMPRSS2. Optionally, the second therapeutic agent is a second antibody or antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008. In some embodiments, the second antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) comprised within a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 220. In some embodiments, the second antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 214, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 216, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 218, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 222, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 224. In some embodiments, the second antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 220. In some embodiments, the second antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 226, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 228.

[0040] In one aspect, the disclosure provides an isolated antibody or antigen-binding fragment thereof that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, wherein the isolated antibody or antigen-binding fragment comprises three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 212, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 220.

[0041] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 214, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 216, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 218, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 222, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 126, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 224. Optionally, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212. Optionally, the isolated antibody or antigen-binding fragment thereof comprises a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 220. Optionally, the isolated antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 220.

[0042] In one aspect, the disclosure provides an isolated antibody that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, the isolated antibody comprising an immunoglobulin constant region, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 212, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 220.

[0043] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 214, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 216, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 218, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 222, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 126, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 224. In some embodiments, the isolated antibody comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 220. In some embodiments, the isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 226, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 228. In some embodiments, the isolated antibody comprises an immunoglobulin constant region that is an IgG1 constant region. In some embodiments, the isolated antibody is a recombinant antibody. In some embodiments, the isolated antibody is multispecific.

[0044] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody as discussed above and a pharma- ceutically acceptable carrier or diluent. Optionally, the pharmaceutical composition further comprises a second therapeutic agent. Optionally, the second therapeutic agent is selected from the group consisting of a second antibody or antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, an anti-inflammatory agent, an anti-malarial agent, and an antibody or antigen-binding fragment thereof that binds to TMPRSS2. Optionally, the second therapeutic agent is a second antibody or antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008. Optionally, the second antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) comprised within a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 887, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 895. In some cases, the second antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 889, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 891, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 893, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 897, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 899. In some cases, the second antibody or antigen-binding fragment thereof comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 887, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 895. In some cases, the second antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 901, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 903.

[0045] In one aspect, the disclosure provides an isolated antibody that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, the isolated antibody comprising an immunoglobulin constant region, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 270, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 278.

[0046] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 272, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 274, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 276, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 280, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 282. In some embodiments, the antibody comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 270, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 278. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 284, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 286. In some embodiments, the immunoglobulin constant region is an IgG1 constant region. In some embodiments, the antibody is a recombinant antibody. In some embodiments, the antibody is multispecific.

[0047] In one aspect, the disclosure provides a pharmaceutical composition comprising an isolated antibody as discussed above and a pharma- ceutically acceptable carrier or diluent.

[0048] In some cases, the pharmaceutical composition further comprises a second therapeutic agent. In some cases, the second therapeutic agent is selected from the group consisting of a second antibody or antigen-binding fragment thereof that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, an anti-inflammatory agent, an anti-malarial agent, and an antibody or antigen-binding fragment thereof that binds to TMPRSS2.

[0049] In some embodiments, the second therapeutic agent is a second antibody or antigen-binding fragment thereof that binds to the SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008. In some embodiments, the second antibody or antigen-binding fragment thereof comprises three heavy chain CDRs (HCDR1, HCDR2, and HCDR3) comprised within an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 220. In some embodiments, the second antibody or antigen-binding fragment thereof comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 214, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 216, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 218, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 222, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 126, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 224. In some cases, the second antibody or antigen-binding fragment thereof comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 212, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 220. In some cases, the second antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 226, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 228.

[0050] In one aspect, the disclosure provides a method for the production of a SARS-CoV-2 spike protein comprising: a) a first isolated antibody that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, the first isolated antibody comprising an immunoglobulin constant region, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 887, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 895; and b) a first isolated antibody that binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, the first isolated antibody comprising an immunoglobulin constant region, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 887, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 895. and a second isolated antibody that binds to SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, wherein the second isolated antibody comprises an immunoglobulin constant region, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 1030, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 1038.

[0051] In some embodiments, the first isolated antibody comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 889, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 891, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 893, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 897, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 164, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 899. Optionally, the first isolated antibody comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 887, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 895. Optionally, the first isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 901, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 903. In some embodiments, the first isolated antibody comprises an immunoglobulin constant region that is an IgG1 constant region. Optionally, the first isolated antibody is a recombinant antibody. Optionally, the first isolated antibody is multispecific.

[0052] In some embodiments, the pharmaceutical composition further comprises a pharma- ceutically acceptable carrier or diluent.

[0053] In some embodiments, the second isolated antibody comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1032, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 1034, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 1036, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1040, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 1042, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 1044. Optionally, the second isolated antibody comprises an HCVR comprising the amino acid sequence set forth in SEQ ID NO: 1030, and an LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1038. Optionally, the second isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1048, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 1048.

[0054] In some embodiments, the pharmaceutical composition further comprises a third isolated antibody.

[0055] In some embodiments, a third isolated antibody binds to a SARS-CoV-2 spike protein comprising the amino acid sequence set forth in SEQ ID NO: 1008, and the third isolated antibody comprises an immunoglobulin constant region, three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 1010, and three light chain complementarity determining regions (CDRs) (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 1018. In some embodiments, the third isolated antibody comprises an HCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1012, an HCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 1014, an HCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 1016, an LCDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1020, an LCDR2 comprising the amino acid sequence set forth in SEQ ID NO: 1022, and an LCDR3 comprising the amino acid sequence set forth in SEQ ID NO: 1024. In some cases, the third isolated antibody comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 1010, and a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 1018. In some cases, the third isolated antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 1026, and a light chain comprising the amino acid sequence set forth in SEQ ID NO: 1028.

[0056] In one aspect, a method is provided for treating or preventing infection with omicron-variant SARS-CoV-2 in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) in Table 4 that comprises an amino acid sequence having at least about 90% sequence identity with a HCVR, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) in Table 4 that comprises an amino acid sequence having at least about 90% sequence identity with a LCVR.

[0057] In one aspect, a method is provided for preventing in a subject in need thereof one or more COVID-19 symptoms resulting from infection with an omicron variant SARS-CoV-2, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) in Table 4 that comprises an amino acid sequence having at least about 90% sequence identity with a HCVR, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) in Table 4 that comprises an amino acid sequence having at least about 90% sequence identity with a LCVR.

[0058] In some embodiments of the method, preventing comprises pre-exposure prophylaxis. In some embodiments of the method, preventing comprises post-exposure prophylaxis.

[0059] In some cases, the antibody or antigen-binding fragment comprises (a) an immunoglobulin heavy chain variable region comprising HCDR1, HCDR2, and HCDR3 of an antibody of Table 4, and / or (b) an immunoglobulin light chain variable region comprising LCDR1, LCDR2, and LCDR3 of an antibody of Table 4.

[0060] In some cases, the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, and HCDR3 of an antibody of Table 4, and the LCDR1, LCDR2, and LCDR3 of that antibody of Table 4.

[0061] In some embodiments of the method, the subject is administered one or more additional therapeutic agents. Optionally, the one or more additional therapeutic agents are an antiviral agent or a vaccine. Optionally, the one or more additional therapeutic agents are selected from the group consisting of an anti-inflammatory agent, an anti-malarial agent, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S. In some embodiments, the anti-inflammatory agent is an antibody such as sarilumab, tocilizumab, or gimsilumab. Optionally, the antibody that binds to CoV-S is casirivimab or imdevimab.

[0062] In some embodiments of the method, the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of a second antibody of Table 4.

[0063] In some embodiments of the method, the administering comprises injecting the antibody or antigen-binding fragment into the subject's body. Optionally, the antibody or antigen-binding fragment is injected subcutaneously, intravenously, or intramuscularly into the subject's body.

[0064] In one aspect, the disclosure provides a method for treating or preventing one or more COVID-19 symptoms resulting from infection with SARS-CoV-2 in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 362, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 370.

[0065] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 364, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 366, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 368, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 372, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 374. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 362. In some embodiments, the antibody or antigen-binding fragment comprises a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 370. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 376. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 378.

[0066] In some embodiments, administering comprises administering the antibody or antigen-binding fragment via injection. Optionally, the injection is intravenous. Optionally, the injection is subcutaneous.

[0067] In some embodiments, administering comprises administering 300 mg of the antibody or antigen-binding fragment thereof. In some embodiments, administering comprises administering 600 mg of the antibody or antigen-binding fragment thereof. In some embodiments, administering comprises administering 1200 mg of the antibody or antigen-binding fragment thereof.

[0068] In some embodiments, administering comprises administering two doses of the antibody or antigen-binding fragment thereof. Optionally, each of the two doses comprises 300 mg of the antibody or antigen-binding fragment thereof. Optionally, the two doses are administered 4 weeks apart, 5 weeks apart, 6 weeks apart, 7 weeks apart, 8 weeks apart, 9 weeks apart, 10 weeks apart, 11 weeks apart, 12 weeks apart, 13 weeks apart, 14 weeks apart, 15 weeks apart, or 16 weeks apart. Optionally, the two doses are administered 8-16 weeks apart. Optionally, the two doses are administered 12 weeks apart. Optionally, the two doses are administered 8 weeks apart.

[0069] In some embodiments, the administering reduces the SARS-CoV-2 viral load in the subject.

[0070] In some embodiments, administration of the antibody or antigen-binding fragment thereof occurs prior to SARS-CoV-2 infection.

[0071] In one aspect, the disclosure provides a method for treating or preventing one or more COVID-19 symptoms resulting from infection with SARS-CoV-2 in a subject in need thereof, comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof comprising three heavy chain complementarity determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) comprised within a heavy chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 362, and three light chain CDRs (LCDR1, LCDR2, and LCDR3) comprised within a light chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 370, wherein said administering comprises administering to the subject two doses of the antibody or antigen-binding fragment thereof, each dose comprising 300 mg of the antibody or antigen-binding fragment thereof.

[0072] In some embodiments, the administering is subcutaneous.

[0073] In some embodiments, HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 364, HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 366, HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 368, LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 372, LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 106, and LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 374. In some embodiments, the antibody or antigen-binding fragment comprises a HCVR comprising the amino acid sequence set forth in SEQ ID NO: 362. In some embodiments, the antibody or antigen-binding fragment comprises a LCVR comprising the amino acid sequence set forth in SEQ ID NO: 370. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 376. In some embodiments, the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 378.

[0074] In one aspect, the disclosure provides a polynucleotide encoding an antibody or antigen-binding fragment thereof, or the HCVR and / or LCVR, or the heavy and / or light chain of the antibody or antigen-binding fragment thereof, as described above or discussed herein. The disclosure further provides one or more vectors comprising the polynucleotides described above or discussed herein, as well as host cells comprising the antibody or antigen-binding fragment, one or more polynucleotides, and / or one or more vectors described above or discussed herein.

[0075] In some embodiments, the administering comprises administering 600 mg of the antibody or antigen-binding fragment thereof. In some embodiments, the administering comprises administering 300 mg of the antibody or antigen-binding fragment thereof.

[0076] In any of the various embodiments of the antibodies or antigen-binding fragments thereof described above or discussed herein, the antibody or antigen-binding fragment may neutralize the omicron variant of SARS-CoV-2. In various embodiments, the omicron variant is selected from BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, or BA.4 / BA.5.

[0077] In various embodiments, any of the features or components of the embodiments described above or discussed herein may be combined, and such combinations are encompassed within the scope of the present disclosure. Any specific value described above or discussed herein may be combined with another associated value described above or discussed herein to recite a range having values ​​representing the upper and lower limits of the range, and such ranges are encompassed within the scope of the present disclosure. [Brief description of the drawings]

[0078] [Figure 1-1] Cross-competition between 41 anti-SARS-CoV-2-S monoclonal antibodies upon binding to immobilized anti-SARS-Cov-2 RBD-MMH (SEQ ID NO: 1069). Red: pre-bound mAb-1 reduced the binding of mAb-2 to SARS-CoV-2 RBD-MMH by more than 50%, and binding to SARS-CoV-2 RBD-MMH was also reduced by more than 50% when the binding order of mAb-1 and mAb-2 was reversed. Yellow: pre-bound mAb-1 reduced the binding of mAb-2 to SARS-CoV-2 RBD-MMH by more than 50%, but binding to SARS-CoV-2 RBD-MMH was reduced by less than 50% when the binding order of mAb-1 and mAb-2 was reversed. [Figure 1-2] Continued from Figure 1-1. [Figure 1-3] Continued from Figure 1-2. [Figure 1-4] Continued from Figure 1-3. [Figure 1-5] Continued from Figure 1-4. [Figure 1-6] Continued from Figure 1-5. [Figure 1-7] Continued from Figure 1-6. [Figure 1-8] Continued from Figure 1-7. [Figure 1-9] Continued from Figure 1-8. [Figure 2-1]Cross-competition between 15 anti-SARS-CoV-2-S monoclonal antibodies upon binding to immobilized anti-SARS-Cov-2 RBD-MMH. Red: pre-bound mAb-1 reduced the binding of mAb-2 to SARS-CoV-2 RBD-MMH by more than 50%, and binding to SARS-CoV-2 RBD-MMH was also reduced by more than 50% when the binding order of mAb-1 and mAb-2 was reversed. Yellow: pre-bound mAb-1 reduced the binding of mAb-2 to SARS-CoV-2 RBD-MMH by more than 50%, but binding to SARS-CoV-2 RBD-MMH was reduced by less than 50% when the binding order of mAb-1 and mAb-2 was reversed. [Figure 2-2] Continued from Figure 2-1. [Figure 2-3] Continued from Figure 2-2. [Figure 2-4] Continued from Figure 2-3. [Figure 2-5] Continued from Figure 2-4. [Figure 2-6] Continued from Figure 2-5. [Diagram 2-7] Continued from Figure 2-6. [Figure 2-8] Continued from Figure 2-7. [Figure 2-9] Continued from Figure 2-8. [Diagram 3] We depict the cryo-EM structure of mAb14256, mAb10987, and the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein in complex at 3.9 Å resolution. [Figure 4] Figure 1 shows that mAb14256 binds at the top of the RBD, thereby blocking ACE2 binding. mAb14256 competes with mAb10933 (middle structure) and mAb10985 (not depicted). [Diagram 5] We depict the cryo-EM structure of mAb15160, mAb14315, and the receptor binding domain (RBD) of the SARS-CoV-2 spike glycoprotein in complex at 3.18 Å resolution. [Figure 6]We depict the cryo-EM structures of antigen-binding fragments of mAb1428 and mAb15160 with the receptor binding domains (RBDs) from the Wuhan-Hu-1 and BA.1 strains in complex at 3.3 and 3.4 Å resolution, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] Before the methods are described, it is to be understood that the invention is not limited to the particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0080] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated by reference in their entirety.

[0081] The term "coronavirus" or "CoV" refers to any virus of the Coronaviridae family, including but not limited to MERS-CoV, SARS-CoV, and SARS-CoV-2. SARS-CoV-2 is also known as 2019-nCoV and Wuhan coronavirus. It binds to the human host cell receptor angiotensin-converting enzyme 2 (ACE2) via the viral spike protein. The spike protein also binds to and is cleaved by TMPRSS2, which activates the spike protein for viral membrane fusion.

[0082] The term "CoV-S", also referred to as "S" or "S protein", refers to the spike protein of coronaviruses and may 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 is assembled into trimers that constitute the spike or peplomer on the surface of enveloped coronavirus particles. The protein has two essential functions, host receptor binding and membrane fusion, which are attributed to the N-terminal (S1) and C-terminal (S2) halves of the S protein. CoV-S binds to its cognate receptor via a receptor binding domain (RBD) present in the S1 subunit. The amino acid sequence of the full-length SARS-CoV-2 spike protein is exemplified by the amino acid sequence provided in SEQ ID NO: 1008. The term "CoV-S" includes protein variants of CoV spike proteins isolated from different CoV isolates, as well as recombinant CoV spike proteins or fragments thereof. The term also encompasses, for example, a CoV spike protein or fragment thereof linked to a histidine tag, mouse or human Fc, or a signal sequence such as ROR1.

[0083] The term "coronavirus infection" or "CoV infection" as used herein refers to an infection with a coronavirus, such as SARS-CoV-2, MERS-CoV, or SARS-CoV. The term includes coronavirus respiratory infections, often in the lower respiratory tract. Symptoms can include high fever, dry cough, shortness of breath, pneumonia, gastrointestinal symptoms such as diarrhea, organ failure (renal failure and dysfunction), septic shock, and in severe cases, death.

[0084] virus The present invention includes methods for treating or preventing a viral infection in a subject. The term "virus" includes any virus whose infection in the body of a subject is treatable or preventable by administration of an anti-CoV-S antibody or an antigen-binding fragment thereof (e.g., the infectivity of the virus depends at least in part on CoV-S). In an embodiment of the present invention, a "virus" is any virus that expresses a spike protein (e.g., CoV-S). The term "virus" also includes CoV-S-dependent respiratory viruses, which are viruses that infect the respiratory tissues of a subject (e.g., upper and / or lower respiratory tract, trachea, bronchi, lungs) and are treatable or preventable by administration of an anti-CoV-S antibody or an antigen-binding fragment thereof. For example, in an 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 can include the genera Alphacoronavirus, Betacoronavirus, Gammacoronavirus, and Deltacoronavirus. In some embodiments, the antibodies or antigen-binding fragments provided herein can bind to and / or neutralize alphacoronavirus, betacoronavirus, gammacoronavirus, and / or deltacoronavirus. In certain embodiments, this binding and / or neutralization can be specific to a particular genus of coronavirus or a particular subgroup of a genus. "Viral infection" refers to the entry and proliferation of a virus within the body of a subject.

[0085] Coronavirus virions are spherical with a diameter of approximately 125 nm. The most striking feature of coronaviruses are the club-shaped spiked projections that emanate from the surface of the virion. These spikes are the defining feature of virions, giving them the appearance of a solar corona and inspiring the name coronavirus. Within the virion envelope is the nucleocapsid. Coronaviruses have a helically symmetrical nucleocapsid, which is rarely seen among positive-sense RNA viruses but is much more common in negative-sense RNA viruses. SARS-CoV-2, MERS-CoV, and SARS-CoV belong to the Coronaviridae family. The initial attachment of the virion to the host cell is initiated by the interaction between the S protein and its receptor. The site of the receptor binding domain (RBD) within the S1 region of the coronavirus S protein varies depending on the virus, with some having the RBD at the C-terminus of S1. The S protein / receptor interaction is the primary determinant for coronaviruses to infect host species and also governs the tissue tropism of the virus. Many coronaviruses utilize peptidases as their cellular receptors. After receptor binding, the virus must then gain access to the host cell cytosol. This is generally accomplished by acid-dependent proteolytic cleavage of the S protein by a cathepsin, TMPRRS2, or another protease, followed by fusion of the viral and cellular membranes.

[0086] Coronaviruses described herein also include variant coronaviruses, which in some embodiments are classified by the World Health Organization (WHO) as "variants of interest" or "variants of concern." Variant coronaviruses may have mutations in their spike glycoproteins that may alter the characteristics of the virus, such as the severity or transmissibility of COVID-19. The WHO defines a variant of concern as one associated with one or more of the following changes from the wild type to such an extent that it is of global public health significance: 1) increased transmissibility or deleterious changes in the epidemiology of COVID-19, 2) increased virulence or changes in clinical symptoms, or 3) reduced effectiveness of public health and social measures, including available therapeutics, vaccines, and diagnostics. A variant of interest is defined by the WHO as a SARS-CoV-2 variant that 1) has genetic changes predicted or demonstrated to affect viral characteristics, including immune evasion, treatment evasion, diagnostic evasion, transmissibility, and disease severity, and 2) has been identified as causing significant community transmission or multiple COVID-19 clusters in multiple countries with increasing relative prevalence over time with increasing case numbers, or other epidemiological impacts suggesting an emerging risk to global public health. As of December 13, 2021, there are five variants of concern isolated by the WHO (alpha, beta, gamma, delta, and omicron), and two variants of interest classified by the WHO (lambda and mu). Each variant can be defined by a mutation in its spike glycoprotein compared to the wild-type spike glycoprotein.For example, the Omicron variant (also classified as B.1.1.529) contains the following mutations in its spike glycoprotein: A67V, Δ69-70, T95I, G142D / Δ143-145, Δ211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G4 46S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, and L981F (full-length omicron spike glycoprotein: SEQ ID NO: 1072). Additionally, these variants may have additional lineages that encompass groups of related viruses. For example, as of July 8, 2022, omicron variants have the lineages BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, and BA.4 / BA.5. In some embodiments, the antibodies and antigen-binding fragments thereof described herein can bind to any of these variants and / or lineages. In further embodiments, the antibodies and antigen-binding fragments thereof are capable of neutralizing these variants and / or strains.

[0087] Anti-CoV-S antibodies and antigen-binding fragments The present invention provides antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, that specifically bind to a CoV spike protein or an antigenic fragment thereof.

[0088] The term "antibody" as used herein refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds (i.e., a "complete antibody molecule"), as well as multimers thereof (e.g., IgM). Exemplary antibodies include, for example, those listed in Table 4. Each heavy chain comprises a heavy chain variable region ("HCVR" or "V H ") and the heavy chain constant region (domain C H 1. C H 2, and C H Each light chain comprises a light chain variable region ("LCVR" or "VVR").L ) and light chain constant region (C L ) V H and V L The regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). H and V Lcomprises three CDRs and four FRs arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain CDRs are also referred to as HCDRs or CDR-H and may be numbered as described above (e.g., HCDR1, HCDR2, and HCDR3, or HCDR1, HCDR2, and HCDR3). Similarly, the light chain CDRs are referred to as LCDRs or CDR-L and may be numbered as LCDR1, LCDR2, and LCDR3, or LCDR1, LCDR2, and LCDR3. In certain embodiments of the present invention, the FRs of the antibody (or antigen-binding fragment thereof) are identical to human germline sequences or are naturally or artificially modified. Exemplary human germline sequences include, but are not limited to, VH3-66 and Vk1-33. Thus, the disclosure provides an anti-CoV-S antibody or antigen-binding fragment thereof (e.g., an anti-SARS-CoV-2-S antibody or antigen-binding fragment thereof) comprising the HCDR and LCDR sequences of Table 4 within the VH3-66 or Vk1-33 variable heavy or light chain region.The present disclosure relates to IgKV4-1, IgKV 1-5, 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- a light chain selected from IgLV2-8, IgKV3-11, IgLV2-11, IgLV2-14, IgLV2-23, or IgLV6-57, and a light chain selected from IgHV1-69, IgHV3-64, IgHV4-59, IgHV3-53, IgHV3-48, IgHV4-34, IgHV3-33, IgH Further provided is an anti-CoV-S antibody or antigen-binding fragment thereof (e.g., an anti-SARS-CoV-2-S antibody or antigen-binding fragment thereof) comprising the HCDR and LCDR sequences of Table 4 in a heavy chain combination selected from IgHV3-30, IgHV3-23, IgHV3-20, IgHV1-18, IgHV3-15, IgHV3-11, IgHV3-9, IgHV1-8, IgHV3-7, IgHV2-5, IgHV1-2, IgHV2-70, IgHV3-66, IgHV5-51, IgHV1-46, IgHV4-39, IgHV4-31, IgHV3-30-3, IgHV2-26, or IgHV7-4-1.The present disclosure relates to IgKV4-1, IgKV 1-5, 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- a light chain selected from IgLV2-8, IgKV3-11, IgLV2-11, IgLV2-14, IgLV2-23, or IgLV6-57, and a light chain selected from IgHV1-69, IgHV3-64, IgHV4-59, IgHV3-53, IgHV3-48, IgHV4-34, IgHV3-33, IgH Further provided is an anti-CoV-S antibody or antigen-binding fragment thereof (e.g., an anti-SARS-CoV-2-S antibody or antigen-binding fragment thereof) comprising the HCVR and LCVR sequences of Table 4 in a heavy chain combination selected from 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.

[0089] Typically, the variable domains of both heavy and light immunoglobulin chains contain three hypervariable regions, also called complementarity determining regions (CDRs), located within relatively conserved framework regions (FRs). Generally, from N-terminus to C-terminus, both the light and heavy chain variable domains contain FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. In an embodiment of the invention, the assignment of amino acids to each domain is based on the Sequences of Proteins of Immunological Interest, Kabat, et al., National Institutes of Health, Bethesda, Md.; 5 thed.;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 Chothia, et al., (1989) Nature 342:878-883.

[0090] The present invention includes monoclonal anti-CoV-S antigen binding proteins, e.g., antibodies and antigen-binding fragments thereof, as well as monoclonal compositions comprising a plurality of isolated monoclonal antigen binding proteins. The term "monoclonal antibody" as used herein refers to a population of substantially homogenous antibodies, i.e., the antibody molecules comprising the population are identical in amino acid sequence, except for naturally occurring mutations that may be present in minor amounts. A "plurality" of such monoclonal antibodies and fragments in a composition refers to a concentration of identical (i.e., in amino acid sequence, except for naturally occurring mutations that may be present in minor amounts, as discussed above) antibodies and fragments that exceeds that normally occurring in nature, e.g., in the blood of a host organism, such as a mouse or human.

[0091] In embodiments of the invention, the anti-CoV-S antigen binding protein, e.g., an antibody or antigen binding fragment, comprises a heavy chain constant domain, e.g., of the IgA (e.g., IgA1 or IgA2), IgD, IgE, IgG (e.g., IgG1, IgG2, IgG3 and IgG4), or IgM type. In embodiments of the invention, the antigen binding protein, e.g., an antibody or antigen binding fragment, comprises a light chain constant domain, e.g., of the kappa or lambda type.

[0092] The term "human" antigen-binding protein, such as an antibody, as used herein includes antibodies having variable and constant regions derived from human germline immunoglobulin sequences, whether in a human cell or grafted into a non-human cell, e.g., a mouse cell. See, e.g., US8502018, US6596541, or US5789215. The human mAbs of the invention may include, for example, in the CDRs, and in particular in CDR3, amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include mAbs in which CDR sequences derived from the germline of another mammalian species (e.g., a mouse) have been grafted onto human FR sequences. The term includes antibodies recombinantly produced in a non-human mammal or in the cells of a non-human mammal. The term is not intended to include antibodies isolated from or generated in a human subject. See below.

[0093] The present invention includes anti-CoV-S chimeric antigen binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of use thereof. As used herein, a "chimeric antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, the first and second antibodies being from different species. (US4816567, and Morrison et al., (1984) Proc. Natl. Acad. Sci. USA 81:6851-6855).

[0094] The present invention includes anti-CoV-S hybrid antigen binding proteins, e.g., antibodies and antigen-binding fragments thereof, and methods of use thereof. As used herein, a "hybrid antibody" is an antibody having a variable domain from a first antibody and a constant domain from a second antibody, where the first and second antibodies are from different animals, or the variable domain, but not the constant region, is from the first animal. For example, the variable domain may be taken from an antibody isolated from a human and expressed with the constant constant region not isolated from the antibody. An exemplary hybrid antibody is described in Example 1, which refers to PCR products derived from antibody heavy and light chain variable regions cloned into an expression vector containing heavy and light chain constant regions, respectively. Hybrid antibodies are synthetic and not naturally occurring because the variable and constant regions they contain are not isolated from a single natural source.

[0095] The term "recombinant" antigen binding protein, such as an antibody or antigen binding fragment thereof, refers to such molecules that are made, expressed, isolated, or obtained by techniques or methods known in the art as recombinant DNA technology, including, for example, DNA splicing and transgenic expression. The term includes antibodies expressed in non-human mammals (including transgenic non-human mammals, e.g., transgenic mice), or cell (e.g., CHO cell) expression systems, or non-human cell expression systems, or isolated from recombinant combinatorial human antibody libraries. In some embodiments, a recombinant antibody shares sequences with an antibody isolated from an organism (e.g., mouse or human), but has been expressed via recombinant DNA technology. Such antibodies may have different post-translational modifications (e.g., glycosylation) than antibodies isolated from an organism.

[0096] In some embodiments, the antibody disclosed herein lacks fucose in its constant region glycosylation.Methods for measuring fucose in antibody compositions are described in the art, for example, U.S. Patent No. 8,409,838 (Regeneron Pharmaceuticals), which is incorporated herein by reference.In some embodiments, fucose is undetectable in a composition comprising a population of antibody molecules.In some embodiments, the antibody lacking fucose has enhanced ADCC activity.

[0097] In some embodiments, antibodies lacking fucose can be produced using a cell line that is deficient in the ability to fucosylate proteins, i.e., the ability to fucosylate proteins is reduced or eliminated. Fucosylation of glycans requires the synthesis of GDP-fucose via the de novo pathway or the salvage pathway, both of which involve the sequential function of several enzymes and lead to the addition of a fucose molecule to the first N-acetylglucosamine (GlcNAc) moiety at the reducing end of the glycan. The two key enzymes of the de novo pathway involved in the production of GDP-fucose are GDP-D-mannose-4,6-dehydratase (GMD) and GDP-keto-6-deoxymannose-3,5-epimerase,4-reductase (FX). In the absence of fucose, these two de novo pathway enzymes (GMD and FX) convert mannose and / or glucose to GDP-fucose, which is then transported into the Golgi complex where nine fucosyl-transferases (FUT1-9) act in concert to fucosylate the first GlcNAc molecule of the glycan. However, in the presence of fucose, the salvage pathway enzymes, fucose kinase and GDP-fucose pyrophosphorylase, convert fucose to GDP-fucose.

[0098] Cell lines deficient in the ability to fucosylate proteins have been described in the art. In some embodiments, the cell line deficient in the ability to fucosylate proteins is a mammalian cell line (e.g., a CHO cell line such as CHO K1, DXB-11 CHO, Veggie-CHO, etc.) that contains one or more mutations or genetic modifications of endogenous FUT1-9 resulting in the absence of one or more functional fucosyl-transferases. In some embodiments, the mammalian cell line contains a mutation of the endogenous FUT8 gene (e.g., a FUT8 knockout cell line in which the FUT8 gene is disrupted, resulting in the absence of α1,6-fucosyltransferase in the cell line), as described in U.S. Pat. No. 7,214,775 (Kyowa Hakko Kogyo Co., Ltd.) and U.S. Pat. No. 7,737,725 (Kyowa Hakko Kirin Co., Ltd.), which are incorporated herein by reference. In some embodiments, the mammalian cell line comprises a mutation or genetic modification of an endogenous GMD gene that results in the lack of functional GMD in the cell line, e.g., a GMD knockout cell line in which the GMD gene has been disrupted, as described in U.S. Pat. No. 7,737,725 (Kyowa Hakko Kirin Co., Ltd), which is incorporated herein by reference. In some embodiments, the mammalian cell line comprises a mutation or genetic modification of an endogenous Fx gene that results in the lack of functional Fx protein. In some embodiments, the mammalian cell line is an Fx knockout cell line in which the endogenous Fx gene has been disrupted (see, e.g., U.S. Pat. No. 7,737,725 (Kyowa Hakko Kirin Co., Ltd), which is incorporated herein by reference). In some embodiments, the mammalian cell line comprises a mutation in an endogenous Fx mutation that confers a temperature-sensitive phenotype (as described in U.S. Pat. No. 8,409,838 (Regeneron Pharmaceuticals), which is incorporated herein by reference). In some embodiments, the mammalian cell line that is deficient in the ability to fucosylate proteins is a cell line selected on the basis of resistance to a particular lectin, for example, lentil lectin.See, for example, US Pat. No. 8,409,838 (Regeneron Pharmaceuticals), which is incorporated herein by reference.

[0099] Recombinant anti-CoV-S antigen binding proteins, e.g., antibodies and antigen binding fragments, disclosed herein can also be produced in an E. coli / T7 expression system. In this embodiment, a nucleic acid encoding an anti-CoV-S antibody immunoglobulin molecule of the invention (e.g., as found in Table 4) can be inserted into a pET-based plasmid and expressed in an E. coli / T7 system. For example, the invention includes a method for expressing an antibody or an antigen binding fragment thereof or an immunoglobulin chain thereof in a host cell (e.g., a bacterial host cell such as E. coli, e.g., BL21 or BL21DE3), comprising expressing T7 RNA polymerase in a cell that also comprises a polynucleotide encoding an immunoglobulin chain operably linked to a T7 promoter. For example, in an embodiment of the invention, a bacterial host cell, e.g., E. coli, comprises a polynucleotide encoding a T7 RNA polymerase gene operably linked to a lac promoter, and expression of the polymerase and chains is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). See US4952496 and US5693489, or Studier & Moffatt, Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes, J. Mol. Biol. 1986 May 5;189(1):113-30.

[0100] There are several methods for producing recombinant antibodies known in the art. One example of a method for the recombinant production of antibodies is disclosed in US4816567.

[0101] Transformation can be by any known method for introducing polynucleotides (e.g., DNA or RNA, including mRNA) into a host cell. Methods for the introduction of 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 in liposomes, lipid nanoparticle technology, biolistec injection, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral vectors such as lentivirus or adeno-associated virus. Methods for transforming cells are well known in the art. See, for example, U.S. Patent Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455. In some embodiments, the antibody or antigen-binding fragment thereof of the present disclosure can be introduced into a subject in a nucleic acid form (e.g., DNA or RNA, including mRNA) such that the subject's own cells produce the antibody. The disclosure further provides modifications to the nucleotide sequences encoding the anti-CoV-S antibodies described herein that result in increased antibody expression, increased antibody stability, increased nucleic acid (e.g., mRNA) stability, or improved affinity or specificity of the antibody for the CoV spike protein.

[0102] Thus, the invention includes recombinant methods for making anti-CoV-S antigen binding proteins, such as antibodies or antigen binding fragments thereof, or immunoglobulin chains thereof of the invention, comprising: (i) introducing one or more polynucleotides (e.g., comprising any one or more of the nucleotide sequences of any one or more of the sequences of Table 5) encoding, for example, the light and / or heavy immunoglobulin chains, or CDRs of an antigen binding protein of Table 4, e.g., where the polynucleotides are in a vector and / or integrated into a host cell chromosome and / or operably linked to a promoter; (ii) culturing a host cell (e.g., CHO or Pichia or Pichia pastoris) under conditions favorable for expression of the polynucleotides; and (iii) optionally isolating the antigen binding protein (e.g., antibody or fragment) or chain from the host cell and / or the medium in which the host cell is grown. For example, a polynucleotide can be integrated into a host cell chromosome through targeted insertion using a vector such as an adeno-associated virus (AAV), e.g., after chromosomal breakage using a gene editing system (e.g., CRISPR (e.g., CRISPR-Cas9), TALEN, megaTAL, zinc finger, or Argonaute). Targeted insertion can be at a host cell locus, such as, for example, an albumin or immunoglobulin genomic locus. Alternatively, insertion can be at a random locus, e.g., using a vector such as a lentivirus. When making an antigen binding protein (e.g., an antibody or antigen binding fragment) that comprises more than one immunoglobulin chain, e.g., an antibody that comprises two immunoglobulin heavy chains and two immunoglobulin light chains, co-expression of the chains in a single host cell leads to association of the chains, e.g., within the cell, or on the cell surface, or outside the cell if such chains are secreted, to form the antigen binding protein (e.g., an antibody or antigen binding fragment). The methods include those in which only heavy or only light immunoglobulin chains (e.g., any of those disclosed herein, including mature fragments and / or variable domains thereof) are expressed. Such chains are useful, for example, as intermediates in the expression of antibodies or antigen-binding fragments comprising such chains.For example, the invention also includes anti-CoV-S antigen binding proteins, such as antibodies and antigen binding fragments thereof, that comprise a heavy chain immunoglobulin (or a variable domain thereof, or including CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence set forth in Table 5, and a light chain immunoglobulin (or a variable domain thereof, or including CDRs thereof) encoded by a nucleotide sequence set forth in Table 5, that are the products of such production methods, and optionally the purification methods described herein. For example, in some embodiments, the product of the method is an anti-CoV-S antigen binding protein that is an antibody or fragment thereof, that comprises a HCVR that comprises an amino acid sequence set forth in Table 4, and a LCVR that comprises an amino acid sequence set forth in Table 4, wherein the HCVR and LCVR sequences are selected from a single antibody listed in Table 4. In some embodiments, the product of the method is an anti-CoV-S antigen binding protein that is an antibody or fragment thereof, that comprises HCDR1, HCDR2, and HCDR3 that comprise an amino acid sequence set forth in Table 4, and LCDR1, LCDR2, and LCDR3 that comprise an amino acid sequence set forth in Table 4, wherein the six CDR sequences are selected from a single antibody listed in Table 4. In some embodiments, the product of this method is an anti-CoV-S antigen binding protein that is an antibody or fragment thereof, comprising a heavy chain comprising the HC amino acid sequence set forth in Table 4, and a light chain comprising the LC amino acid sequence set forth in Table 4.

[0103] Eukaryotic and prokaryotic host cells, including mammalian cells, can be used as hosts for the expression of anti-CoV-S antigen binding proteins. Such host cells are well known in the art and many are available from the American Type Culture Collection (ATCC). These host cells include, among others, Chinese hamster ovary (CHO) cells, NS0, SP2 cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, 3T3 cells, HEK-293 cells, and several other cell lines. Mammalian host cells include human, mouse, rat, dog, monkey, pig, goat, cow, horse, and hamster cells. Other cell lines that can be used 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 species, Saccharomyces cerevisiae, Saccharomyces species, Hansenula polymorpha, Kluyveromyces species, Kluyveromyces lactis, Candida albicans, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, Chrysosporium The present invention includes isolated host cells (e.g., CHO cells) containing an antigen binding protein such as those in Table 4, or a polynucleotide encoding such a polypeptide.

[0104] The polynucleotides discussed herein may encode all or part of the antibodies or antigen-binding fragments discussed throughout this disclosure. In some cases, a single polynucleotide may encode both the HCVR and LCVR of an antibody or antigen-binding fragment (e.g., defined in terms of the CDRs contained within the HCVR and LCVR whose respective amino acid sequences are defined, defined in terms of the amino acid sequences of the CDRs of the HCVR and LCVR, respectively, or defined via the amino acid sequences of the HCVR and LCVR, respectively), or the HCVR and LCVR may be encoded by separate polynucleotides (i.e., a pair of polynucleotides). In the latter case where the HCVR and LCVR are encoded by separate polynucleotides, the polynucleotides may be combined in a single vector or may be included in separate vectors (i.e., a pair of vectors). In either case, the host cell used to express the polynucleotide or vector may contain the full complement of component parts to generate the antibody or antigen-binding fragment thereof. For example, the host cell may contain separate vectors, each encoding the HCVR and LCVR of the antibody or antigen-binding fragment thereof discussed above or herein. Similarly, one or more polynucleotides and one or more vectors can be used to express the full-length heavy and full-length light chains of an antibody described above or discussed herein. For example, a host cell can contain a single vector having polynucleotides encoding both the heavy and light chains of an antibody, or the host cell can contain separate vectors having polynucleotides encoding the heavy and light chains, respectively, of an antibody described above or discussed herein.

[0105] In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the HCDRs of SEQ ID NOs: 214, 216, and 218 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 213, 215, and 217). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the LCDRs of SEQ ID NOs: 222, 126, and 224 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 221, 125, and 223). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the amino acid sequence of SEQ ID NO: 212 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 211). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the amino acid sequence of SEQ ID NO: 220 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 219). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 226 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 225). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 228 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 227).In one embodiment, the disclosure provides a host cell comprising a pair of polynucleotides or a pair of vectors encoding the HCVR and LCVR, or the HC and LC, respectively, as discussed in this paragraph, and a pair of polynucleotides and / or a pair of vectors for encoding an antibody designated mAb14315 or an antigen-binding fragment thereof.

[0106] In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the HCDRs of SEQ ID NOs: 364, 366, and 368 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 363, 365, and 367). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the LCDRs of SEQ ID NOs: 372, 106, and 374 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 371, 105, and 373). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the amino acid sequence of SEQ ID NO: 362 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 361). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the amino acid sequence of SEQ ID NO: 370 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 369). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 376 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 375). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 378 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 377).In one embodiment, the disclosure provides a host cell comprising a pair of polynucleotides or a pair of vectors encoding the HCVR and LCVR, or the HC and LC, respectively, as discussed in this paragraph, and a pair of polynucleotides and / or a pair of vectors for encoding the antibody designated mAb15160 or an antigen-binding fragment thereof.

[0107] In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the HCDRs of SEQ ID NOs: 495, 497, and 499 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 494, 496, and 498). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the LCDRs of SEQ ID NOs: 503, 505, and 507 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 502, 504, and 506). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the amino acid sequence of SEQ ID NO: 493 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 492). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the amino acid sequence of SEQ ID NO:501 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:500). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:509 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:508). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a light chain (LC) comprising the amino acid sequence of SEQ ID NO:511 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:510).In one embodiment, the disclosure provides a host cell comprising a pair of polynucleotides or a pair of vectors encoding the HCVR and LCVR, or the HC and LC, respectively, as discussed in this paragraph, and a pair of polynucleotides and / or a pair of vectors for encoding the antibody designated mAb14284 or an antigen-binding fragment thereof.

[0108] In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the HCDRs of SEQ ID NOs: 889, 891, and 893 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 888, 890, and 892). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the LCDRs of SEQ ID NOs: 897, 164, and 899 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 896, 163, and 898). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the amino acid sequence of SEQ ID NO: 887 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 886). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the amino acid sequence of SEQ ID NO:895 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:894). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:901 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:900). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a light chain (LC) comprising the amino acid sequence of SEQ ID NO:903 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:902).In one embodiment, the disclosure provides a host cell comprising a pair of polynucleotides or a pair of vectors encoding the HCVR and LCVR, or the HC and LC, respectively, as discussed in this paragraph, and a pair of polynucleotides and / or a pair of vectors for encoding the antibody designated mAb14256 or an antigen-binding fragment thereof.

[0109] In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the HCDRs of SEQ ID NOs: 495, 497, and 499 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 494, 496, and 498). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the LCDRs of SEQ ID NOs: 503, 505, and 507 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 502, 504, and 506). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the amino acid sequence of SEQ ID NO: 493 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 492). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the amino acid sequence of SEQ ID NO:501 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:500). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO:1075 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:1074). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a light chain (LC) comprising the amino acid sequence of SEQ ID NO:511 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO:510).In one embodiment, the disclosure provides a host cell comprising a pair of polynucleotides or a pair of vectors encoding the HCVR and LCVR, or the HC and LC, respectively, as discussed in this paragraph, and a pair of polynucleotides and / or a pair of vectors for encoding the antibody designated mAb17090 or an antigen-binding fragment thereof.

[0110] In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the HCDRs of SEQ ID NOs: 364, 366, and 368 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 363, 365, and 367). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the LCDRs of SEQ ID NOs: 372, 106, and 374 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NOs: 371, 105, and 373). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a HCVR comprising the amino acid sequence of SEQ ID NO: 362 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 361). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a LCVR comprising the amino acid sequence of SEQ ID NO: 370 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 369). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a heavy chain (HC) comprising the amino acid sequence of SEQ ID NO: 1077 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 1076). In an exemplary embodiment, the disclosure provides a polynucleotide (or a vector comprising a polynucleotide, or a host cell comprising a polynucleotide or vector) encoding a light chain (LC) comprising the amino acid sequence of SEQ ID NO: 378 (e.g., in one embodiment, the polynucleotide comprises SEQ ID NO: 377).In one embodiment, the disclosure provides a host cell comprising a pair of polynucleotides or a pair of vectors encoding the HCVR and LCVR, or the HC and LC, respectively, as discussed in this paragraph, and a pair of polynucleotides and / or a pair of vectors for encoding the antibody designated mAb15160_2 or an antigen-binding fragment thereof.

[0111] The term "specifically binds" refers to a binding affinity of at least about 10 as measured, for example, by a real-time label-free biolayer interferometry assay, such as an Octet® HTX biosensor, at 25° C. or 37° C., by surface plasmon resonance, such as BIACORE™, or by solution affinity ELISA. -8 K of M D The term "mAb" refers to an antigen binding protein (e.g., mAb) that has binding affinity for an antigen, such as a CoV-S protein (e.g., SARS-CoV-2-S), represented as: The present invention includes antigen binding proteins that specifically bind to a CoV-S protein.

[0112] The term "antigen-binding portion" or "antigen-binding fragment" of an antibody or antigen-binding protein and equivalents as used herein includes any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Non-limiting examples of antigen-binding fragments include (i) Fab fragments, (ii) F(ab')2 fragments, (iii) Fd fragments, (iv) Fv fragments, (v) single-chain Fv (scFv) molecules, (vi) dAb fragments, and (vii) a minimal recognition unit consisting of amino acid residues mimicking a hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR) such as a CDR3 peptide) or a constrained FR3-CDR3-FR4 peptide. Other engineered molecules such as domain-specific antibodies, single domain antibodies, domain deleted antibodies, chimeric antibodies, CDR-grafted 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 immunopharmaceuticals (SMIPs), and shark variable IgNAR domains are also encompassed within the expression "antigen-binding fragment" as used herein. In an embodiment of the invention, the antigen-binding fragment comprises three or more CDRs (e.g. HCDR1, HCDR2, and HCDR3, or LCDR1, LCDR2, and LCDR3) of an antibody of Table 4.

[0113] Antigen-binding fragments of antibodies, in embodiments of the invention, will contain at least one variable domain. A variable domain may be of any size or amino acid composition and will generally contain at least one CDR adjacent to or in frame with one or more framework sequences. L Domain associated with V H For antigen-binding fragments containing domains, V H Domain and V L The domains may be positioned relative to each other in any suitable configuration. For example, the variable region may be a dimer and may have a V H -V H , V H -V L Or VL -V L Alternatively, the antigen-binding fragment of the antibody may comprise a monomeric V H Or V L It may include a domain.

[0114] In certain embodiments, an antigen-binding fragment of an antibody may comprise at least one variable domain covalently linked to at least one constant domain. Non-limiting exemplary configurations of variable and constant domains that may be found within an antigen-binding fragment of an antibody of the invention include: (i) a V H -C H 1, (ii) V H -C H 2. (iii) V H -C H 3. (iv) V H -C H 1-C H 2. (v) V H -C H 1-C H 2-C H 3. (vi) V H -C H 2-C H 3. (vii) V H -C L , (viii) V L -C H 1, (ix) V L -C H 2. (x)V L -C H 3. (xi) V L -C H 1-C H 2. (xii) V L -C H 1-C H 2-C H 3. (xiii) V L -C H 2-C H 3, and (xiv) V L -C LIn any configuration of variable and constant domains, including any of the exemplary configurations listed above, the variable and constant domains may be either directly linked to each other or linked by a complete or partial hinge or linker region. The hinge region may consist of at least two (e.g., 5, 10, 15, 20, 40, 60, or more) amino acids that provide a flexible or semi-flexible connection between adjacent variable and / or constant domains in a single polypeptide molecule. Furthermore, antigen-binding fragments of antibodies of the invention may be linked to each other and / or to one or more monomeric V H Or V L It may comprise homodimers or heterodimers (or other multimers) of any of the variable and constant domain configurations listed above in non-covalent association (eg, via disulfide bonds) between the domains.

[0115] 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.

[0116] In specific embodiments, the antibodies or antibody fragments of the invention may be conjugated to a moiety, such as a ligand or therapeutic moiety (an "immunoconjugate"), such as an antiviral drug, a second anti-influenza antibody, or any other therapeutic moiety useful for treating a viral infection, e.g., influenza virus infection. See below.

[0117] The invention also provides a complex comprising an anti-CoV-S antigen-binding protein, e.g., an antibody or antigen-binding fragment as discussed herein, complexed with a CoV-S polypeptide or antigenic fragment thereof, and / or with a secondary antibody or antigen-binding fragment thereof (e.g., a detectably labeled secondary antibody) that specifically binds to the anti-CoV-S antibody or fragment. In an embodiment of the invention, the antibody or fragment is in vitro (e.g., immobilized on a solid substrate) or in the body of a subject. In an embodiment of the invention, the CoV-S is in vitro (e.g., immobilized on a solid substrate) or on the surface of a cell or in the body of a subject. Immobilized anti-CoV-S antibodies and antigen-binding fragments thereof that are covalently bound to an insoluble substrate material (e.g., glass or polysaccharides such as agarose or sepharose, e.g., beads or other particles thereof) are also part of the invention, and optionally the immobilized antibody is complexed with CoV-S or an antigenic fragment thereof or a secondary antibody or fragment thereof.

[0118] "Isolated" antigen binding proteins, antibodies or antigen binding fragments thereof, polypeptides, polynucleotides, and vectors are at least partially free of other biological molecules from the cell or cell culture in which they are produced. Such biological molecules include nucleic acids, proteins, other antibodies or antigen binding fragments, lipids, carbohydrates, or other substances such as cell debris and growth medium. An isolated antibody or antigen binding fragment may further be at least partially free of expression system components such as biological molecules from a host cell or its growth medium. In general, the term "isolated" is not intended to refer to the complete absence of such biological molecules, or the absence of water, buffers, or salts, or the absence of components of a pharmaceutical formulation that includes the antibody or fragment.

[0119] The term "epitope" refers to a specific antigen-binding site of an antigen-binding protein, known as a paratope, e.g., an antigenic determinant (e.g., on a CoV-S polypeptide) that interacts with a variable region of an antibody molecule. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions on an antigen and have different biological effects. The term "epitope" also refers to a site on an antigen to which B cells and / or T cells respond. The term also refers to the region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of structural epitopes and have residues that are directly involved in the affinity of the interaction. Epitopes may be linear or conformational, i.e., composed of nonlinear amino acids. In certain embodiments, epitopes may include determinants that are chemically active surface groups of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, may have specific three dimensional structural and / or specific charge characteristics.

[0120] Methods for determining the epitope of an antigen-binding protein, e.g., an antibody or fragment or a polypeptide, include alanine scanning mutation analysis, peptide blot analysis (Reineke (2004) Methods Mol. Biol. 248:443-63), peptide truncation analysis, crystallographic studies, and NMR analysis. In addition, methods such as epitope excision, epitope extraction, and chemical modification of antigens can be employed (Tomer (2000) Prot. Sci. 9:487-496). Another method that can be used to identify amino acids within a polypeptide with which an antigen-binding protein (e.g., an antibody or fragment or a polypeptide) (e.g., coversin) interacts is hydrogen / deuterium exchange detected by mass spectrometry. In general terms, the hydrogen / deuterium exchange method involves labeling a protein of interest with deuterium, followed by binding an antigen-binding protein, e.g., an antibody or fragment or a polypeptide, to the deuterium-labeled protein. The CoV-S protein / antigen-binding protein complex is then transferred to water, and exchangeable protons in amino acids protected by the antibody complex undergo deuterium to hydrogen back-exchange at a slower rate than exchangeable protons in amino acids that are not part of the interface. As a result, amino acids that form part of the protein / antigen-binding protein may retain deuterium and therefore exhibit a relatively high mass compared to amino acids that are not included in the interface. After dissociation of the antigen-binding protein (e.g., an antibody or fragment or a polypeptide), the target protein undergoes protease cleavage and mass spectrometry analysis, thereby revealing deuterium-labeled residues that correspond to the specific amino acids with which the antigen-binding protein interacts. See, for example, Ehring (1999) Analytical Biochemistry 267:252-259, Engen and Smith (2001) Anal. Chem. 73:256A-265A.

[0121] The term "compete" as used herein refers to an antigen binding protein (e.g., an antibody or antigen binding fragment thereof) that binds to an antigen (e.g., CoV-S) and inhibits or blocks the binding of another antigen binding protein (e.g., an antibody or antigen binding fragment thereof) to the antigen. The term also includes competition between two antigen binding proteins, e.g., antibodies, in both orientations, i.e., a first antibody that binds to and blocks the binding of a second 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, a first and a second antigen binding protein (e.g., an antibody) may bind to different, but overlapping, epitopes, with the binding of one inhibiting or blocking the binding of the second antibody, e.g., via steric hindrance. Competition between antigen binding proteins (e.g., antibodies) may be measured by methods known in the art, e.g., 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 the spike protein receptor binding domain (RBD) monomer), are competing for the same epitope, or are competing but have diverse microepitopes (e.g., identified through HDX). In an embodiment of the invention, competition between a first and a second anti-CoV-S antigen binding protein (e.g., antibody) is determined by measuring the ability of an immobilized first anti-CoV-S antigen binding protein (e.g., antibody) (initially not complexed to a CoV-S protein) to bind to a soluble CoV-S protein complexed with a second anti-CoV-S antigen binding protein (e.g., antibody). A reduction in the ability of the first anti-CoV-S antigen binding protein (e.g., antibody) to bind to the complexed CoV-S protein relative to the uncomplexed CoV-S protein indicates that the first and second anti-CoV-S antigen binding proteins (e.g., antibodies) compete. The degree of competition can be expressed as a percentage of reduction in binding.Such competition can be measured, for example, using a real-time label-free biolayer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.), ELISA (enzyme-linked immunosorbent assay), or SPR (surface plasmon resonance).

[0122] Binding competition between anti-CoV-S antigen-binding proteins (e.g., monoclonal antibodies (mAbs)) can be determined using a real-time label-free biolayer interferometry assay on an Octet RED384 biosensor (Pall ForteBio Corp.). For example, to determine competition between two anti-human CoV-S monoclonal antibodies, anti-CoV-S can first be captured on an Octet biosensor tip (Pall ForteBio Corp., no. 18-5060) coated with anti-hFc antibodies by immersing the tip in a solution of anti-human CoV-S mAb (hereafter referred to as "mAb1"). As a positive control for blocking, the biosensor tip with captured antibody can then be saturated with a known blocking isotype control mAb (hereafter referred to as "blocking mAb") by immersing it in a solution of blocking mAb. To determine whether mAb2 competes with mAb1, the biosensor tip can then be subsequently immersed in a co-complexed solution of pre-incubated CoV-S polypeptide and a second anti-CoV-S mAb (hereafter referred to as "mAb2") for a period of time, and binding of mAb1 to the CoV-S polypeptide can be determined. The biosensor tip can be washed with buffer between all steps of the experiment. Real-time binding responses can be monitored during the course of the experiment, and the binding responses at the end of all steps can be recorded.

[0123] For example, in embodiments of the invention, the competitive assay is carried out at 25° C. and a pH of about 7, eg, 7.4, in the presence of, eg, buffers, salts, detergents, and non-specific proteins (eg, bovine serum albumin).

[0124] Typically, an antibody or antigen-binding fragment of the invention, modified in any way, retains the ability to specifically bind to CoV-S, e.g., retains at least 10% of its CoV-S binding activity (compared to the parent antibody) when that activity is expressed on a molar basis. Preferably, an antibody or antigen-binding fragment of the invention retains at least 20%, 50%, 70%, 80%, 90%, 95%, or 100% or more of the CoV-S binding affinity as the parent antibody. It is also contemplated that an antibody or antigen-binding fragment of the invention may include conservative or non-conservative amino acid substitutions (referred to as "conservative variants" or "functionally conservative variants" of an antibody) that do not substantially alter its biological activity.

[0125] "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 mAb8029 V H , V L , HC, or LC) refers to a polypeptide comprising an amino acid sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical or similar to a reference amino acid sequence described herein (e.g., SEQ ID NO: 2, 10, 18, 20, 22, 30, 38, 40, 42, 50, 58, or 60), and when the comparison is performed by the BLAST algorithm, the parameters of the algorithm are selected to produce the largest match between the respective sequences over the entire length of the respective reference sequences (e.g., expectation threshold: 10, word size: 3, maximum match within query range: 0, BLOSUM 62 matrix, gap cost:existence 11,extension 1,conditional composition score matrix adjustment).

[0126] A "variant" of a polynucleotide refers to a polypeptide comprising a nucleotide sequence that is at least about 70-99.9% (e.g., 70, 72, 74, 75, 76, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9%) identical to a reference nucleotide sequence described herein (e.g., SEQ ID NO: 1, 9, 17, 19, 21, 29, 37, 39, 41, 49, 57, or 59), and where the comparison is performed by the BLAST algorithm, the parameters of the algorithm are selected to produce maximum matches between the respective sequences over the entire length of the respective reference sequences (e.g., expectation threshold: 10, word size: 28, maximum match within query range: 0, match / mismatch score: 1, -2, gap cost: linear).

[0127] Anti-CoV-S antigen binding proteins, such as antibodies and antigen binding fragments thereof, in embodiments of the invention comprise a heavy chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to the HCVR amino acids set forth in Table 4, and / or a light chain immunoglobulin variable region having at least 70% (e.g., 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) amino acid sequence identity to the LCVR amino acids set forth in Table 4.

[0128] In addition, variant anti-CoV-S antigen binding proteins can include polypeptides comprising an amino acid sequence as described herein, except for one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) mutations, such as, for example, missense mutations (e.g., conservative substitutions), nonsense mutations, deletions, or insertions. For example, the invention includes antigen binding proteins comprising immunoglobulin heavy chain variants comprising the LCVR amino acid sequences described in Table 4, but with one or more of such mutations, and / or immunoglobulin light chain variants comprising the HCVR amino acid sequences described in Table 4, but with one or more of such mutations. In an embodiment of the invention, the variant anti-CoV-S antigen binding protein comprises an immunoglobulin light chain comprising LCDR1, LCDR2, and LCDR3, in which one or more (e.g., one, or two, or three) of such CDRs have such a mutation (e.g., a conservative substitution), and / or an immunoglobulin heavy chain comprising HCDR1, HCDR2, and HCDR3, in which one or more (e.g., one, or two, or three) of such CDRs have such a mutation (e.g., a conservative substitution). The substitution may be in the CDR, framework, or constant region.

[0129] The invention further provides variant anti-CoV-S antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, that include one or more variant CDRs described herein (e.g., any one or more of LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and / or HCDR3) that have, e.g., at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% sequence identity or similarity to the heavy and light chain CDRs of Table 4.

[0130] The present invention also relates to the corresponding V H , V LHC, or LC. H and V L , or HC and LC, but where the LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 of such immunoglobulins are not variant and comprise the CDR amino acid sequences set out in Table 4. Thus, in such embodiments, the CDRs within the variant antigen binding proteins are not themselves variant.

[0131] Conservatively modified variants Anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present invention. "Conservatively modified variants" or "conservative substitutions" refer to variants in which there is a substitution of one or more amino acids in a polypeptide by other amino acids having similar properties (e.g., charge, side chain size, hydrophobicity / hydrophilicity, backbone conformation and rigidity, etc.). Such changes can frequently occur without significantly impairing the biological activity of the antibody or fragment. Those skilled in the art recognize that, in general, single amino acid substitutions in non-essential regions of a polypeptide do not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin / Cummings Pub. Co., p. 224 (4 th (See, e.g., U.S. Pat. No. 6,333,363, Ed.). In addition, substitutions of structurally or functionally similar amino acids are unlikely to significantly impair biological activity.

[0132] Examples of groups of amino acids with side chains containing similar chemical properties include: 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine, 2) aliphatic-hydroxyl side chains: serine and threonine, 3) amide-containing side chains: asparagine and glutamine, 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan, 5) basic side chains: lysine, arginine, and histidine, 6) acidic side chains: aspartic acid and glutamic acid, and 7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid, and asparagine-glutamine. Alternatively, a conservative substitution is any change that has a positive value in the PAM250 log-likelihood matrix, as disclosed in Gonnet et al. (1992) Science 256:1443 45.

[0133] Function-conservative variants of anti-CoV-S antibodies and antigen-binding fragments thereof are also part of the present invention. Any of the variants of anti-CoV-S antibodies and antigen-binding fragments thereof (discussed herein) may be "function-conservative variants". Such function-conservative variants may also be characterized as conservatively modified variants in some cases. "Function-conservative variants" as used herein refers to variants of anti-CoV-S antibodies or antigen-binding fragments thereof in which one or more amino acid residues have been altered without significantly altering one or more functional properties of the antibody or fragment. In embodiments of the present invention, the function-conservative variant anti-CoV-S antibodies or antigen-binding fragments thereof of the present invention comprise variant amino acid sequences and exhibit one or more of the following functional properties: inhibiting the growth of coronaviruses (e.g., SARS-CoV-2, SARS-CoV, and / or MERS-CoV) in ACE2- and / or TMPRSS2-expressing cells (e.g., Calu-3 cells); -Does not significantly bind to MDCK / Tet-on cells that do not express ACE2 and / or TMPRSS2; limiting the spread of coronavirus infection (e.g., by SARS-CoV-2, SARS-CoV, and / or MERS-CoV) in cells, e.g., Calu-3, in vitro; and / or Optionally, when optionally combined with a second therapeutic agent, protecting a mouse engineered to express human TMPRSS2 and / or ACE2 proteins from death caused by a coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV), e.g., where the mouse is infected with an otherwise lethal virus. Optionally, when optionally combined with a second therapeutic agent, protecting a mouse engineered to express human TMPRSS2 and / or ACE2 proteins from weight loss caused by a coronavirus infection (e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV), e.g., where the mouse has been infected with a dose of virus that would otherwise cause weight loss.

[0134] A "neutralizing" or "antagonist" anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment, refers to a molecule that inhibits the activity of CoV-S to any detectable extent, e.g., inhibits the ability of CoV-S to bind to a receptor such as ACE2, be cleaved by a protease such as TMPRSS2, or mediate viral entry or replication in a host cell.

[0135] Table 4 shows the heavy chain or V H (or a variant thereof) and light chain or V L (or a variant thereof) or its CDRs (HCDR1 (or a variant thereof), HCDR2 (or a variant thereof), and HCDR3 (or a variant thereof) H and its CDRs (LCDR1 (or a variant thereof), LCDR2 (or a variant thereof), and LCDR3 (or a variant thereof)) L"Amino acid sequence" refers to antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, including, for example, immunoglobulin chains, variable regions, and / or CDRs comprising the specific amino acid sequences set out below.

[0136] The antibodies described herein also include V H is fused to wild type IgG4 (e.g., where residue 108 is S) or to an IgG4 variant (e.g., where residue 108 is P).

[0137] The antibodies and antigen-binding fragments of the invention include immunoglobulin chains comprising the amino acid sequences described herein, as well as intracellular and in vitro post-translational modifications to the antibodies. For example, the invention includes antibodies and antigen-binding fragments that specifically bind to CoV-S, comprising the heavy and / or light chain amino acid sequences (e.g., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and / or LCDR3) described herein, as well as antibodies and fragments in which one or more amino acid residues are glycosylated, one or more Asn residues are deamidated, one or more residues (e.g., Met, Trp, and / or His) are oxidized, the N-terminal Gln is pyroglutamic acid (pyroE), and / or the C-terminal lysine is missing. The amino acid and nucleotide sequences of exemplary anti-SARS-CoV-2-spike protein (SARS-CoV-2-S) antibodies are shown in the following table of exemplary sequences (Table 1). [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] [Table 1-10]

[0138] Administration of antibodies The invention provides methods for administering an anti-CoV-S antigen binding protein of the invention, e.g., those in Table 4, comprising introducing the antigen binding protein into the body of a subject (e.g., a human). For example, the method comprises puncturing the body of the subject with a needle of a syringe and injecting the antigen binding protein into the subject's body, e.g., into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0139] The invention provides a container (e.g., a plastic or glass vial, hollow needle or syringe cylinder, e.g., with a cap or a chromatography column) comprising an anti-CoV-S antigen binding protein of the invention, e.g., those in Table 4.

[0140] The present invention also provides an injection device comprising one or more antigen binding proteins (e.g., antibodies or antigen binding fragments) that specifically bind to CoV-S, e.g., those in Table 4, or a pharmaceutical composition thereof. The injection device may be packaged in a kit. An injection device is a device that introduces a substance into a subject's body via a parenteral route, e.g., intramuscular, subcutaneous, or intravenous. For example, the injection device may be a syringe (e.g., pre-filled with a pharmaceutical composition, such as an autoinjector), e.g., comprising a cylinder or barrel for holding the fluid to be injected (e.g., comprising an antibody or fragment or pharmaceutical composition thereof), a needle for puncturing the skin and / or blood vessel for injection of the fluid, and a plunger for pushing the fluid from the cylinder through the hole in the needle. In an embodiment of the present invention, the injection device comprising an antigen binding protein from the combination of the present invention, e.g., an antibody or antigen binding fragment thereof, or a pharmaceutical composition thereof, is an intravenous (IV) injection device. Such a device may include an antigen binding protein or pharmaceutical composition thereof in a cannula or trocar / needle, which may be attached to a tube that may be attached to a bag or reservoir for holding a fluid (e.g., saline) to be introduced into the subject's body through the cannula or trocar / needle. The antibody or fragment or pharmaceutical composition thereof may be introduced into the device, in embodiments of the invention, when the trocar and cannula are inserted into a subject's vein and the trocar is removed from the inserted cannula. The IV device may be inserted, for example, into a peripheral vein (e.g., in the hand or arm), into the superior or inferior vena cava, or into the right atrium (e.g., central IV), or into the subclavian, internal jugular, or femoral vein and advanced toward the heart until it reaches, for example, the superior or right vena cava (e.g., central venous line). In embodiments of the invention, the injection device is an autoinjector, jet injector, or external infusion pump. A jet injector introduces the antibody or fragment or pharmaceutical composition thereof into the subject's body using a high-pressure narrow liquid jet that penetrates the epidermis. An external infusion pump is a medical device that delivers an antibody or fragment, or a pharmaceutical composition thereof, in controlled amounts to the body of a subject. External infusion pumps can be electrically or mechanically powered.Different pumps work in different ways, for example, syringe pumps hold the fluid in a syringe reservoir and a moveable piston controls the fluid delivery, elastic pumps hold the fluid in a stretchy balloon reservoir and pressure from the elastic walls of the balloon drives the fluid delivery, in peristaltic pumps a set of rollers are clamped into a long flexible tube that pushes the fluid forward, and multi-channel pumps can deliver fluid from multiple reservoirs at multiple speeds.

[0141] Preparation of human antibodies Methods for generating human antibodies in transgenic mice are known in the art. Any such known methods can be used in the context of the present invention to generate human antibodies that specifically bind to CoV-S. Antibodies against CoV-S can be generated using immunogens including any one of the following: In certain embodiments of the present invention, the antibodies of the present invention are obtained from mice immunized with full-length native CoV-S, or with live attenuated or inactivated virus, or with DNA encoding the protein or fragments thereof. Alternatively, CoV-S proteins or fragments thereof can be produced, modified, and used as immunogens using standard biochemical techniques. In one embodiment of the present invention, the immunogen is a recombinantly produced CoV-S protein or fragment thereof. In certain embodiments of the present invention, the immunogen can be a CoV-S polypeptide vaccine. In certain embodiments, one or more booster injections can be administered. In certain embodiments, the immunogen can be a recombinant CoV-S polypeptide expressed in E. coli or any other eukaryotic or mammalian cell, such as Chinese Hamster Ovary (CHO) cells.

[0142] Using VELOCIMMUNE® technology (see, e.g., US 6,596,541, Regeneron Pharmaceuticals, VELOCIMMUNE®) or any other known method for generating monoclonal antibodies, high affinity chimeric antibodies against CoV-S with human variable regions and mouse constant regions can first be isolated. VELOCIMMUNE® technology involves the generation of transgenic mice with genomes that include human heavy and light chain variable regions operably linked to endogenous mouse constant region loci such that the mice produce antibodies containing human variable regions and mouse constant regions in response to antigenic challenge. DNA encoding the heavy and light chain variable regions of the antibody is isolated and operably linked to DNA encoding the human heavy and light chain constant regions. The DNA is then expressed in cells capable of expressing fully human antibodies.

[0143] Generally, VELOCIMMUNE® mice are loaded with an antigen of interest and lymphoid cells (such as B cells) are collected from the mice that express antibodies. The lymphoid cells can be fused with a myeloma cell line to prepare an immortalized hybridoma cell line, which is screened and selected to identify a hybridoma cell line that produces an antibody specific to the antigen of interest. DNA encoding the variable regions of the heavy and light chains can be isolated and combined with the desired isotype constant regions of the heavy and light chains. Such antibody proteins can be produced in cells such as CHO cells. Alternatively, DNA encoding the antigen-specific chimeric antibody or the variable domains of the light and heavy chains can be isolated directly from the antigen-specific lymphocytes.

[0144] First, a high affinity chimeric antibody with human variable regions and mouse constant regions is isolated. As in the experimental section below, the antibody is characterized and selected for desirable properties including affinity, selectivity, epitope, etc. The mouse constant region is replaced with the desired human constant region to generate a fully human antibody of the invention, e.g., wild-type or modified IgG1 or IgG4. While the constant region selected can vary according to the specific application, the high affinity antigen binding and target specificity properties reside in the variable region.

[0145] Anti-coronavirus spike protein antibodies, including Fc variants According to certain embodiments of the invention, there are provided anti-CoV-S antigen binding proteins, e.g., antibodies or antigen binding fragments, comprising an Fc domain that includes one or more mutations that enhance or decrease antibody binding to the FcRn receptor, e.g., at acidic pH compared to neutral pH. For example, the invention provides a method for the preparation of a C-terminal ... H 2 or C HThe present invention includes anti-CoV-S antibodies that contain mutations in the 3 region that increase the affinity of the Fc domain for FcRn in acidic environments (e.g., in endosomes where the pH ranges from about 5.5 to about 6.0). Such mutations can result in an increased serum half-life of the antibody when administered to an animal. Non-limiting examples of such Fc modifications include, for example, modifications at positions 250 (e.g., E or Q), 250 and 428 (e.g., L or F), 252 (e.g., L / Y / F / W or T), 254 (e.g., S or T), and 256 (e.g., S / R / Q / E / D or T), or modifications at positions 428 and / or 433 (e.g., H / L / R / S / P / Q or K) and / or 434 (e.g., A, W, H, F, or Y [N434A, N434W, N434H, N434F or N434Y]), or modifications at positions 250 and / or 428, or modifications at positions 307 or 308 (e.g., 308F, V308F), and 434. In one embodiment, the modifications include 428L (e.g., M428L) and 434S (e.g., N434S) modifications, 428L, 259I (e.g., V259I), and 308F (e.g., V308F) modifications, 433K (e.g., H433K) and 434 (e.g., 434Y) modifications, 252, 254, and 256 (e.g., 252Y, 254T, and 256E) modifications, 250Q and 428L modifications (e.g., T250Q and M428L), and 307 and / or 308 modifications (e.g., 308F and / or 308P). In yet another embodiment, the modifications include 265A (e.g., D265A) and / or 297A (e.g., N297A) modifications.

[0146] For example, the present invention provides peptides including 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). 307A, 380A and 434A (e.g., T307A, E380A and N434A); and 433K and 434F (e.g., H433K and N434F). In particular, the antibodies designated mAb17090 and mAb15160_2 each contain M252Y, S254T, and T256E modifications in the heavy chain constant region compared to mAb14286 and mAb15160, respectively.

[0147] The VF domain mutations described herein, including any possible combination of the above-mentioned Fc domain mutations. H and / or V L Anti-CoV-S antigen binding proteins, such as antibodies and antigen-binding fragments thereof, are contemplated within the scope of the present invention, including

[0148] The present invention also relates to a method for the preparation of a compound according to the present invention. H and chimeric heavy chain constant (C H ) region, an anti-CoV-S antigen-binding protein, antibody or antigen-binding fragment thereof, H A region can be a C region of more than one immunoglobulin isotype. H For example, the antibodies of the present invention may include segments derived from C regions derived from human IgG1, human IgG2, or human IgG4 molecules. H C derived from a human IgG1 molecule, a human IgG2 molecule, or a human IgG4 molecule in combination with part or all of the C3 domain. H Chimeric C containing part or all of the 2 domains H According to certain embodiments, the antibodies of the invention may comprise a chimeric C region having a chimeric hinge region.H For example, the chimeric hinge may comprise an "upper hinge" amino acid sequence (amino acid residues 216-227 according to EU numbering) derived from a human IgG1, IgG2, or IgG4 hinge region in combination with a "lower hinge" sequence (amino acid residues 228-236 according to EU numbering) derived from a human IgG1, IgG2, or IgG4 hinge region. According to certain embodiments, the chimeric hinge region comprises amino acid residues derived from a human IgG1 upper hinge or a human IgG4 upper hinge, and amino acid residues derived from a human IgG2 lower hinge. The chimeric C described herein may be H Antibodies comprising the region, in certain embodiments, exhibit modified Fc effector functions without adversely affecting the therapeutic or pharmacokinetic properties of the antibody (see, e.g., WO2014 / 022540).

[0149] Immunoconjugates The present invention encompasses anti-CoV-S antigen binding proteins, e.g., antibodies or antigen binding fragments, conjugated to another moiety, e.g., a therapeutic moiety, such as a toxoid or antiviral drug for treating influenza virus infection ("immunoconjugates"). In an embodiment of the invention, the anti-CoV-S antibody or fragment is conjugated to any of the additional therapeutic agents described herein. As used herein, the term "immunoconjugate" refers to an antigen binding protein, e.g., an antibody or antigen binding fragment, that is chemically or biologically bound to a radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide or protein, or therapeutic agent. The antigen binding protein may be bound to a radiopharmaceutical, cytokine, interferon, target or reporter moiety, enzyme, peptide, or therapeutic agent anywhere along the molecule, so long as it is capable of binding to its target (CoV-S). Examples of immunoconjugates include antibody drug conjugates and antibody toxin fusion proteins. In one embodiment of the present invention, the agent may be a second, different antibody that specifically binds to CoV-S. The type of therapeutic moiety that can be conjugated to the anti-CoV-S antigen binding protein (e.g., antibody or fragment) takes into account the condition to be treated and the desired therapeutic effect to be achieved. See, for example, Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug Delivery", Controlled Drug Delivery (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”, Immunol. Rev., 62:119-58 (1982).

[0150] multispecific antibodies The present invention includes anti-CoV-S antigen binding proteins, e.g., antibodies and antigen binding fragments thereof, as well as methods of use and methods of making such antigen binding proteins. The term "anti-CoV-S" antigen binding protein, e.g., antibody or antigen binding fragment, includes multispecific (e.g., bispecific or biparatopic) molecules comprising at least one first antigen binding domain that specifically binds to CoV-S (e.g., an antigen binding domain from an antibody in Table 4), and at least one second antigen binding protein that binds to a different antigen or epitope in CoV-S that is different from that of the first antigen binding domain. In some embodiments, both the first antigen binding domain and the second antigen binding domain are selected from the antigen binding domains in Table 4. In an embodiment of the invention, the first and second epitopes overlap. In another embodiment of the invention, the first and second epitopes do not overlap. For example, in embodiments of the invention, the multispecific antibody is a bispecific IgG antibody (e.g., IgG1 or IgG4) comprising a first antigen-binding domain that specifically binds CoV-S comprising the heavy and light immunoglobulin chains of an antibody in Table 4, and a second antigen-binding domain that specifically binds to a different epitope of CoV-S. In some embodiments, the bispecific IgG antibody (e.g., IgG1 or IgG4) comprises a first antigen-binding domain that specifically binds CoV-S, and a second binding domain that binds to a host cell protein, e.g., ACE2 or TMPRSS2.

[0151] The antibodies in Table 4 each comprise the CDR-H and CDR-L, V and VD of these antibodies (including their variants as described herein). H and V L or a multispecific molecule, such as an antibody or antigen-binding fragment, comprising an HC and an LC.

[0152] In an embodiment of the invention, an antigen-binding domain that specifically binds to CoV-S that may be comprised in a multispecific molecule comprises: (1) (i) a heavy chain variable domain sequence comprising the HCDR1, HCDR2, and HCDR3 amino acid sequences set forth in Table 4; and (ii) a light chain variable domain sequence comprising the LCDR1, LCDR2, and LCDR3 amino acid sequences set forth in Table 4; or (2) (i) a heavy chain variable domain sequence comprising an amino acid sequence set forth in Table 4, and (ii) a light chain variable domain sequence comprising an amino acid sequence set forth in Table 4; or (3) (i) a heavy chain immunoglobulin sequence comprising an amino acid sequence set forth in Table 4, and (ii) a light chain immunoglobulin sequence comprising an amino acid sequence set forth in Table 4.

[0153] In an embodiment of the invention, the multispecific antibody or fragment comprises more than two different binding specificities (e.g. a trispecific molecule), e.g. one or more additional antigen binding domains, which are the same as or different from the first and / or second antigen binding domain.

[0154] In one embodiment of the invention, the bispecific antigen-binding fragment comprises a first scFv (e.g., V of Table 4) having binding specificity for a first epitope (e.g., CoV-S). H and V L For example, in an embodiment of the invention, the first and second scFvs include a linker, e.g., a peptide linker (e.g., (GGGGS) n (SEQ ID NO: 834), 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 a bispecific IgG antibody that contains the heavy and light chain CDRs of Table 4 and the heavy and light chain CDRs of another antibody that binds to a different epitope.

[0155] Treatment method The invention provides methods for treating or preventing a viral infection (e.g., a coronavirus infection) by administering a therapeutically effective amount of an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment (e.g., of Table 4) to a subject (e.g., a human) in need of such treatment or prevention.

[0156] Coronavirus or influenza virus infections can be treated or prevented in a subject by administering to the subject an anti-CoV-S antigen binding protein of the invention. Exemplary coronaviruses include SARS-CoV-2, which can further include variants such as alpha, beta, gamma, delta, and omicron.

[0157] An effective or therapeutically effective dose of an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment (e.g., of Table 4), for treating or preventing a viral infection refers to an amount of the antibody or fragment sufficient to alleviate one or more signs and / or symptoms of an infection in a treated subject, whether by inducing a reduction or elimination of such signs and / or symptoms, or by inhibiting the progression of such signs and / or symptoms. The dosage may vary depending on the age and size of the subject to which it is administered, the target disease, condition, route of administration, and the like. In an embodiment of the invention, an effective or therapeutically effective dose of an antibody or antigen-binding fragment thereof of the invention for treating or preventing a viral infection in, for example, an adult human subject, is about 0.01 to about 200 mg / kg, e.g., up to about 150 mg / kg. In an embodiment of the invention, the dosage is up to about 10.8 or 11 grams (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 grams). Depending on the severity of the infection, the frequency and duration of treatment may be adjusted. In certain embodiments, the antigen binding protein of the invention may be administered in an initial dose, followed by one or more secondary doses. In certain embodiments, the initial dose may be followed by administration of a second dose or multiple subsequent doses of the antibody or antigen binding fragment thereof in an amount that may be about the same as the initial dose or a smaller dose, with the subsequent doses separated by at least 1 to 3 days, at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 12 weeks, or at least 14 weeks.

[0158] In some embodiments, mAb10933 and mAb10987 (casirivimab and imdevimab, respectively) can be administered to a subject in a method of prophylaxis, e.g., pre-exposure prophylaxis, against symptomatic COVID-19. In some embodiments, the subject is immunocompromised, e.g., has an immunodeficiency, such as a primary or secondary immunodeficiency. In some embodiments, the subject has not mounted an effective response to COVID-19 vaccination. Exemplary criteria associated with immune compromise include active or recent treatment for solid tumors and hematological malignancies, receiving a solid organ transplant or recent hematopoietic stem cell transplant, severe primary immunodeficiency, progressive or untreated HIV infection, active treatment with high-dose corticosteroids, alkylating agents, antimetabolites, tumor necrosis (TNF) blockers, and other biologic agents that are immunosuppressive or immunomodulatory, as well as chronic medical conditions such as asplenia and chronic kidney disease, some patients with these conditions may be associated with various degrees of immunodeficiency. In some embodiments, the subject meets one or more of the following criteria: A solid organ transplant (SOT) or hematopoietic cell transplant (HSCT) recipient receiving any immunosuppressant medication b. Active hematologic malignancy or hematologic malignancy for which therapy has been completed within 3 months Solid organ malignancies undergoing active treatment with cT-cell or B-cell immunosuppressive therapy d. Moderate or severe primary immunodeficiency (hypogammaglobulinemia, common variable immunodeficiency, severe combined immunodeficiency, etc.) e. HIV and CD4 < 200 cells / microliter f. Patients with rheumatic diseases, autoimmune diseases, or multiple sclerosis undergoing immunosuppressive therapy that modulates Th1, Th17, or B cell responses. g. Receiving any of the following immunosuppressants for more than 3 weeks: -T-cell suppressive agents (e.g., ≥ 5 mg prednisone equivalent / day for > 3 weeks), proteasome inhibitors (e.g., bortezomib, lenalidomide), alemtuzumab, antithymocyte globulin, CAR-T therapy, calcineurin inhibitors) -Alkylating agents - Purine analogs such as fludarabine or cladribine -B cell depleting agents such as rituximab and ocrelizumab -mTOR inhibitors -Antimetabolites such as mycophenolate -JAK inhibitors

[0159] Exemplary dosing regimens include the following: Simultaneous administration of mAb10933 and mAb10987 combination therapy, 1200 mg (600 mg per mAb) on day 1, then 600 mg (300 mg per mAb) subcutaneously (SC) every 4 weeks (Q4W); Concurrent administration of mAb10933 and mAb10987 combination therapy, 300 mg (150 mg per mAb) SC Q4W; and Coadministration of mAb10933 and mAb10987 combination therapy, 300 mg (150 mg per mAb) SC Q12W.

[0160] As used herein, the term "subject" refers to a mammal (e.g., rat, mouse, cat, dog, cow, sheep, horse, goat, rabbit), preferably a human, in need of prevention and / or treatment of a disease or disorder, such as, for example, a viral infection or cancer. The subject may have a viral infection, e.g., influenza infection, or be prone to developing an infectious disease. Subjects prone to developing an infectious disease or may be at increased risk of contracting an infectious disease (e.g., of coronavirus or influenza virus) include subjects with a weakened immune system due to an autoimmune disease, subjects undergoing immunosuppressive therapy (e.g., after organ transplantation), subjects suffering from human immunodeficiency syndrome (HIV) or acquired immune deficiency syndrome (AIDS), subjects with a form of anemia that depletes or destroys white blood cells, subjects undergoing radiation or chemotherapy, or subjects suffering from an inflammatory disease. In addition, very young subjects (e.g., 5 years old or younger) or elderly subjects (e.g., 65 years old or older) are at increased risk. Additionally, a subject may be at risk for contracting a viral infection due to proximity to an outbreak of the disease, e.g., the subject lives in a densely populated city or in close proximity to a subject with confirmed or suspected viral infection, or due to occupational choices, e.g., hospital workers, pharmaceutical researchers, travelers to affected areas, or frequent flyers.

[0161] "Treat" or "treating" means administering an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment, of the invention (e.g., of Table 4) to a subject who has one or more signs or symptoms of a disease or infection, e.g., a viral infection, where the antigen binding protein is effective when administered to the subject in an effective or therapeutically effective amount or dose (as discussed herein).

[0162] The invention also encompasses prophylactic administration of an anti-CoV-S antigen binding protein, e.g., an antibody or antigen binding fragment thereof, of the invention (e.g., of Table 4) to a subject at risk of such infection to prevent viral infection. Passive antibody-based immunoprophylaxis has proven an effective strategy to prevent subjects from viral infection. See, e.g., Berry et al., Passive broad-spectrum influenza immunoprophylaxis. Influenza Res Treat. 2014; 2014: 267594. Epub 2014 Sep 22, and Jianqiang et al., Passive immune neutralization strategies for prevention and control of influenza A infections, Immunotherapy. 2012 February; 4(2): 175-186; Prabhu et al., Antivir Ther. 2009; 14(7): 911-21, Prophylactic and therapeutic efficacy of a chimeric monoclonal antibody specific for H5 hemagglutinin against lethal H5N1 influenza. "Prevent" or "preventing" means administering an anti-CoV-S antigen binding protein, e.g., an antibody or antigen binding fragment, of the invention (e.g., Table 4) to a subject to inhibit the appearance of a disease or infection (e.g., a viral infection) in the body of the subject, where the antigen binding protein is effective when administered to the subject in an effective or therapeutically effective amount or dose (discussed herein). As used herein, prevention can be pre-exposure prevention (e.g., administration of an antibody or antigen binding fragment described herein to an individual prior to exposure to SARS-CoV-2 virus), or post-exposure prevention (e.g., administration of an antibody or antigen binding fragment described herein to an individual prior to exposure to SARS-CoV-2 virus). In some embodiments, post-exposure prevention can prevent one or more symptoms of COVID-19 despite infection with SARS-CoV-2.

[0163] In an embodiment of the invention, a sign or symptom of a viral infection in a subject is the survival or proliferation of the virus within the subject's body, for example, as determined by a viral titer assay (e.g., coronavirus spread in embryonated eggs or a coronavirus spike protein assay). Other signs and symptoms of viral infection are discussed herein.

[0164] As noted above, in some embodiments, the subject may be a non-human animal, and the antigen binding proteins (e.g., antibodies and antigen-binding fragments) discussed herein may be used in veterinary settings to treat and / or prevent disease in non-human animals (e.g., cats, dogs, pigs, cows, horses, goats, rabbits, sheep, and the like).

[0165] The present invention provides methods for treating or preventing a viral infection (e.g., a coronavirus infection), or for inducing regression or elimination of, or inhibiting the progression of, at least one sign or symptom of a viral infection, such as: Feeling fever or chills, ·cough, ·sore throat, Runny or stuffy nose, ·sneeze, Muscle or body pain, ·headache, Fatigue (tiredness), ·vomiting, ·diarrhea, ·Respiratory infections, · Chest discomfort, ·shortness of breath, Bronchitis, and / or ·pneumonia The signs or symptoms are secondary to a viral infection, and the method is in a subject (e.g., a human) in need of the method by administering to the subject a therapeutically effective amount of an anti-CoV-S antigen binding protein (e.g., of Table 4), for example, by injection of the protein into the subject's body.

[0166] Combinations and Pharmaceutical Compositions To prepare pharmaceutical compositions of anti-CoV-S antigen binding proteins, e.g., antibodies and antigen-binding fragments thereof (e.g., of Table 4), the antigen binding protein is mixed with a pharma- ceutically acceptable carrier or excipient. For example, 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 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 See, for example, Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY. In an embodiment of the invention, the pharmaceutical composition is sterile. Such compositions are part of the present invention.

[0167] The scope of the invention includes dry, e.g., lyophilized compositions comprising an anti-CoV-S antigen binding protein (e.g., of Table 4), e.g., an antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof comprising a pharma- ceutically acceptable carrier, but substantially devoid of water.

[0168] In a further embodiment of the invention, the additional therapeutic agent administered to the subject in association with an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, disclosed herein (e.g., of Table 4) is selected from the group consisting of antibodies, antibodies, and antibodies to CoV-S antigen binding fragments, as described in Physicians' Desk Reference 2003 (Thomson Healthcare; 57 th The vaccine is administered to subjects according to the ClinicalTrials.gov / pubmed / 123441 (Nov. 1, 2002)).

[0169] The mode of administration can vary, including oral, rectal, transmucosal, intestinal, parenteral, intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, insufflation, topical, dermal, transdermal, or intraarterial.

[0170] The invention provides methods for administering an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment thereof (e.g., of Table 4), comprising introducing the protein into the body of a subject. For example, the method comprises puncturing the body of the subject with a needle of a syringe and injecting the antigen binding protein into the body of the subject, e.g., into a vein, artery, tumor, muscle tissue, or subcutaneous tissue of the subject.

[0171] The present invention provides anti-CoV-S antigen binding proteins, e.g., antibodies or antigen-binding fragments thereof, polypeptides (e.g., HC, LC, V, etc., of Table 4). H , or V L ), or any of the polynucleotides or vectors described herein (e.g., of Table 5), or pharmaceutical compositions thereof comprising a pharma- ceutical carrier, are provided.

[0172] In embodiments of the invention, an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment thereof, of the invention (e.g., of Table 4) is administered in conjunction with one or more additional therapeutic agents. The additional therapeutic agents include, but are not limited to, an anti-inflammatory agent, an anti-malarial agent, a second antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and a second antibody or antigen-binding fragment thereof that specifically binds to CoV-S (e.g., an antibody described herein or in U.S. Pat. No. 10,787,501, which is specifically incorporated herein by reference in its entirety). In some embodiments, the anti-malarial agent is chloroquine or hydroxychloroquine. In some embodiments, the anti-inflammatory agent is an antibody, such as sarilumab, tocilizumab, or gimsilumab. In some embodiments, the additional therapeutic agent is a second antibody or antigen-binding fragment disclosed herein, e.g., of Table 4. In some cases, the antibody that binds to CoV-S is casirivimab or imdevimab. In certain embodiments, one, two, three, four, or more antibodies or antigen-binding fragments thereof in Table 4 can be administered in combination (e.g., simultaneously or sequentially). In particular, the combination of antibodies can be selected such that the antibodies do not cross-compete, as described in Example 7. In some embodiments, mAb14256 is administered in combination with mAb14315. In some embodiments, mAb15151 is administered in combination with mAb14315. In some embodiments, an antibody (e.g., one antibody or two antibodies) of the present disclosure can be combined with an antibody (e.g., one antibody or two antibodies) described in U.S. Patent No. 10,787,501 (the '501 patent). In certain embodiments, mAb10987 of the '501 patent is administered in combination with mAb14256 and / or mAb15151 of the present disclosure. In certain embodiments, mAb10933 and mAb10987 of the '501 patent are administered in combination with mAb14256 of the present disclosure. In certain embodiments, mAb10985 and mAb10987 of the '501 patent are administered in combination with mAb15151 of the present disclosure.In certain embodiments, mAb10985 of the '501 patent is administered in combination with mAb14315 and / or mAb15151 of the present disclosure. In certain embodiments, mAb15160 of the present disclosure is administered in combination with mAb10987 of the '501 patent. In certain embodiments, mAb15160 of the present disclosure is administered in combination with mAb10985 of the '501 patent. In certain embodiments, mAb15160 of the present disclosure is administered in combination with mAb10985 and mAb10987 of the '501 patent. In certain embodiments, mAb15160 of the present disclosure is administered in combination with any one, two, or three of mAb14256, mAb14315, and mAb15151, optionally further administered with mAb10987 and / or mAb10985. In certain embodiments, any one, two, three, or four of mAb14256, mAb14315, mAb15151, and mAb15160 are administered in combination with i) mAb10933, ii) mAb10987, or iii) mAb10933 and mAb10987. In certain embodiments, any combination of one, two, three, four, five, six, or seven of mAb10933, mAb10987, mAb10985, mAb14256, mAb1435, mAb15151, and mAb15160 are administered in combination. In certain embodiments, an antibody or antigen-binding fragment thereof selected from mAb10987, mAb14284, mAb14315, and mAb17090 is combined with an antibody or antigen-binding fragment thereof selected from mAb10933, mAb14256, mAb15160, and mAb15160_2. In certain embodiments, the combination comprises mAb10987 and mAb10933. In certain embodiments, the combination comprises mAb10987 and mAb14256. In certain embodiments, the combination comprises mAb10987 and mAb15160. In certain embodiments, the combination comprises mAb10987 and mAb15160_2. In certain embodiments, the combination comprises mAb14284 and mAb10933.In certain embodiments, the combination comprises mAb14284 and mAb14256. In certain embodiments, the combination comprises mAb14284 and mAb15160. In certain embodiments, the combination comprises mAb14284 and mAb15160_2. In certain embodiments, the combination comprises mAb14315 and mAb10933. In certain embodiments, the combination comprises mAb14315 and mAb14256. In certain embodiments, the combination comprises mAb14315 and mAb15160. In certain embodiments, the combination comprises mAb14315 and mAb15160_2. In certain embodiments, the combination comprises mAb17090 and mAb10933. In certain embodiments, the combination comprises mAb17090 and mAb14256. In certain embodiments, the combination comprises mAb17090 and mAb15160. In certain embodiments, the combination comprises mAb17090 and mAb15160_2. In any of the combinations discussed above or herein, the antibody or antigen-binding fragment may be defined by the CDRs contained within the HCVR and LCVR sequences identified in Table 4, by the heavy and light chain CDR sequences identified in Table 4, by the HCVR and LCVR sequences identified in Table 4, or by the full-length heavy and light chain sequences identified in Table 4, each of which is encompassed within the present disclosure. Certain exemplary combinations of two antibodies are provided below. In some embodiments, the antibody that specifically binds to TMPRSS2 is H1H7017N, as described in International Patent Publication No. WO / 2019 / 147831. [Table 2] [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8]

[0173] 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 may be combined. The invention includes compositions comprising two (or more) anti-SARS-CoV-2-S antibodies or antigen-binding fragments comprising variable domains from a human subject, where the two (or more) antibodies or antigen-binding fragments are derived from different subjects (e.g., two different human subjects). Antibody variable regions derived from human B cells are discussed, for example, in Examples 1 and 2 (Table 6), which describe variable domains cloned from such B cells being combined with constant regions not derived from those B cells to produce hybrid antibodies.

[0174] In some embodiments, the additional therapeutic agent is an antiviral agent and / or a vaccine. As used herein, the term "antiviral agent" refers to any anti-infective agent or therapy used to treat, prevent, or ameliorate a viral infection in a subject. The term "antiviral agent" includes, but is not limited to, cationic steroid antibacterial agents, leupeptin, aprotinin, ribavirin, or interferon-alpha 2b. Methods for treating or preventing a viral (e.g., coronavirus) infection in a subject in need thereof by administering an antibody or antigen-binding fragment of Table 4 in association with an additional therapeutic agent are part of the present invention.

[0175] For example, in embodiments of the invention, the additional therapeutic agent is a vaccine, such as a coronavirus vaccine. In embodiments of the invention, the vaccine is an inactivated / killed virus vaccine, a live attenuated virus vaccine, or a viral subunit vaccine.

[0176] For example, in embodiments of the invention, the additional therapeutic agent is [ka] [ka] See Shen et al. Biochimie 142:1-10 (2017).

[0177] In an embodiment of the invention, the antiviral agent is an antibody or antigen-binding fragment that specifically binds to a coronavirus, e.g., SARS-CoV-2, SARS-CoV, or MERS-CoV. Exemplary anti-CoV-S antibodies include H4sH15188P, H1H15188P, H1H15211P, H1H15177P, H4sH15211P, H1H15260P2, H1H15259P2, H1H15203P, H4sH15260P2, H4sH15231P2, H1H15237P2, H1H15208P, H4sH15231P2, H1H15237P2, H1H15208P, H4sH15231P2, H1H15237P2, H4sH15231P2, H4s ... , H1H15228P2, H1H15233P2, H1H15264P2, H1H15231P2, H1H15253P2, H1H15215P, and H1H15249P2, or antigen-binding fragments thereof, for example, the antibody or fragment can be a light chain immunoglobulin (e.g., V, VL ... L or its light chain), and a heavy chain comprising HCDR1, HCDR2, and HCDR3 (e.g., V H or its heavy chain).

[0178] In certain embodiments of the invention, the additional therapeutic agent is not aprotinin, leupeptin, a cationic steroid antibacterial agent, an influenza vaccine (e.g., killed, live, attenuated whole virus or subunit vaccine), or an antibody against influenza virus (e.g., an anti-hemagglutinin antibody).

[0179] The term "in association with" indicates that a component, an anti-CoV-S antigen binding protein of the invention, e.g., an antibody or antigen-binding fragment thereof, along with another agent, may be formulated in a single composition or formulated separately in two or more compositions (e.g., a kit), e.g., for simultaneous delivery. Each component may be administered to a subject at a different time than when the other component is administered, e.g., each administration may be non-concurrent (e.g., separately or sequentially), spaced apart over a given period of time. Furthermore, the separate components may be administered to a subject by the same route or by different routes (e.g., an anti-CoV-S antibody or antigen-binding fragment thereof).

[0180] kit Further provided are kits comprising one or more components, including but not limited to, an anti-CoV-S antigen binding protein, e.g., an antibody or antigen-binding fragment, as discussed herein (e.g., of Table 4), in association with one or more additional components, including but not limited to, an additional therapeutic agent as discussed herein. The antigen binding protein and / or the additional therapeutic agent may be formulated as a single composition or separately in two or more compositions, e.g., together with a pharma- ceutically acceptable carrier in a pharmaceutical composition.

[0181] In one embodiment of the invention, the kit comprises an anti-CoV-S antigen binding protein of the invention (e.g., of Table 4), e.g., an antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, in one container (e.g., in a sterile glass or plastic vial) and an additional therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0182] In another embodiment, the kit comprises in a single common container a combination of the invention comprising an anti-CoV-S antigen binding protein of the invention (e.g., of Table 4), e.g., an antibody or antigen-binding fragment thereof, or a pharmaceutical composition thereof, optionally in combination with one or more additional therapeutic agents co-formulated in a pharmaceutical composition.

[0183] Where the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration (e.g., an injection device). For example, the kit can include one or more hypodermic needles or other injection devices discussed herein that include an anti-CoV-S antigen binding protein of the invention (e.g., of Table 4), e.g., an antibody or antigen-binding fragment thereof.

[0184] The kit may include a package insert containing information regarding the pharmaceutical compositions and dosage forms in the kit. Generally, such information assists patients and physicians in effectively and safely using the enclosed pharmaceutical compositions and dosage forms. For example, the following information regarding the combination of the present invention may be provided in the package insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, cautions, side effects, overdosage, proper dosage and administration, method of delivery, suitable storage conditions, references, manufacturer / distributor information, and patent information.

[0185] Diagnostic Uses of Antibodies Anti-CoV-S antigen binding proteins of the invention (e.g., of Table 4), e.g., antibodies or antigen-binding fragments thereof, can be used to detect and / or measure CoV-S in a sample. An exemplary assay for CoV-S can include, for example, contacting a sample with an anti-CoV-S antigen binding protein of the invention, which is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate CoV-S from the sample. The presence of the anti-CoV-S antigen binding protein complexed with CoV-S indicates the presence of CoV-S in the sample. Alternatively, an unlabeled anti-CoV-S antibody can be used in combination with a secondary antibody that is itself detectably labeled. The detectable label or reporter molecule can be, for example, 3 H, 14 C. 32 P, 35 S, or 125 The antigen binding protein may be a radioisotope such as I, a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate or rhodamine, or an enzyme such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure CoV-S in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS). Thus, the present invention includes a method for detecting the presence of spike protein polypeptide in a sample, comprising contacting the sample with an anti-CoV-S antigen binding protein and detecting the presence of CoV-S / anti-CoV-S antigen binding protein, the presence of a complex indicating the presence of CoV-S.

[0186] The anti-CoV-S antigen binding proteins of the invention (e.g., of Table 4) may be used in Western blot or immunoprotein blot procedures to detect the presence of CoV-S or a fragment thereof in a sample. Such methods form part of the invention and include, for example, the following steps: For example, providing a membrane or other solid substrate comprising a sample to be tested for the presence of CoV-S, optionally including transferring proteins from the sample to be tested for the presence of CoV-S (e.g. from PAGE or SDS-PAGE electrophoretic separation of proteins in the sample) onto the membrane or other solid substrate using methods known in the art (e.g. semi-dry blotting or tank blotting), and contacting the membrane or other solid substrate to be tested for the presence of CoV-S or a fragment thereof with an anti-CoV-S antigen binding protein of the invention.

[0187] Such membranes may take the form of, for example, nitrocellulose or vinyl-based (e.g., polyvinylidene fluoride (PVDF)) membranes onto which proteins to be tested for the presence of CoV-S have been transferred (e.g., after electrophoretic separation in a gel) in a non-denaturing PAGE (polyacrylamide gel electrophoresis) gel or SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gel. Prior to contacting the membrane with the anti-CoV-S antigen-binding protein, the membrane is optionally blocked, for example, with non-fat dry milk or equivalent, to bind to non-specific protein binding sites on the membrane. (2) washing the membrane one or more times to remove unbound anti-CoV-S antigen-binding protein and other unbound material; and (3) Detecting bound anti-CoV-S antigen-binding proteins.

[0188] Detection of bound antigen-binding protein indicates that CoV-S protein is present on the membrane or substrate and in the sample. Detection of bound antigen-binding protein can be by binding the antigen-binding protein with a directly labeled secondary antibody (anti-immunoglobulin antibody) and then detecting the presence of the secondary antibody label.

[0189] The anti-CoV-S antigen binding proteins disclosed herein (e.g., antibodies and antigen-binding fragments (e.g., of Table 4)) can also be used for immunohistochemistry. Such methods form part of the invention and include, for example: contacting the tissue to be tested for the presence of CoV-S proteins with an anti-CoV-S antigen-binding protein of the invention; and Detecting antigen-binding proteins on or in tissues.

[0190] If the antigen binding protein itself is detectably labeled, it can be directly detected. Alternatively, the antigen binding protein can be bound by a directly labeled secondary antibody, and the label then detected. EXAMPLES

[0191] The following examples are presented to provide those skilled in the art with a complete disclosure and description of how to make and use the disclosed methods and compositions, and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, room temperature is about 25°C, and pressure is at or near atmospheric pressure.

[0192] Example 1: Generation of human antibodies against the SARS-CoV-2 spike protein (SARS-CoV-2-S) Human antibodies against SARS-CoV-2-spike protein (SARS-CoV-2-S) were generated in VELOCIMMUNE® mice containing DNA encoding human immunoglobulin heavy and kappa light chain variable regions, or human immunoglobulin heavy and lambda light chain variable regions. Each mouse was immunized with a vector expressing the SARS-CoV-2-S receptor binding domain (RBD) (amino acids 1-1273 of NCBI Accession No. (MN908947.3), SEQ ID NO: 1008) followed by a booster with the SARS-CoV-2-S vector or SARS-CoV-2-S protein. Antibody immune responses were monitored by SARS-CoV-2-S specific immune assays. Anti-SARS-CoV-2-S antibodies were isolated directly from antigen-positive mouse B cells without fusion with myeloma cells, as described in U.S. Pat. No. 7,582,298, expressly incorporated herein by reference in its entirety. Using this method, fully human anti-SARS-CoV-2-S antibodies (i.e., antibodies bearing human variable and human constant domains) were obtained.

[0193] Antibody variable regions were also isolated from human blood samples. Whole blood was received from patients 6–8 weeks after a laboratory-confirmed PCR-positive test for SARS-CoV-2 and symptomatic COVID-19 disease. Ammonium chloride-based lysis buffer (Life Technologies) was used to lyse red blood cells and enrich for B cells by negative selection. Single B cells that bound the SARS-CoV-2 spike protein were isolated by fluorescence-activated cell sorting (FACS). Isolated B cells were plated in single wells and mixed with antibody light and heavy chain variable region-specific PCR primers. cDNA for each single B cell was synthesized via a reverse transcriptase (RT) reaction. Each resulting RT product was then split and transferred into 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 chain variable region leader sequence, or a 5' degenerate primer specific to the antibody light chain variable region leader sequence and mouse heavy chain constant region, and a 3' primer specific to the antibody constant region to form an amplicon. The amplicon was then amplified again by PCR using a 5' degenerate primer specific to the antibody heavy chain variable region framework 1, or a 5' degenerate primer specific to the antibody light chain variable region framework 1, and a 3' primer specific to the antibody constant region to generate an amplicon for cloning. The PCR products from the antibody heavy and light chains were cloned into an expression vector containing the heavy and light chain constant regions, respectively, thereby producing an expression vector of a hybrid antibody. The expression vector expressing the full-length heavy and light chain pairs was transfected into CHO cells to produce the antibody protein for testing.

[0194] The biological properties of exemplary antibodies generated according to the methods of this example are described in detail in the Examples set forth below.

[0195] Example 2: Heavy and light chain variable region amino acid and nucleotide sequences Table 4 lists the amino acid sequence identifiers for the heavy and light chain variable regions and CDRs, and the heavy and light chain sequences of exemplary anti-SARS-CoV-2-S antibodies. The corresponding nucleic acid sequence identifiers are listed in Table 5. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

[0196] The antibodies disclosed herein have fully human variable regions, but can have mouse constant regions (e.g., mouse IgG1 Fc or mouse IgG2 Fc (a or b isotypes)), or human constant regions (e.g., human IgG1 Fc or human IgG4 Fc). As will be appreciated by one of skill in the art, an antibody with a particular Fc isotype may be converted to an antibody with a different Fc isotype (e.g., an antibody with a mouse IgG1 Fc may be converted to an antibody with a human IgG4 Fc, etc.), but in either case, the variable domains (including the CDRs) indicated by the numerical identifiers shown in Tables 4 and 5 will remain the same, and the binding characteristics to the antigen are expected to be the same or substantially similar regardless of the nature of the constant domains.

[0197] As described above, antibodies were obtained by direct isolation from antigen-positive VELOCIMMUNE® mouse B cells or derived from variable regions cloned from antigen-positive human B cells. A summary of these sources is shown in Table 6. [Table 6-1] [Table 6-2]

[0198] Example 3: Biacore binding kinetics of purified anti-SARS-CoV-2-S monoclonal antibodies A real-time surface plasmon resonance-based T200 / Biacore 8K biosensor was used to determine the equilibrium dissociation constants (K ) of different SARS-COV-2 RBD reagents binding to purified CHOt anti-SARS-COV-2 monoclonal antibodies (mAbs). D ) was determined. All binding studies were performed at 25°C and 37°C in a running buffer of 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, and 0.05% v / v surfactant Tween-20, pH 7.4 (HBS-ET). A Biacore CM5 sensor chip surface was first derivatized by amine coupling with a mouse anti-human Fc specific mAb (Regeneron, mAb2567) to capture the anti-SARS-COV-2 mAb. Binding studies were performed with the human SARS-COV-2 RBD ectodomain expressed with a C-terminal myc-myc-hexahistidine tag (SARS-COV-2 RBD-mmH) (monomeric RBD) (SEQ ID NO: 1069), the SARS-COV-2 RBD ectodomain expressed with a C-terminal mouse IgG2a Fc (SARS-COV-2 RBD mFc) (dimeric RBD) (SEQ ID NO: 1070), and the SARS-CoV2 spike ecto foldon trimer expressed with a C-terminal myc-myc-hexahistidine (SARS-CoV2 spike ECD foldon) (trimeric RBD). The use of these reagents allowed for testing the ability of antibodies to bind to monomeric, dimeric, and trimeric RBD peptides, respectively.

[0199] Three concentrations, 50 nM, 12.5 nM, and 3.12 nM, of hSARS-COV-2 RBD-mmH, SARS-COV-2 RBD mFc, and SARS-CoV2 spike ECD Foldon prepared in HBS-ET running buffer were injected for 1.5–3 min at a flow rate of 50 μL / min, while the dissociation of the different SARS-COV-2 RBD reagents bound by mAb was monitored for 5–8 min in HBS-ET running buffer. At the end of each cycle, the SARS-COV-2 RBD mAb capture surface was regenerated using a 12 s injection of 20 mM phosphate onto the mouse anti-human Fc specific mAb surface. Association rates (k) were calculated by fitting the real-time binding sensorgrams to a 1:1 binding model with mass transport limitations using BiaEvaluation software v3.1, or Biacore Insight Evaluation software v2.0. or curve fitting software. a ) and dissociation rate (k d The binding / dissociation equilibrium constant (K D ) and dissociation half-life (t1 / 2) were calculated from the kinetic rates as follows:

number

[0200] The binding kinetic parameters of different anti-SARS-COV-2 mAbs binding to the monomeric, dimeric, and trimeric SARS-COV-2 RBD of the present invention at 25° C. and 37° C., respectively, are shown in Tables 7 to 12. The isotype control mAb1932 was also used as a control. [Table 7-1] [Table 7-2] [Table 7-3] NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions. ≦1.00E-05 indicates that no dissociation is observed under the current experimental conditions, and k d Values ​​were 1.00E-05s while fitting real-time binding sensorgrams -1 Indicates that the position has been manually fixed. [Table 8-1] [Table 8-2] NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions. ≦1.00E-05 indicates that no dissociation is observed under the current experimental conditions, and k d Values ​​were 1.00E-05s while fitting real-time binding sensorgrams -1 Indicates that the position has been manually fixed. [Table 9-1] [Table 9-2] [Table 9-3] NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions. [Table 10-1] [Table 10-2] [Table 10-3] NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions. [Table 11-1] [Table 11-2] NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions. [Table 12-1] [Table 12-2] [Table 12-3] hBST: Human B cell sorting technology NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions.

[0201] Example 4: Neutralization of SARS-CoV-2 wild-type and variant spike proteins To test whether anti-SARS-CoV-2 spike protein antibodies could neutralize SARS-CoV-2 variants, these antibodies were screened against a panel of VSV pseudotyped viruses expressing wild-type and variant spike proteins.

[0202] Generation of recombinant VSV Non-replicating pseudoparticles were generated using a VSV genome encoding the firefly luciferase gene and the GFP gene in place of the native viral glycoprotein (VSV-G). Infectious particles complemented with VSV-G (VSV-ΔG-Fluc-2A-GFP / VSV-G) were harvested and produced using standard techniques with minor modifications. HEK293T cells (ATCC CRL-3216) were plated on polylysine-treated plates and incubated overnight in glutamine-free DMEM (Life Technologies), 10% fetal bovine serum (Life Technologies), and 1% penicillin / streptomycin / L-glutamine (Life Technologies). The next day, cells were transfected with VSV genome clones driven by the T7 promoter and helper plasmids expressing VSV-N, VSV-P, VSV-G, VSV-L, and T7 RNA polymerase using Lipofectamine LTX reagent (Life Technologies). After 48 hours, the transfected cells were co-cultured with BHK-21 cells (ATCC CCL-10) transfected with VSV-G using the SE cell line 4D-Nucleofector X kit L (Lonza) in glutamine-free DMEM (Life Technologies), 3% fetal bovine serum (Life Technologies), and 1% penicillin / streptomycin / L-glutamine (Life Technologies). Cells were monitored for GFP expression or cytopathic effect (CPE), indicative of viral replication. Virus was then plaque purified, propagated, and titered in BHK-21 cells transiently expressing VSV-G. Fully replicating VSV-SARS-CoV-2-S virus was generated by replacing the VSV glycoprotein with the native SARS-CoV-2 sequence encoding residues 1 to 1255 of the spike protein (NCBI accession number MN908947.3). VSV-SARS-CoV-2-spike virus was harvested as described above, but instead, HEK293T cells were co-cultured with BHK-21 cells transfected with both VSV-G and hACE2.VSV-SARS-CoV-2-S virus was plaque purified, titered in Vero cells (ATCC CCL-81) and propagated in Vero E6 cells (ATCC CRL-1586). After harvest, both virus stocks were clarified by centrifugation at 3000xg for 5 min, concentrated 10-fold in sucrose buffer, aliquoted, and frozen at -80°C.

[0203] VSV pseudotyping Non-replicating pseudoparticles were generated as previously described (Baum et al., Science 2020). Human codon-optimized SARS-CoV-2 spike (NCBI accession number MN908947.3) was cloned into an expression plasmid. A total of 1.2 × 107 HEK293T cells (ATCC CRL-3216) were seeded overnight in 15 cm dishes in glutamine-free DMEM (Life Technologies) and 10% heat-inactivated fetal bovine serum (Life Technologies), as well as penicillin-streptomycin-L-glutamine (Life Technologies). The next day, cells were transfected with 15 μg of spike expression plasmid with Lipofectamine LTX (Life Technologies) according to the manufacturer's protocol. 24 hours after transfection, cells were washed with phosphate-buffered saline (PBS) and infected with VSV-ΔG-Fluc-2A-GFP / VSV-G virus diluted in 10 mL of Opti-MEM (Life Technologies) at an MOI of 1. Cells were incubated for 1 hour at 37°C with 5% CO2. Cells were washed three times with PBS to remove residual input virus and overlaid with glutamine-containing DMEM (Life Technologies) containing 0.7% IgG-free BSA (Sigma), sodium pyruvate (Life Technologies), and gentamicin (Life Technologies). After 24 hours at 37°C with 5% CO2, the supernatant containing the pseudoparticles was collected, clarified by centrifugation at 3000xg for 5 minutes, aliquoted, and frozen at -80°C. Using site-directed mutagenesis, variants were cloned into the spike expression plasmid and pseudoparticles were produced as described above. Neutralization assay using VSV-based pseudoparticles.

[0204] Vero cells (ATCC:CCL-81) were seeded in 96-well black clear bottom tissue culture dishes (Corning:3904) at 20,000 cells / well in glutamine-free DMEM medium (Life Technologies) containing 10% heat-inactivated fetal bovine serum (Life Technologies) and 1X penicillin / streptomycin / L-glutamine (Life Technologies) 24 hours prior to the assay. Cells were allowed to reach approximately 85% confluence before use in the assay. Antibodies were diluted to 2X assay concentration in infection medium containing glutamine-containing DMEM (Life Technologies), 0.7% low IgG BSA (Sigma), 1X sodium pyruvate (Life Technologies), and 0.5% gentamicin (Life Technologies) and diluted 3-fold in infection medium for an 11-point dilution curve in the assay starting at 3 μg / mL (20 nM). Antibody dilutions were mixed 1:1 with pseudoparticles for 30 minutes at room temperature before being added onto the Vero cells. Cells were incubated at 37°C, 5% CO2 for 24 hours. Supernatant was removed from cells prior to lysis with 100 μL of Glo lysis buffer (Promega). 100 μL of resuspended Bright Glo substrate (Promega) was then added and luminescence was read on a Spectramax i3x (Molecular Devices). Exported values ​​were analyzed using GraphPad Prism (v8.4.1).

[0205] The half-maximal inhibitory concentration (IC50) of each monoclonal antibody against VSV-SARS-CoV-2 spike protein (S)-expressing pseudoviruses encoding the Wuhan-Hu-1 (NCBI Accession No. MN908947.3) sequence of the spike protein (S-wt) or the D614G spike protein variant (SEQ ID NO: 1071) was determined in Vero cells (Table 13). The majority of the antibodies showed neutralizing potency in the picomolar range (pM), some in the nanomolar (nM) range, and some were non-neutralizing. In addition, IC50s were measured for individual monoclonal antibodies tested against certain variants of concern / notable VSV-SARS-CoV-2 spike protein (S)-expressing pseudoviruses (Table 14), including Omicron variants (individual mutations in Omicron in Table 15, the complete set of Omicron mutations in Table 16, and comparison with and combinations with mAb10933 and mAb10987 in Table 17). In the Omicron neutralization assay, mAb14315 had slightly reduced neutralization, while mAb15160 was unaffected. Based on these results, the combinations mAb14315 / mAb15160 and mAb15160 / mAb14256 maintain effective neutralization of Omicron variants. In addition, mAb14284, mAb14235, and mAb14287 were potent neutralizers and were unaffected by omicron variants, while mAb15151 had a half-log decrease in neutralization of omicron variants, yet maintained potency based on its potent neutralizing properties. As shown in the table, the lower the fold change from wt or D614G, the less impact the omicron mutation has on neutralization. Additional mAb15160 and mAb14284 neutralization data are presented in Tables 18 and 19, showing that these antibodies are potent neutralizers of different variants, including the omicron lineage. [Table 13-1] [Table 13-2] The underlined IC50 values ​​were determined using the D614G spike protein. [Table 14] The complete B.1.1.7 (H69del, V70del, Y145del, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), B.1.351 (D80Y, D215Y, L241del, L242del, A243del, L242del, K417N, E484K, N501Y, D614G, A701V), and P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F) variants were evaluated. Key RBD residues from the B.1.429 (L452R) and B.1.526 (E484K) lineages were evaluated. Fold reduction in potency (FC) was calculated relative to wt or D614G controls from the same assay. NA: No activity ND: Not decided [Table 15] Italics: IC50 values ​​of each antibody against wt or D614G control spike protein Fold reduction in potency (FC) was calculated relative to wt or D614G controls from the same assay. ND: Not decided [Table 16] [Table 17] [Table 18] [Table 19] The omicron spike protein used contains SEQ ID NO: 1073, the following mutations: A67V, Δ69-70, T95I, G142D / Δ143-145, Δ211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F. NC: No activity, IC50 not calculated FC: fold change calculated relative to D614G control Bold and underlined values: fold change is at least this value calculated as highest assay concentration / D614G IC50, the highest concentration in the assay was 2.00E-08. [Table 20] a Amino acid substitutions versus Wuhan lineage S protein: B.1.1.529 / BA.1 (A67V, del69-70, T95I, G142D / del143-145, del211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F) b IC generated by antibodies in the presence of specific variants 50 Values ​​are expressed as the IC generated by the antibody in the presence of a reference virus from the same assay. 50 The fold reduction relative to the reference (ref) D614G virus was calculated by dividing by the value. c I C 50In cases where it was not possible to calculate the value accurately, the highest antibody concentration tested (20 nM) was calculated using the IC 50 The change in mAb potency in the presence of each mutant was calculated by dividing by the value. The actual fold change is at least the change in the value shown. Abbreviations: NC, not calculated due to poor or lack of neutralization [Table 21-1] [Table 21-2] [Table 21-3] [Table 21-4] [Table 21-5] aComplete sequences and / or key residues of the S protein from the variants under surveillance; Complete S protein sequences: B.1.1.7 (H69del, V70del, Y145del, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), B.1.351 (D80Y, D215G, L241del, L242del, A243del, K417N, E484K, N501Y, D614G, A701V), P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G , H655Y, T1027I, V1176F), B.1.617.2(T19R, G142D, E156G, F157del, R158d el, L452R, T478K, D614G, P681R, D950N), B.1.617.1(T95I, G142D, E154K, L 452R, E484Q, D614G, P681R, Q1071H), BA.1(A67V, del69-70, T95I, G142D / del143-145, del211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T5 47K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981 F), BA.1.1(BA.1+R346K), BA.2(T19I, del24-26, A27S, G142D, V213G, G339 D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E 484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K; major S protein residues: AY.1 (K417N+L452R+T478K), AY4.2 (Y145H+A222V+L452R+T478K), B.1.427 / B.1.429 (L452R), B.1.526 (E484K), B.1.621 (R346K+E484K+N501Y), AV.1 (N439K+E484K), B.1.619 / B.1.619.1 / B.1.625(N440K+E484K), C.1.2(Y449H+E484K+N501Y). . b I C 50 In cases where it was not possible to calculate values ​​accurately, the highest antibody concentration tested (20 nM) was used as the calculated IC for casirivimab, imdevimab, or casirivimab + imdevimab in the presence of reference pVSV-SARS-CoV-2-S pseudoparticles. 50 The change in mAb potency in the presence of each mutant was calculated by dividing by the value. c IC of neutralization of this variant by mAb15160 and imdevimab 50 and IC 90 Values ​​were compared to different reference viral datasets for casirivimab and casirivimab + imdevimab. del: deletion, ins: insertion, NA: not applicable, NC: not calculated due to poor or absent neutralization, NT: not tested Fold change calculated relative to D614G control [Table 22-1] [Table 22-2] [Table 22-3] *Values ​​shown for the indicated variant / reference virus represent the geometric mean from at least three replicate assays. aThe complete S protein sequences and / or key residues of the variants under surveillance were evaluated. The key residues evaluated and the lineage they represent are shown in the table. The complete sequences are indicated by footnote "a" in the table and contain the following substitutions in the Wuhan-Hu-1 S protein reference sequence: B.1.1.7 (H69del, V70del, Y145del, N501Y, A570D, D614G, P681H, T716I, S982A, D1118H), B.1.351 (D80Y, D215G, L241del, L242del, A243del, K417N, E484K, N501Y, D614G, A701V), P.1 (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V1176F), B.1.617.2(T19R, G142D, E156G, F157del, R158del, L452R, T478K, D614G, P68 1R, D950N), B.1.617.1(T95I, G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H), BA.1(A67V, del69-70, T95I, G142D / del1 43-145, del211 / L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q 498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F), BA.1.1(BA.1+R346K), BA.2(T19I, del24-26, A27S, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E 484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K), BA.2.12.1(BA.2+L452Q), BA.3 (G142D, G339D, S371F, S373P, S375F, D405N, K417N, D614G, H655Y, N679K, P681H, D796Y, Q954H, N969K), BA.4 / BA.5 (both lineages have identical S protein sequences [T19I, L24del, P25del, P26del, A27S, H69del, V70del, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478 K, E484A, F486V, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K]). b I C 50 In cases where it was not possible to calculate the value accurately, the highest antibody concentration tested (20 nM) was taken as the IC calculated for the antibody in the presence of reference pVSV-SARS-CoV-2-S pseudoparticles. 50 The change in mAb potency in the presence of each mutant was calculated by dividing by the value. del: deletion, ins: insertion, NC: not calculated due to poor or absent neutralization mAb14284 was tested at concentrations ranging from approximately 300 fM to 20 nM. IC determined for antibody in the presence of specific variants 50 Values ​​are the IC determined for the antibodies in the presence of reference pseudoparticles (pVSV-SARS-CoV-2 pseudotyped with the D614G mutation) from the same assay. 50 Fold changes compared to the reference (reference) virus were calculated by dividing the values.

[0206] Example 5: Anti-SARS-CoV-2 antibodies block RBD binding to hACE2 as determined by ELISA An ELISA-based blocking assay was used to determine the ability of anti-SARS-CoV-2 antibodies to block the binding of the SARS-COV-2 spike protein receptor-binding domain (RBD) to its receptor, human angiotensin-converting enzyme 2 (hACE2).

[0207] The SARS-CoV-2 protein used in this assay consisted of the receptor binding domain (RBD) portion of the SARS-CoV-2 spike protein (amino acids Arg319-Phe541) expressed with a C-terminal myc-myc-6-histidine tag (SARS-CoV-2 RBD.mmh). The human ACE-2 protein used in the experiments, containing a portion of the human ACE-2 extracellular domain (amino acids Leu18-Ser740) and a C-terminal 10-histidine tag (hACE-2-10His, ACE2 NCBI accession number Q9BYF1), was purchased from R&D Systems.

[0208] The experiment was carried out using the following procedure: hACE-2-10His receptor was coated at 2 μg / ml in PBS on 96-well microtiter plates overnight at 4 °C. A 0.5% (w / v) solution of BSA in PBS was used to subsequently block non-specific binding sites. In other microtiter plates, a constant amount of 300 pM of SARS-CoV-2 RBD.mmh protein was combined for 1 h with anti-SARS-COV-2-S antibodies or an irrelevant human IgG1 antibody control at dilutions ranging from 1.7 pM to 100 nM in PBS + 0.5% BSA. A fixed concentration of SARS-CoV-2 RBD was chosen to be close to the concentration that produces 50% of maximum binding (EC50 value) to plate-adhered hACE-2. The antibody-protein complex was transferred to the hACE-2-10His-coated microtiter plate. After 1 hour incubation at room temperature, wells were washed and plate-bound SARS-COV-2 RBD.mmh was detected using a polyclonal goat anti-myc antibody conjugated with horseradish peroxidase (HRP) (NovusBio). Plates were then developed using TMB substrate solution (BD Biosciences, no. 51-2606KC and no. 51-2607KC) according to the manufacturer's recommended procedure, and absorbance at 450 nm was measured on a Victor X5 plate reader (PerkinElmer, Shelton CT).

[0209] Binding data was analyzed using a sigmoidal dose-response model within Prism™ software (GraphPad). The calculated IC50 value, defined as the concentration of antibody required to block 50% of SARS-CoV-2 RBD binding to plate-coated hACE2, was used as an indicator of blocking potency. The blocking rate of anti-SARS-CoV-2-S antibodies at a given concentration was calculated based on the following formula:

number

[0210] Antibodies that blocked binding by 50% or less at the highest concentration tested were classified as non-blocking agents and no IC50 values ​​were reported for those antibodies.

[0211] A blocking ELISA was used to evaluate the ability of anti-SARS-CoV-2 antibodies to block SARS-CoV-2 RBD binding to human ACE2. In this assay, 300 pM of SARS-COV-2 RBD.mmh was titrated with a broad range of concentrations of anti-SARS-COV-2-S antibodies. Inhibition of SARS-COV-2 RBD binding to hACE-2 in the presence of SARS-COV-2 antibodies was evaluated. Plate-bound SARS-COV-2 RBD.mmh was detected with an HRP-conjugated goat anti-myc antibody. The IC50 and maximum blockade at the highest tested concentration of anti-SARS-COV-2 antibodies are summarized in Table 20.

[0212] Of the 56 antibodies tested, 42 demonstrated antibody concentration-dependent blocking of SARS-COV-2 RBD binding to hACE-2, with maximal blockage ranging from 61% to approximately 100% at the highest antibody concentration tested (100 nM). IC50 values ​​of the identified blocking antibodies ranged from 102 pM to 19.7 nM. The remaining 14 antibodies showed less than 50% blocking activity at the highest concentration tested and were classified as non-blockers. An isotype control mAb 193281 was also used as a control. [Table 23-1] [Table 23-2] Nbl: Non-blocking-blocking% is less than 50%.

[0213] Example 6: pH Sensitivity of Anti-SARS-CoV-2-S Monoclonal Antibody Binding to Monomeric SARS-CoV-2-S RBD Reagent Measured at 37°C The dissociation rate constants (k) of 56 purified anti-SARS-CoV-2-S monoclonal antibodies at neutral and acidic pH conditions were determined using real-time surface plasmon resonance (SPR)-based Biacore T200 or Biacore 4000 biosensors. d ) was determined. All binding studies were performed at 37 °C using running buffers of PBS containing 0.05% v / v detergent Tween-20 at pH 7.4, pH 6.0, and pH 5.0 (PBS-T-pH 7.4, -pH 6.0, and -pH 5.0, respectively). The Biacore CM5 sensor chip surface was first derivatized by amine coupling with a mouse anti-human Fc specific mAb (Regeneron, mAb2567) to capture anti-SARS-CoV-2 monoclonal antibodies. The ligand investigated for binding to anti-SARS-CoV-2 monoclonal antibodies was a recombinant protein containing the SARS-COV-2 RBD extracellular domain expressed with a myc-myc-hexahistidine C-terminal tag (SARS-COV-2 RBD-MMH, SEQ ID NO: 1069). A solution of 90 nM SARS-COV-2 RBD-MMH was prepared in PBS-T-pH 7.4 buffer and then injected over the antibody surface for 3 min at a flow rate of 25 μL / min, followed by a dissociation phase of bound SARS-COV-2 RBD-MMH using running buffer at pH 7.4, pH 6.0, or pH 5.0 for 8 min. Isotype control mAb1932 was also used as a control.

[0214] Dissociation rate constants (k) in the three pH running buffers were determined by fitting the real-time binding sensorgrams to a 1:1 binding model using Scrubber 2.0c curve fitting software.d The dissociation half-life (t1 / 2) was determined as follows: k d Calculated from the values:

number

[0215] Resulting k for binding of monomeric SARS-COV-2 RBD-MMH to 56 anti-SARS-CoV-2 monoclonal antibodies captured on anti-hFc surfaces at pH 6.0 and pH 5.0 at 37°C. d and t1 / 2 values ​​are summarized in Tables 21 and 22, respectively, and results at pH 7.4 are listed in both tables for comparison. [Table 24-1] [Table 24-2] [Table 24-3] NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions. [Table 25-1] [Table 25-2] [Table 25-3] NB: No binding was observed under the current experimental conditions (NB). IC: The observed binding did not fit a binding simulation model, and no binding kinetic parameters were determined under the current experimental conditions.

[0216] Example 7: Cross-competition between anti-SARS-CoV-2-S antibodies Binding competition between 41 SARS-CoV-2-S monoclonal antibodies (mAbs) was determined using a real-time label-free biolayer interferometry (BLI) assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was performed at 25°C in a pH 7.4 buffer solution (HBS-EBT) containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant Tween-20, and 1 mg / mL BSA, with shaking of the sample plate at a speed of 1000 rpm. To assess whether the mAbs compete with each other for binding to their respective epitopes on the SARS-COV-2 RBD extracellular domain expressed with a C-terminal myc-myc-hexahistidine (SARS-COV-2 RBD-MMH, SEQ ID NO: 1069) tag, approximately 0.52-0.63 nm of SARS-COV-2 RBD-MMH was first captured on an Octet biosensor tip (Fortebio Inc, no. 18-5122) coated with an anti-penta-His antibody by immersing the biosensor tip in a well containing a 10 μg / mL solution of SARS-COV-2 RBD-MMH for 90 seconds. The biosensor tip with the captured SARS-COV-2 RBD-MMH was then saturated with the first anti-SARS-CoV-2 monoclonal antibody (mAb-1) by immersing the tip in a well containing a 50 μg / mL solution of mAb-1 for 4 minutes. The tip was then immersed in a well containing 50 μg / mL of a second anti-SARS-CoV-2 monoclonal antibody (mAb-2) for 4 minutes. Between steps, the tip was rinsed with HBS-ETB. Real-time binding responses were monitored during the entire course of the experiment, and binding responses were recorded at the end of all steps. The response of mAb-2 binding to SARS-COV-2 RBD-MMH pre-complexed with mAb-1 was compared to the mAb-2 binding signal where mAb-1 was a non-binding isotype control, and the percent reduction in mAb-2 binding was calculated. mAb-1 and mAb-2 were classified as competing mAbs if pre-binding of mAb-1 resulted in a >50% reduction in mAb-2 binding and reversing the order of binding of the mAb pair also showed a >50% reduction in binding.Figure 1 displays the results from the cross-competition study of anti-SARS-CoV-2 mAbs in a matrix format. Table 23 lists the competing mAbs for each anti-SARS-CoV-2 mAb included in the experiment. [Table 26-1] [Table 26-2] [Table 26-3] [Table 26-4] [Table 26-5] [Table 26-6] [Table 26-7] [Table 26-8] [Table 26-9] [Table 26-10]

[0217] Binding competition between 15 additional anti-SARS-Cov-2-S monoclonal antibodies was performed substantially as described above. Competition was determined using a real-time label-free biolayer interferometry (BLI) assay on an Octet HTX biosensor platform (Pall ForteBio Corp.). The entire experiment was carried out at 25°C in a pH 7.4 buffer (HBS-EBT) containing 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% v / v surfactant Tween-20, and 1 mg / mL BSA, with the plate shaking at a speed of 1000 rpm. To assess whether the mAbs compete with each other for binding to their respective epitopes on the SARS-COV-2 RBD extracellular domain expressed with a C-terminal myc-myc-hexahistidine (SARS-COV-2 RBD-MMH, SEQ ID NO: 1069) tag, approximately 0.51 nm of SARS-COV-2 RBD-MMH was first captured on an Octet biosensor tip (Fortebio Inc, no. 18-5122) coated with an anti-penta-His antibody by immersing the biosensor tip in a well containing 10 μg / mL of SARS-COV-2 RBD-MMH for 90 seconds. The biosensor tip with the captured SARS-COV-2 RBD-MMH was then saturated with a first anti-SARS-CoV-2 monoclonal antibody (mAb-1) by immersing the tip in a well containing 50 μg / mL of mAb-1 solution for 4 minutes. The biosensor tip was then immersed in a well containing 50 μg / mL of a second anti-SARS-CoV-2 monoclonal antibody (mAb-2) for 4 minutes. Between steps, the biosensor tip was rinsed with HBS-ETB buffer. Real-time binding responses were monitored during the entire course of the experiment, and binding responses were recorded at the end of all steps. The response of mAb-2 binding to SARS-COV-2 RBD-MMH precomplexed with mAb-1 was compared to the mAb-2 binding signal where mAb-1 was a non-binding isotype control, and the percent reduction in mAb-2 binding was calculated. mAb-1 and mAb-2 were classified as competing mAbs if pre-binding of mAb-1 resulted in a >50% reduction in mAb-2 binding and reversing the order of binding of the mAb pair also showed a >50% reduction in binding.Figure 2 displays the results from the cross-competition study of anti-SARS-CoV-2 mAbs in a matrix format. Table 24 lists the competing mAbs for each anti-SARS-CoV-2 mAb included in the experiment. [Table 27-1] [Table 27-2] [Table 27-3] [Table 27-4] [Table 27-5] [Table 27-6] [Table 27-7] [Table 27-8] [Table 27-9] [Table 27-10] [Table 27-11] [Table 27-12] [Table 27-13] [Table 27-14] [Table 27-15] [Table 27-16] [Table 27-17] [Table 27-18] [Table 27-19] [Table 27-20] [Table 27-21] [Table 27-22] [Table 27-23] [Table 27-24] [Table 27-25]

[0218] Example 8: Characterization of anti-SARS-CoV-2-S mAbs in an ADCC surrogate assay (FcγR3a Val176 signaling assay) The ability of antibodies targeting the spike protein of SARS-CoV-2, which interacts with FcγR3a, an Fc receptor prominently expressed on NK cells that induces antibody-dependent cell-mediated cytotoxicity (ADCC), was measured in a surrogate bioassay using reporter cells and target cells bound to the antibody. In this assay, engineered Jurkat T cells expressed the reporter gene luciferase under the control of the transcription factor NFAT (NFAT-Luc) along with the high affinity human FcγR3a 176Val allotypic receptor (Jurkat / NFAT-Luc / hFcγR3a 176Val). Target cells were engineered Jurkat T cells expressing human CD20 (used as a positive control with a CD20 targeting human IgG1 antibody) alone or in combination with the full-length SARS-CoV-2 spike protein. Reporter cells were incubated with target cells, and engagement of FcγR3a via the Fc domain of human IgG1 antibodies bound to the target cells led to activation of the transcription factor NFAT in the reporter cells, driving expression of luciferase, which was then measured via a luminescent readout.

[0219] target cell Jurkat / hCD20: Jurkat T cells were engineered to constitutively express full-length human CD20 (amino acids M1-P297 of NCBI accession number NP_690605.1). Jurkat / hCD20 cells were stained for CD20 expression and maintained in RPMI+10%FBS+P / S / G+250g / ml hygromycin growth medium. These cells were used as negative controls.

[0220] Jurkat / hCD20 / SARS-CoV-2 spike: Jurkat / hCD20 T cells were engineered to constitutively express the full-length SARS-CoV-2 spike protein (amino acids M1-T1273 of NCBI accession number YP_009724390.1). Jurkat / hCD20 / SARS-CoV-2 spike cells were selected for high expression of spike protein and maintained in RPMI+10%FBS+P / S / G+1μg / ml puromycin+250μg / ml hygromycin growth medium.

[0221] Reporter T cells Jurkat / NFAT-Luc / FcγR3a 176Val: Jurkat T cells were engineered to stably express a nuclear factor of activated T cells (NFAT) luciferase reporter construct along with the high affinity human FcγR3a 176Val allotype receptor (amino acids M1-K254 of accession number P08637 VAR_003960). Engineered reporter cells were maintained in RPMI1640+10%FBS+P / S / G+0.5μg / ml puromycin+500μg / ml G418 growth medium.

[0222] Assay setup One day before the experiment, Jurkat reporter cells were cultured at 7.5 × 10 in RPMI1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 500 μg / ml G418 growth medium. 5 Jurkat target cells were split at 5 × 10 cells / ml in RPMI 1640 + 10% FBS + P / S / G + 0.5 μg / ml puromycin + 250 μg / ml hygromycin growth medium. 5 cells / ml.

[0223] On the day of the experiment, target and reporter cells were transferred into assay medium (RPMI + 10% FBS + P / S / G) and plated in a 3:2 ratio (3 × 10 4 / well target cells and 2 x 10 4 / well reporter cells).

[0224] Anti-SARS-CoV-2 spike protein antibodies, negative isotype-matched control antibodies, and a positive control antibody for ADCC (anti-CD20) were titrated in 11-point 1:3 serial dilutions ranging from 1.7 pM to 100 nM final concentrations, with the final 12th point containing no antibody. All samples were tested in duplicate. Plates were incubated at 37 °C / 5% CO2 for 5 h, followed by addition of an equal volume of ONE-Glo™ (Promega) reagent to lyse cells and detect luciferase activity. Emitted light was captured in relative light units (RLU) on a multilabel plate reader Envision (PerkinElmer). GraphPad Prism software was used to determine antibody EC50 values ​​from a 4-parameter logistic equation for the 12-point dose-response curves (including background signal). The following formula was used to calculate maximum fold induction:

[0225] Fold induction = maximum mean RLU / mean RLU within the tested dose range for each antibody (background signal = no antibody)

[0226] The EC50 values ​​and fold induction are summarized in Table 25.

[0227] Results using Jurkat / hCD20 / SARS-CoV-2 spike target cells Each of the anti-SARS-CoV-2-S antibodies tested demonstrated greater fold induction than the negative control antibody. EC50 values ​​ranged from 60.4 pM to 3.4 nM, and fold induction values ​​ranged from 3.9-fold to 13.5-fold. The positive CD20 control antibody demonstrated a 29.9-fold induction with an EC50 value of 18.1 pM.

[0228] Results using Jurkat / hCD20 target cells Each of the 41 anti-SARS-CoV-2-S antibodies, except for one (mAb14312), behaved similarly to the negative control antibody, exhibiting minimal activity. mAb14312 had an EC50 value of 18.4 nM and a fold induction value of 2.0. The positive CD20 control antibody showed a fold induction value of 29.4 with an EC50 value of 33.7 pM. [Table 28-1] [Table 28-2] *At high concentrations, the signal was reduced (hook effect) and the dots on the hook were removed before the EC50 was determined. NC: not calculated as data did not fit a four-parameter logistic equation. ND: Not determined because no concentration-dependent response was observed. Fold induction = maximum mean RLU / mean RLU within the tested dose range for each antibody (background signal = no antibody)

[0229] From the initial mAbs tested, a select few were selected for further follow-up with the antibodies described in US Patent No. 10,787,501. The EC50 values ​​and fold induction are summarized in Table 26.

[0230] Results using Jurkat / hCD20 / SARS-CoV-2 spike target cells Each of the freshly purified SARS-CoV-2 spike antibodies demonstrated greater fold induction than the negative control antibody. The EC50 values ​​for the freshly purified SARS-CoV-2 spike antibodies ranged from 188 pM to 575 pM, and the fold induction values ​​ranged from 2.0-fold to 3.2-fold. The positive CD20 control antibody demonstrated a fold induction of 27.0-fold with an EC50 value of 51.8 pM.

[0231] Results using Jurkat / hCD20 target cells Each of the freshly purified SARS-CoV-2 spike antibodies behaved similarly to the negative control antibody, exhibiting minimal activity. In contrast, the positive CD20 control antibody demonstrated a fold induction value of 23.5-fold with an EC50 value of 142 pM. [Table 29] †Antibodies described in U.S. Pat. No. 10,787,501 NC: not calculated as data did not fit a four-parameter logistic equation. ND: Not determined because no concentration-dependent response was observed. Fold induction = maximum mean RLU / mean RLU within the tested dose range for each antibody (background signal = no antibody)

[0232] Subsequent rounds of testing were also performed on the newly purified antibodies. EC50 values ​​and fold induction are summarized in Table 27.

[0233] Results using Jurkat / hCD20 / SARS-CoV-2 spike target cells Each of the newly purified anti-SARS-CoV-2-S antibodies demonstrated greater fold induction than the negative control antibody. EC50 values ​​for the SARS-CoV-2 spike antibodies ranged from 81.7 pM to 905 pM, with fold induction values ​​ranging from 4.2-fold to 13.0-fold. The positive CD20 control antibody demonstrated a fold induction of 46.7-fold with an EC50 value of 74.4 pM.

[0234] Results using Jurkat / hCD20 target cells All freshly purified SARS-CoV-2-S antibodies behaved similarly to the negative control antibody, exhibiting minimal activity. In contrast, the positive CD20 control antibody showed a fold induction value of 62.1 with an EC50 value of 145 pM. [Table 30] †Antibodies described in U.S. Pat. No. 10,787,501 § Antibodies listed in Table 25 and / or Table 26 *At high concentrations, the signal was reduced (hook effect) and the dots on the hook were removed before the EC50 was determined. ND: Not determined because no concentration-dependent response was observed. Fold induction = maximum mean RLU / mean RLU within the tested dose range for each antibody (background signal = no antibody)

[0235] Example 9: Characterization of anti-SARS-CoV-2-S mAbs in the ADCP assay The ability of antibodies targeting the spike protein of SARS-CoV-2 to induce phagocytosis of Jurkat cells engineered to express the SARS-CoV-2 full-length spike protein was evaluated. Macrophages differentiated from monocytes in the presence of macrophage colony-stimulating factor (M-CSF) were used as effector cells in an antibody-dependent cellular phagocytosis (ADCP) assay. IgG1 isotype as a negative control was evaluated in parallel. Phagocytosis was assessed by measuring the number of fluorescently labeled target cells that colocalized with fluorescently labeled macrophages using the Opera Phenix High-Content Screening System.

[0236] target cell Jurkat / hCD20 / SARS-CoV-2 spike: Jurkat T cells were engineered to constitutively express full-length human CD20 (amino acids M1-P297 of NCBI accession number NP_690605.1) as well as full-length SARS-CoV-2 spike protein (amino acids M1-T1273 of NCBI accession number YP_009724390.1). Jurkat / hCD20 / SARS-CoV-2 spike cells were selected for high expression of spike protein and maintained in RPMI+10%FBS+P / S / G+1μg / ml puromycin+250μg / ml hygromycin growth medium.

[0237] Effector cells Macrophages differentiated from monocytes in the presence of M-CSF: Frozen CD14+ monocytes (Lonza) were thawed, resuspended in assay medium (RPMI 1640 supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, and 292 μg / ml L-glutamine, NaPyr, HEPES, and NEAA), supplemented with 100 ng / ml M-CSF, and incubated at 5.2 × 10 cells for differentiation into macrophages over 9 days. 4 Cells were plated per well in clear bottom, collagen-coated 96-well black plates.

[0238] Assay setup One day before the experiment, Jurkat target cells were cultured at 5 × 10 5 cells / ml.

[0239] On the day of the experiment, target cells and monocyte-derived macrophages were incubated for 15 min at 37° C., 5% CO 2 in PBS supplemented with either CellTrace CFSE dye or CellTrace Violet dye, respectively.

[0240] CFSE-labeled target cells were washed and resuspended in assay medium (RPMI 1640 supplemented with 10% FBS, 100 U / ml penicillin, 100 μg / ml streptomycin, and 292 μg / ml L-glutamine, NaPyr, HEPES, NEAA, and 10 μM BME) and cultured at 5 × 10 4 Cells / well were added in duplicate in 96-well U-bottom plates at a density of 1000 x 1000 cells / well. Anti-SARS-CoV-2 spike and IgG1 control antibodies were titrated in assay medium in 1:5 serial dilutions ranging from 51 fM to 20 nM final concentrations and added to the plate. The zero antibody point was represented by 10 fM in GraphPad Prism.

[0241] After 15 min incubation on ice, the mixture of target cells with or without titrated antibody was then transferred to the plates containing violet-labeled macrophages, and the plates were incubated for 30 min at 37° C., 5% CO. The wells were washed three times with PBS, followed by the addition of 4.21% formaldehyde in PBS supplemented with 2.5 μM DRAQ5.

[0242] After 20 minutes of incubation, the wells were washed with PBS and imaged using the Opera Phenix High-Content Screening System in both 488 nm (CFSE-labeled target cells) and 375 nm (violet-labeled macrophages) excitation channels. Image analysis was performed with Harmony software. Image segmentation in the 375 nm excitation channel was used to identify macrophage populations. Image segmentation in the 488 nm excitation channel was used to identify target cells. Phagocytosis was quantified by identifying macrophage populations that contained target cells within them.

[0243] The phagocytosis rate was calculated by comparing the number of phagocytosed macrophages with the total macrophage cell number.

number

[0244] EC 50 For the determination, ADCP% was analyzed using GraphPad Prism using a 4-parameter logistic equation over a 10-point dose-response curve, including a no-antibody control. Maximum (Max) ADCP% was determined as the highest mean ADCP% value measured within the dose range tested. Experiments were performed in duplicate using a single donor, except for the following antibodies, which were performed without replication: mAb10933, mAb10987, mAb10989, mAb1932, mAb10985, mAb14234, mAb10986, and mAb14259.

[0245] EC 50 Values ​​and Max ADCP% are summarized in Table 28.

[0246] Results using Jurkat / hCD20 / SARS-CoV-2 spike cells: Each of the 21 anti-SARS-CoV-2 spike antibodies showed a greater maximum ADCP% compared to the IgG1 control antibody. EC 50The values ​​ranged from 5.22 pM to 1.12 nM, and the maximum ADCP% ranged from 16.72-fold to 39.89-fold. [Table 31] †Antibodies described in U.S. Pat. No. 10,787,501 ND: Not determined because no concentration-dependent response was observed. NC: not calculated as data did not fit a four-parameter logistic equation. Max(ADCP%) is the highest mean ADCP% value within the dose range tested.

[0247] Example 10: Structure determination and interactions of antibody-binding spike proteins To better understand the binding of mAb10987 and mAb14256 to the spike protein RBD, structural analysis was performed via cryo-electron microscopy (cryo-EM). Fab fragments of mAb14256 and mAb10987 were isolated using the FabALACTICA kit (Genovis). 25 μg of mAb14256 Fab and 25 μg of mAb10987 Fab were mixed with 15 μg of SARS-CoV-2-S RBD and incubated on ice for 30 min. For cryo-EM grid preparation, protein samples were diluted to 0.94 mg / mL and 0.15% PMAL-C8 amphipol was added. 3 μL of protein was deposited on a freshly plasma-cleaned UltrAufoil grid (0.6 / 1.0, 300 mesh). Excess solution was blotted using filter paper and plunge-frozen in liquid ethane using a Vitrobot Mark IV. Cryo-EM grids were transferred to a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan). Movies were collected using an EPU (Thermo Fisher) at 105,000x magnification, corresponding to a pixel size of 0.86 Å. A dose rate of 15 electrons per pixel per second was used, and each movie was recorded over 10000 Å. 2 The time required for the experiment was 2 seconds, corresponding to a total dose of about 40 electrons per second.

[0248] All cryo-EM data processing was performed using cryoSPARC v3.2.0. 6,106 aligned micrographs were collected and 5,735 aligned micrographs were selected for further processing based on estimated defocus values ​​and CTF fit resolution. An initial set of particles picked using a blob picker underwent 2D classification to generate templates for template selection. Approximately 1.4 million particles picked by template selection underwent multiple rounds of 2D classification to remove particles containing unbound Fab and incomplete complexes. An ab initio reconstruction using three classes generated two classes containing 301,461 particles corresponding to the complete mAb10987 Fab-mAb14256 Fab-RBD complex. Heterogeneous refinement of these two classes was performed, with one class showing good quality maps while the other showed a high preferred orientation. Heterogeneous refinement of the "good" class of particles containing 189,200 particles was followed by local refinement, resulting in a 3.86 Å resolution (FSC=0.143) map that was used to build the model. Within this map, a model of the RBD (obtained from PDB code 6M0J) was manually placed, along with two Fabs (obtained from previous antibody structures, i.e., mAb10987 from PDB 6XDG and mAb14256 from another mAb14256 model). These models were then manually reconstructed using real space refined against the map using Coot and Phenix.

[0249] Confirming the data described above, single particle cryo-EM of the complex of SARS-CoV-2 spike RBD bound to the Fab fragments of mAb14256 and mAb10987 shows that the two antibodies in this cocktail can bind simultaneously to distinct regions of the RBD (Figure 3, Figure 4). A 3D reconstruction map of the complex with a nominal resolution of 3.9 Å showed that both Fab fragments bind to non-overlapping regions on the surface of the RBD, confirming that they are non-competing antibodies. mAb14256 bound at the top of the RBD and overlapped extensively with the binding site of ACE2. On the other hand, the epitope of mAb10987 was located on the side of the RBD, far away from the mAb14256 epitope, with little or no overlap with the ACE2 binding site. The structure also revealed that mAb14256 competes with mAb10933 and mAb10985, although combinations of these antibodies may still be desirable due to different microepitope binding and the need to bind and neutralize different SARS-CoV-2 spike protein variants.

[0250] Similar techniques were used to investigate the structural basis of mAb15160 binding to the SARS-CoV-2 RBD. Cryo-EM was used to determine the structure of SARS-CoV-2 RBD complexed with antigen-binding (Fab) fragments of mAb15160 and mAb14315. The resulting 3.18 Å resolution structure revealed that mAb15160 binds to a small epitope on the top (i.e., closest to the target cell membrane) surface of the SARS-CoV-2 RBD (Figure 5). Analysis of the binding interface between the SARS-CoV-2 RBD and the antibody Fab fragment indicates that mAb15160 Fab contacts the RBD with both its heavy chain (13 residue-residue interactions) and light chain (13 residue-residue interactions). A total of 13 SARS-CoV-2 RBD residues are involved in mAb15160 binding. The functional impact of K417N, E484A, and Q493R, all present in the Omicron variant, as well as other substitutions within the binding epitope, were evaluated in the neutralization studies discussed above. mAb15160 retained neutralization capacity against the individual mutations K417N and E484A. Although there was an impact on neutralization potency against the individual mutation Q493R (a 5-fold reduction compared to the reference pseudoparticles), the neutralization potency of mAb15160 was within 1.5-fold against pseudoparticles pseudotyped with the complete S protein sequences from the BA.1, BA.1.1, and BA.2 (Omicron) lineages compared to the reference pseudoparticles.

[0251] Similar techniques were used to investigate the structural basis of mAb14284 binding to SARS-CoV-2 Wuhan-Hu-1 and BA.1 (Omicron lineage) RBDs. Cryo-EM was used to determine the structures of antigen-binding (Fab) fragments of mAb14284 and mAb15160 with RBDs from the Wuhan-Hu-1 and BA.1 strains. The resulting 3.3 and 3.4 Å resolution structures revealed that mAb14284 binds to the top patch on both the Wu-1 and BA.1 RBDs in a manner that competes with ACE2 binding, respectively (Figure 6). Analysis of the binding interface between mAb14284 Fab fragment and SARS-CoV-2 RBDs from Wu-1 and BA.1 shows that mAb14284 binds to the same positions on the Wu-1 and BA.1 RBDs, with the same epitope residues located around them, except for four changes (Figure 29, below). The epitope residues mutated in the BA.1 variant (N440K, G446S, Q498R, N501Y) relative to the ancestral Wu-1 strain are accommodated in the structure without major changes at the antibody-antigen interface. [Table 32] SARS-CoV-2 Wuhan-Hu-1 and BA.1 S protein RBD residues that interact with residues of mAb14284 Fab heavy and light chains are shown. A hyphen "-" indicates that no interaction was observed between the indicated RBD residue and the indicated antibody chain. Residues were selected based on the presence of hydrogen bonds / salt bridges (not including hydrogen atoms) with interatomic distances of <4 Å, or hydrophobic surfaces with interatomic distances of <5 Å. An asterisk "*" indicates RBD residues that were mutated in the BA (Omicron) lineage.

[0252] To assess the degree of variability of the mAb14284 epitope across currently circulating SARS-CoV-2 viruses, we utilized sequences of 3,825,870 publicly available SARS-CoV-2 genomes identified between January 1, 2022 and June 16, 2022 (i.e., during the Omicron wave). Sequences collected during this time period likely consist primarily of Omicron sequences, although other circulating variants, such as delta, may also be included in this analysis.

[0253] Between Jan. 1, 2022 and Jun. 16, 2022, the RBD residues bound by mAb14284 were >99.96% conserved relative to the Wuhan-Hu-1 strain at each position, except for four residues that are mutated in the omicron variant (i.e., the BA lineage): N440, G446, Q498, and N501 (Table 30). The frequencies of N440, G446, Q498, and N501 over this monitoring period were 22, 59, 13, and 12%, respectively; instead, N440K, Q498R, and N501Y, all present in the omicron variant, became predominant. While G446 remains the predominant residue during this period, 39% of sequences contain the G446S mutation present in the BA.1 lineage of the omicron variant.

[0254] The functional impact of N440K, G446S, Q498R, and N501Y, as well as other substitutions within the binding epitope identified during conservation analysis (identified below in Table 30), were evaluated in neutralization studies discussed in Example 4. mAb14284 retained neutralization potency against all epitope residue variants tested, with the exception of the K444 mutation. Although individual substitutions of K444L, K444M, K444N, K444Q, and K444T resulted in a >5-fold change compared to the reference pseudoparticles, the neutralization potency of mAb14284 was within 1.5-fold against pseudoparticles pseudotyped with K444R, the most prevalent (i.e., 0.0141%) K444 variant (Table 19). [Table 33] A total of 3,825,870 publicly available SARS-CoV-2 genome sequences identified between January 1, 2022 and June 16, 2022 were analyzed against the reference (Wuhan-Hu-1) SARS-CoV-2 genome. Data collected during this period likely consisted primarily of omicron sequences, although other circulating variants such as delta may also be included in this analysis. The frequency (freq) of variants within the mAb14284 epitope (as determined by structural studies described herein) was calculated. Amino acid (AA) positions indicate residues in the SARS-CoV-2 S protein RBD (shown in Table 29). For each AA position within the epitope, The frequencies of the reference residue and the two most frequent variant residues are shown. "Del" indicates a deletion at that AA position. The functional impact of the most predominant circulating epitope residues assessed as of June 16, 2022 (N439K, N439V, N440D, N440K, L441F, L441Q, K444L, K444M, K444N, K444Q, K444R, K444T, V445A, V445T, G446D, G446R, G446S, G446V, G447V, N448S, Y449D, Y449F, Y449H, Y449N, Y449S, Q498H, Q498R, T500A, T500F, T500N, N501T, N501Y) is summarized in Table 19.

[0255] Example 11: In vitro VSV-SARS-CoV-2-S viral escape mutant selection in the presence of mAb15160 To assess the selection of SARS-CoV-2 escape mutants, Vero cells were infected with replicating VSV-SARS-CoV-2-S (Wuhan-Hu-1 strain) and incubated with a range of concentrations of mAb15160 or IgG1 isotype control for 4 days. Infected cells were monitored for virus-induced cytopathic effect (CPE) as a readout of viral replication. Loss of neutralization efficacy, assessed by observable CPE in the presence of increasing concentrations of antibody, indicated potential selection of escape mutants. Virus-containing supernatants were collected from wells with the highest mAb concentration and observable CPE and further passaged in the presence of the same mAb. After passaging, virus-containing supernatants were subjected to RNA-seq analysis and all amino acid changes in the S protein were designated as "putative escape mutations" if the amino acid change was present in the anti-SARS-CoV-2 S protein mAb-treated sample but not present in either the input virus sample (inoculum) or the control sample (IgG1 control or no mAb control).

[0256] CPE in ≥90% of cases was observed in the presence of 0.016–50 μg / mL mAb15160 within the first passage. Deep sequencing of the viral genome after the first passage under selection with mAb15160 identified two putative escape mutations in the SARS-CoV-2 RBD-binding epitope, mAb15160-F486V and Y489H. Notably, none of these individual mutations were found in the prominent omicron and delta variants, or any other previous variants of concern, indicating there is no current clinical concern for these mutations.

[0257] The functional impact of each putative escape mutant on mAb15160 activity was assessed in a neutralization assay as discussed above. F486V and Y489H reduced the neutralization activity of mAb15160 by >531- and 122-fold, respectively, compared to the reference pseudoparticles, confirming that F486V and Y489H are escape mutants of mAb15160.

[0258] Example 12: In vitro effector function activity of mAb15160 The ability of mAb15160 to mediate antibody-dependent cellular phagocytosis (ADCP) of fluorescently labeled target cells engineered to express full-length (FL) SARS-CoV-2 S protein (Jurkat / hCD20 / SARS-CoV-2 S FL) was assessed in the presence of fluorescently labeled primary MDM effector cells from two independent donors differentiated with macrophage colony-stimulating factor (MCSF). Results showed that mAb15160 exerted an EC 50 The anti-CD20 IgG1 control mediated concentration-dependent ADCP of Jurkat / hCD20 / SARS-CoV-2 S FL cells with 0.01% glycerol (0.01% glycerol) values ​​(Table 31), while no ADCP was observed in parental cells that do not express SARS-CoV-2 S protein. The anti-CD20 IgG1 control mediated ADCP of both Jurkat / hCD20 / SARS-CoV-2 S FL and Jurkat / hCD20 cell lines, while the IgG1 isotype control did not mediate ADCP of either Jurkat / hCD20 / SARS-CoV-2 S FL or Jurkat / hCD20 cell lines, demonstrating the ability of the effector cells used in the assay to phagocytose each of the cell lines tested. [Table 34] a Maximum (max) ADCP is defined as the highest mean percentage ADCP value observed within the concentration range tested (0 nM to 20 nM, where 0 nM is the no-antibody control). ND, not determined because no concentration-dependent response was observed

[0259] mAb15160 was evaluated for its ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) against Jurkat / hCD20 / SARS-CoV-2 S FL and Jurkat / hCD20 target cells using human primary natural killer (NK) cells from three independent donors as effector cells. Results demonstrated that mAb15160 mediated ADCC against Jurkat / hCD20 / SARS-CoV-2 S FL target cells but not SARS-CoV-2-negative parental cells, with variable activity shown for each NK donor (donor #3 > donor #2, donor #1 showed no activity (Table 32)). The positive control anti-CD20 IgG1 mediated ADCC of both Jurkat / hCD20 / SARS-CoV-2 S FL and Jurkat / hCD20 cell lines, while the IgG1 control did not mediate ADCC of either the Jurkat / hCD20 / SARS-CoV-2 S FL or Jurkat / hCD20 cell lines, demonstrating the cytotoxic potential of all NK cells used in the assay against each tested cell line. [Table 35] a Maximum (max) cytotoxicity is defined as the highest mean percentage cytotoxicity value observed within the concentration range tested. NC, not calculated as data did not fit a four-parameter logistic equation. ND, not determined as no concentration-dependent increase was observed.

[0260] mAb15160 was evaluated for its ability to mediate activation of FCGR3A signaling in a surrogate ADCC reporter bioassay using Jurkat T cells engineered to express a nuclear factor of activated T cells-luciferase (NFAT-Luc) reporter gene and human FCGR3A (Jurkat / NFAT-Luc / FCGR3A) in the presence of Jurkat / hCD20 / SARS-CoV-2 S FL target cells. Engagement of FCGR3A via the Fc domain of human IgG1 antibody bound to target cells leads to activation of NFAT, driving expression of luciferase, which is then measured via a luminescent readout. Consistent with the results from the ADCC assay using primary NK cells, results from the ADCC surrogate reporter assay showed that mAb15160 inhibited 2.97×10 -10 M's EC 50 We demonstrate that IgG1 mediates a concentration-dependent increase in luciferase activity from reporter cells expressing FCGR3A in the presence of Jurkat / hCD20 / SARS-CoV-2 S FL target cells. The IgG1 negative control did not activate FCGR3A in the presence of Jurkat / hCD20 / SARS-CoV-2 S FL or Jurkat / hCD20 cell lines.

[0261] mAb15160 was evaluated for its ability to mediate CDC on Jurkat / hCD20 / SARS-CoV-2 S FL and Jurkat / hCD20 target cells in the presence of 5% pooled (i.e., from several donors) NHS. The results demonstrate that mAb15160 does not mediate CDC on Jurkat / hCD20 / SARS-CoV-2 S FL or Jurkat / hCD20 cells in the presence of 5% NHS at antibody concentrations ranging from 0.95 pM to 1 μM. The NHS used in the assay was evaluated on the same target cell lines for its ability to mediate CDC using a positive control, anti-CD20 IgG1. In the presence of NHS, anti-CD20 IgG1 mediated CDC on target cells in a concentration-dependent manner. In the absence of NHS, anti-CD20 IgG1 did not mediate lysis on any of the target cell lines tested.

[0262] Example 13: In vitro VSV-SARS-CoV-2-S viral escape mutant selection in the presence of mAb14284 To assess SARS-CoV-2 escape mutant selection, Vero cells were infected with replicating pseudotyped VSV-SARS-CoV-2-S (Wuhan-Hu-1 strain) and incubated with a range of concentrations of mAb14284 or IgG1 isotype control for 4 days. Infected cells were monitored for virus-induced cytopathic effect (CPE) as a readout of viral replication. Loss of neutralization efficacy, assessed by observable CPE in the presence of increasing concentrations of antibody, indicated potential selection of escape mutants. Virus-containing supernatants were collected from wells with the highest mAb concentration and observable CPE and further passaged in the presence of the same mAb. After passaging, virus-containing supernatants were subjected to RNA-seq analysis and all amino acid changes in the S protein were designated as "putative escape mutations" if the amino acid change was present in the mAb14284-treated samples but not in the IgG1 control samples.

[0263] CPE in ≧90% of cases was observed in the presence of 0.016 to 50 μg / mL mAb14284 within the first passage (Table 33). Deep sequencing of the viral genome after the first passage under selection with mAb14284 identified three putative escape mutations in the SARS-CoV-2 RBD binding epitope, REGN14284-K444E, K444N, and V445D (Table 34).

[0264] The functional impact of the putative escape mutant K444N on mAb14284 activity was assessed in the neutralization assay discussed in Example 4. The K444N variant resulted in a complete loss of mAb14284 neutralization activity compared to the reference pseudoparticles (Table 19), confirming that K444N is an escape mutant of mAb14284.

[0265] Functional assays of K444E and V445D are ongoing. However, the K444L, K444M, K444N, K444Q, K444T, V445A, and V445T mutations all resulted in a reduction or complete loss of mAb14284 neutralizing activity, which was retained for the K444R mutation (Table 19). [Table 36] Vero cells were infected with VSV-SARS-CoV-2-S virus for 4 days in the presence of mAb14284 or IgG1 isotype control mAb at a range of concentrations (total 0.016-50 μg / mL). Cells were screened for viral replication by monitoring virus-induced CPE. Virus-containing supernatants were collected from wells with the highest antibody concentration and detectable viral replication (≥20% CPE). Deep sequencing of viral RNA was performed to determine the identity of putative escape mutations. No antibody controls (0 μg / mL) were sequenced to monitor for tissue culture adaptation. Bold and underlined text indicates sequenced samples. [Table 37] Viral RNA from wells with the highest mAb concentration showing detectable CPE was isolated 4 days after infection. Deep sequencing was performed to identify changes in the S protein sequence relative to the Wuhan-Hu-1 reference sequence. Only RBD variants are summarized in this table.

[0266] Example 14: In vitro effector function activity of mAb14284 IgG antibodies bound to target cells can mediate effector functions through their constant (Fc) regions by interacting with specific FcGRs expressed on macrophages and natural killer (NK) cells, resulting in antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC), respectively, or by binding to complement proteins, resulting in complement-dependent cytotoxicity (CDC), resulting in the destruction of the target cell.

[0267] A series of in vitro studies were performed to evaluate the ability of mAb14284 to mediate ADCP, ADCC, and CDC against target cells expressing full-length (FL) SARS-CoV-2 S protein from the Wuhan-Hu-1 strain (AA 1-1273) and the BA.1 and BA.2 omicron lineages (AA 1-1270). The target cell lines were also engineered to stably express human CD20 (hCD20) to allow for the simultaneous testing of an anti-CD20 IgG1 positive control antibody.

[0268] Specifically, mAb14284 was evaluated for its ability to 1) mediate ADCP of target cells using monocyte-derived macrophages (MDMs) as effector cells, 2) mediate ADCC of target cells using primary natural killer (NK) cells as effector cells, 3) activate FCGR3A signaling in an ADCC surrogate reporter assay, and 4) mediate CDC of target cells in the presence of normal human serum (NHS).

[0269] The ability of mAb14284 to mediate ADCP of fluorescently labeled Jurkat / hCD20 target cells expressing FL Wu-1, BA.1, or BA.2 S proteins was assessed in the presence of fluorescently labeled primary MDM effector cells from two independent donors differentiated with macrophage colony-stimulating factor (MCSF). mAb14284 exhibited EC 50 The anti-CD20 IgG1 control mediated concentration-dependent ADCP of Jurkat / hCD20 cells expressing SARS-CoV-2 Wu-1, BA.1, or BA.2 S proteins with values ​​(Table 35) whereas no ADCP was observed in parental cells not expressing SARS-CoV-2 S protein. The anti-CD20 IgG1 control mediated ADCP of all tested cell lines whilst the IgG1 isotype control did not mediate ADCP of any of the cell lines tested, demonstrating the ability of the effector cells used in the assay to phagocytose each cell line evaluated. [Table 38] a Maximum (max) ADCP is defined as the highest mean percentage ADCP value observed within the concentration range tested (0 nM to 20 nM, where 0 nM is the no-antibody control). NC, not calculated as data did not fit a four-parameter logistic equation. ND, not determined as no concentration-dependent increase was observed.

[0270] mAb14284 was evaluated for its ability to mediate ADCC against Jurkat / hCD20 target cells expressing FL Wu-1, BA.1, or BA.2 S proteins using pooled human primary NK cells from three donors as effector cells. Results showed that mAb14284 had EC 50 The results demonstrate that the NK cells mediated concentration-dependent ADCC against Jurkat / hCD20 expressing SARS-CoV-2 Wu-1, BA.1, or BA.2 S proteins, but not against the parental cells that do not express SARS-CoV-2 S protein, with values ​​(Table 36). The IgG1 control did not mediate ADCC against any of the cell lines tested, while the positive control anti-CD20 IgG1 mediated ADCC against all cell lines tested, demonstrating the cytotoxic potential of the NK cells used in the assay against each cell line evaluated. [Table 39] a Maximum (max) cytotoxicity is defined as the highest mean percentage cytotoxicity value within the concentration range tested. NC, not calculated as data did not fit a four-parameter logistic equation. ND, not determined as no concentration-dependent increase was observed.

[0271] mAb14284 was evaluated for its ability to mediate activation of FCGR3A signaling in a surrogate ADCC reporter bioassay using Jurkat T cells engineered to express a nuclear factor of activated T cells-luciferase (NFAT-Luc) reporter gene and human FCGR3A (Jurkat / NFAT-Luc / FCGR3A) in the presence of Jurkat / hCD20 target cells expressing FL SARS-CoV-2 Wu-1, BA.1, or BA.2 S proteins. Engagement of FCGR3A through the Fc domain of human IgG1 antibody bound to target cells leads to activation of NFAT, driving expression of luciferase, which is then measured via a luminescent readout.

[0272] Consistent with the results from the ADCC assay using primary NK cells, the results from the ADCC surrogate reporter assay showed that mAb14284 inhibited NK cell proliferation at levels above picomolar EC 50 We demonstrate that IgG1 mediates a concentration-dependent increase in luciferase activity from reporter cells expressing FCGR3A in the presence of SARS-CoV-2 Wu-1, BA.1, or BA.2 S protein-expressing target cells, but not against parental cells that do not express SARS-CoV-2 S protein. The IgG1 negative control did not activate FCGR3A in the presence of any of the cell lines evaluated.

[0273] mAb14284 was evaluated for its ability to mediate CDC on Jurkat / hCD20 / SARS-CoV-2 Wu-1 S, Jurkat / hCD20 / SARS-CoV-2 BA.1 S, Jurkat / hCD20 / SARS-CoV-2 BA.2 S, and Jurkat / hCD20 target cells in the presence of 5% pooled (i.e., from several donors) NHS. The results demonstrate that mAb14284 did not mediate CDC on parental Jurkat / hCD20 cells or on Jurkat / hCD20 cells expressing full-length SARS-CoV-2 Wu.1, BA.1, or BA.2 S proteins in the presence of 5% NHS at antibody concentrations ranging from 0.95 pM to 1 μM.

[0274] The NHS used in the assay was evaluated for its ability to mediate CDC on the same target cell lines using a positive control, anti-CD20 IgG1. In the presence of NHS, anti-CD20 IgG1 mediated CDC on target cells in a concentration-dependent manner. In the absence of NHS, anti-CD20 IgG1 did not mediate lysis on any of the target cell lines tested.

[0275] Example 15: Phase 3 clinical trial evaluating the safety and efficacy of mAb15160 After obtaining safety and tolerability data from healthy volunteers treated with mAb15160, a Phase 3 randomized, double-blind, placebo-controlled study will be initiated to evaluate the safety and efficacy of mAb15160 to prevent symptomatic COVID-19 (Table 37). This is an event-driven study expected to enroll approximately 5,000 participants ≥18 years of age who are PCR negative for SARS-CoV-2 at the time of randomization, have risk factors for severe SARS-CoV-2 infection, and have no risk factors. Study participants will be randomized in a 1:1 ratio to one of two treatment arms (300 mg of mAb15160 administered once every 12 weeks (Q12W), or placebo) administered as a subcutaneous (SC) injection over a 6-month period.

[0276] The primary endpoint of this study is symptomatic RT-qPCR-confirmed SARS-CoV-2 infection. Secondary endpoints include safety and tolerability of repeated SC injections of mAb15160. Safety assessments will include, but are not limited to, adverse events occurring during treatment, vital signs, and collection of safety laboratory tests. Blood will also be collected for drug concentration, immunogenicity, and serum neutralization titer measurements. Administration of mAb15160 will result in a reduction in the rate of symptomatic SARS-CoV-2 infection compared to the placebo group. [Table 40] Q12W = 1 dose every 12 weeks

[0277] Example 16: Phase 3 clinical trial evaluating the safety and efficacy of mAb15160 in pre-exposure prophylaxis This is a Phase 3 randomized, double-blind, placebo-controlled study in adults and adolescents to evaluate the safety and efficacy of mAb15160 as pre-exposure prophylaxis to prevent COVID-19. This event-driven study will enroll approximately 5,000 participants ≥12 years of age. Study participants will be randomized in a 1:1 ratio to one of two treatment arms (mAb15160 or placebo) administered as a subcutaneous injection over a 6-month period. The study consists of three periods: a 1-14 day screening period, a 6-month prophylaxis and efficacy evaluation period (EAP), and a 3-month follow-up period (see Table 38). Administration of mAb15160 results in a reduction in the rate of symptomatic SARS-CoV-2 infection compared to the placebo group.

[0278] Screening / Criteria After participants provide informed consent, they are assessed for study eligibility. A screening visit will occur up to 14 days prior to the randomization and baseline visits, and for some subjects, the screening, randomization, and baseline visits may occur on the same day. To be included in the study, participants will preferably have a negative SARS-CoV-2 test result from a reverse transcription polymerase chain reaction (RT-PCR) assay from a sample collected within 72 hours of the baseline visit. Randomization will be stratified for treatment group assignment by age group classification (≧12 years to <18 years, ≧18 years to ≦65 years, >65 years), immunocompromised status (yes / no), and prior vaccination (yes / no). Approximately 20% or more of the enrolled participants have an immunocompromised status. Treatment and Efficacy Evaluation Period (EAP)

[0279] On Day 1, after evaluating available laboratory results and completing baseline evaluations, sample collection, and randomization, all participants will receive their first dose of study drug, which will be administered as a 2.5 ml subcutaneous (SC) injection in a blinded fashion. Study participants will be randomized in a 1:1 ratio to one of two treatment arms: mAb15160 (300 mg administered SC once every 8 weeks (SC Q8W)) or placebo administered SC once every 8 weeks. [Table 41-1] [Table 41-2] [Table 41-3] [Table 41-4] [Table 42-1] [Table 42-2]

[0280] All references cited herein are incorporated by reference to the same extent as if each individual publication, database entry (e.g., Genbank sequence or GeneID entry), patent application, or patent was specifically and individually indicated to be incorporated by reference. This statement of incorporation by reference is intended by the applicant to refer to any and all individual publications, database entries (e.g., Genbank sequence or GeneID entry), patent application, or patent, even if such citation is not immediately adjacent to the specific statement of incorporation by reference. The inclusion of a specific statement of incorporation by reference within this specification, if any, does not in any way weaken this general statement of incorporation by reference. The citation of references herein is not intended as an admission that the references are relevant prior art, nor does it constitute any admission as to the contents or dates of these publications or documents.

Claims

1. An isolated recombinant antibody or antigen-binding fragment thereof that specifically binds to the coronavirus spike protein (CoV-S), wherein the antibody has one or more of the following properties, namely, (a) EC less than about 10 -8 nM binds to CoV-S; 50 ​ (b) shows an extended survival in an animal infected with a coronavirus after said administration compared to an animal infected with an equivalent coronavirus that has not been administered; and / or (c) three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within an HCV that includes an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 550, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within an LCVR that includes an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 558, the antibody or antigen-binding fragment.

2. (a) a heavy-chain variable region including HCDR1, HCDR2, and HCDR3, each including the amino acid sequences of SEQ ID NOs: 552, 554, and 556, respectively, and / or a light-chain variable region including LCDR1, LCDR2, and LCDR3, each including the amino acid sequences of SEQ ID NOs: 84, 14, and 560, respectively; and / or (b) a heavy-chain immunoglobulin variable region including an amino acid sequence having at least 90% amino acid sequence identity with an HCV including the amino acid sequence of SEQ ID NO: 550, and / or a light-chain immunoglobulin variable region including an amino acid sequence having at least 90% amino acid sequence identity with an LCVR including the amino acid sequence of SEQ ID NO: 558: and / or (c) HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3, each including SEQ ID NOs: 552, 554, 556, 84, 14, and 560, respectively; and / or (d) an immunoglobulin including an HCV including the amino acid sequence of SEQ ID NO: 550 and an LCVR including the amino acid sequence of SEQ ID NO: 558 The antibody or antigen-binding fragment according to claim 1, comprising.

3. (a) the antibody or antigen-binding fragment is multispecific; and / or (b) includes any one or more of the following properties, namely, (i) inhibits the growth of the coronavirus, (ii) binds to the surface of the coronavirus, (iii) limits the spread of in vitro cellular coronavirus infection, and (iv) protect a mouse engineered to express human ACE2 or TMPRSS2 protein from death and / or weight loss caused by coronavirus infection; and / or, (c) the CoV-S is SARS-CoV-2-S; and / or (d) the antibody or antigen-binding fragment comprises a VH3-66 or Vk1-33 variable domain sequence, The antibody or antigen-binding fragment according to claim 1 or 2. **Claim 4** The antibody or antigen-binding fragment according to any one of claims 1 to 3, comprising a heavy chain constant region having the modifications of 252Y, 254T, and 256E. **Claim 5** A complex comprising the antibody or antigen-binding fragment according to any one of claims 1 to 4 bound to a CoV-S polypeptide, optionally wherein the CoV-S is SARS-CoV-2-S. **Claim 6** A polypeptide comprising: (a) the HCDR1, HCDR2, and HCDR3 of the HCVR domain of an antibody or antigen-binding fragment comprising an HCVR having the amino acid sequence of SEQ ID NO: 550, or (b) the LCDR1, LCDR2, and LCDR3 of the LCVR domain of an immunoglobulin chain comprising an LCVR having the amino acid sequence of SEQ ID NO:

558. **Claim 7** A polynucleotide comprising: (a) the polypeptide according to claim 6, (b) the antibody or antibody-binding fragment thereof according to any one of claims 1 to 4, (c) an HCVR comprising the CDR sequences contained in the HCVR having the amino acid sequence of SEQ ID NO: 550, (d) an LCVR comprising the CDR sequences contained in the LCVR having the amino acid sequence of SEQ ID NO: 558, (e) an HCVR comprising the HCDR1, HCDR2, and HCDR3 sequences set forth in SEQ ID NO: 552, 554, and 556, respectively, (f) an LCVR comprising the LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 84, 14, and 560, respectively, (g) an HCVR and an LCVR comprising the CDR sequences contained in the HCVR / LCVR pair of SEQ ID NO: 550 / 558, (h) an HCVR and an LCVR comprising the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences set forth in SEQ ID NO: 552, 554, 556, 84, 14, and 560, respectively, (i) an HCVR comprising the HCVR sequence of SEQ ID NO: 550, (j) an LCVR comprising the LCVR sequence of SEQ ID NO: 558, (k) An HCV-R and an LC-VR, each containing the HCV-R and LC-VR sequences of SEQ ID NOs: 550 and 558, (l) A heavy chain (HC) containing the HC sequence of SEQ ID NO: 562, (m) A light chain (LC) containing the LC sequence of SEQ ID NO: 564, or (n) A heavy chain (HC) and a light chain (LC), each containing the HC and LC sequences of SEQ ID NOs: 562 and 564, A polynucleotide encoding the same. **Claim 8** A vector containing the polynucleotide according to claim 7, or an antibody or antigen-binding fragment according to any one of claims 1 to 4, or a host cell containing the polypeptide according to claim 7. **Claim 9** A pair of polynucleotides, wherein (a) The first polynucleotide encodes (i) An HCV-R containing the CDR sequences included in the HCV-R having the amino acid sequence of SEQ ID NO: 550, (ii) An HCV-R containing the HCDR1, HCDR2, and HCDR3 sequences described in SEQ ID NOs: 552, 554, and 556, respectively, (iii) An HCV-R containing the HCV-R sequence of SEQ ID NO: 550, or (iv) A heavy chain (HC) containing the HC sequence of SEQ ID NO: 562 and (b) The second polynucleotide encodes (i) An LC-VR containing the CDR sequences included in the LC-VR having the amino acid sequence of SEQ ID NO: 558, (ii) An LC-VR containing the LCDR1, LCDR2, and LCDR3 sequences described in SEQ ID NOs: 84, 14, and 560, respectively, (iii) An LC-VR containing the LC-VR sequence of SEQ ID NO: 550, or (iv) A light chain (LC) containing the LC sequence of SEQ ID NO: 564 A pair of polynucleotides. **Claim 10** A pair of vectors, each containing the first and second polynucleotides according to claim 9, or a host cell containing the pair of vectors. **Claim 11** A method for producing an antibody or antigen-binding fragment according to any one of claims 1 to 4, comprising: (a) Introducing into a host cell one or more polynucleotides encoding the antibody or antigen-binding fragment; (b) Culturing the host cell under conditions favorable for the expression of the one or more polynucleotides; and (c) Optionally, isolating the antibody or antigen-binding fragment from the host cell and / or the medium in which the host cell is grown. ​ Optionally, the host cell is a Chinese hamster ovary cell and / or the one or more polynucleotides are the one or more polynucleotides according to claim 7 or the pair of polynucleotides according to claim 9, method.

12. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 4, a pharmaceutically acceptable carrier, and optionally, a further therapeutic agent, and further optionally, (a) the further therapeutic agent is an antiviral drug or a vaccine; and / or (b) the further therapeutic agent is selected from the group consisting of an anti-inflammatory agent, an antimalarial agent, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S, optionally (i) the antimalarial agent is chloroquine or hydroxychloroquine and / or the anti-inflammatory agent is an antibody, optionally the antibody is sarilumab, tocilizumab, or gemtuzumab, and / or the antibody or antigen-binding fragment that specifically binds to CoV-S is casirivimab or imdevimab; or (ii) the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of the second antibody in Table 4, pharmaceutical composition.

13. A container or injection device comprising an antibody or antigen-binding fragment or composition according to any one of claims 1 to 4 or claim 12.

14. A pharmaceutical composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 4 for treating or preventing in a subject in need of treatment or prevention of infection by a coronavirus, optionally (a) the coronavirus is selected from the group consisting of SARS-CoV-2, SARS-CoV, and MERS-CoV; and / or (b) the pharmaceutical composition is to be administered in combination with one or more further therapeutic agents, optionally (i) the one or more further therapeutic agents are an antiviral drug or a vaccine; or (ii) the one or more additional therapeutic agents are optionally selected from the group consisting of anti-inflammatory agents that are antibodies such as sarilumab, tocilizumab, or gemtuzumab, antimalarial agents such as chloroquine or hydroxychloroquine, an antibody or antigen-binding fragment thereof that specifically binds to TMPRSS2, and an antibody or antigen-binding fragment thereof that specifically binds to CoV-S such as casirivimab or imdevimab, or the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of the second antibody in Table 4; and / or (c) A pharmaceutical composition, wherein the antibody or antigen-binding fragment is injected subcutaneously, intravenously, or intramuscularly into the body of the subject.

15. A pharmaceutical composition for treating or preventing infection by Omicron variant SARS-CoV-2 in a subject in need thereof, or for preventing one or more COVID-19 symptoms resulting from infection by Omicron variant SARS-CoV-2 in a subject in need thereof, comprising an antibody or antigen-binding fragment thereof comprising three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within a heavy-chain variable region (HCVR) comprising an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 550, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within a light-chain variable region (LCVR) comprising an amino acid sequence having at least about 90% sequence identity with SEQ ID NO: 558, optionally (a) Preventing one or more COVID-19 symptoms resulting from infection by Omicron variant SARS-CoV-2 includes pre-exposure prophylaxis or post-exposure prophylaxis; and / or (b) The antibody or antigen-binding fragment thereof (i) an immunoglobulin heavy-chain variable region comprising HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 552, 554, and 556, respectively, and / or an immunoglobulin light-chain variable region comprising LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 84, 14, and 560, respectively; or (ii) HCDR1, HCDR2, and HCDR3 of SEQ ID NOs: 552, 554, and 556, respectively, and / or LCDR1, LCDR2, and LCDR3 of SEQ ID NOs: 84, 14, and 560, respectively, is included; and / or (c) The pharmaceutical composition is to be administered in combination with one or more additional therapeutic agents, optionally (i) the one or more additional therapeutic agents are antiviral drugs or vaccines; or (ii) the one or more additional therapeutic agents are anti-inflammatory agents that are optionally antibodies such as sarilumab, tocilizumab, or gemtuzumab, optionally antimalarial agents such as chloroquine or hydroxychloroquine, antibodies or antigen-binding fragments thereof that specifically bind to TMPRSS2, and optionally antibodies or antigen-binding fragments thereof that specifically bind to CoV-S such as casirivimab or imdevimab, or the antibody or antigen-binding fragment comprises the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 sequences of the second antibody in Table 4; and / or (d) The antibody or antigen-binding fragment is to be injected into the body of the subject, optionally, the antibody or antigen-binding fragment is to be injected subcutaneously, intravenously, or intramuscularly into the body of the subject, pharmaceutical composition.

16. A pharmaceutical composition for treating or preventing in a subject in need of treatment or prevention of one or more COVID-19 symptoms resulting from infection with SARS-CoV-2, comprising an antibody or antigen-binding fragment thereof comprising three heavy-chain complementarity-determining regions (CDRs) (HCDR1, HCDR2, and HCDR3) contained within the heavy-chain variable region (HCVR) comprising the amino acid sequence set forth in SEQ ID NO: 550, and three light-chain CDRs (LCDR1, LCDR2, and LCDR3) contained within the light-chain variable region (LCVR) comprising the amino acid sequence set forth in SEQ ID NO: 558, optionally (a) the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 552, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 554, the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 556, the LCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 84, the LCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 14, and the LCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 560; (b) the HCVR comprises the amino acid sequence set forth in SEQ ID NO: 550 and / or the LCVR comprises the amino acid sequence set forth in SEQ ID NO: 558; ​ (c) the antibody or antigen-binding fragment thereof comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 562, and / or the antibody or antigen-binding fragment thereof comprises a light chain comprising the amino acid sequence set forth in SEQ ID NO: 564; (d) the pharmaceutical composition is to be administered the antibody or antigen-binding fragment via injection, optionally, the injection is intravenous or subcutaneous; (e) the pharmaceutical composition is to be administered in a regimen comprising: (i) administering 300 mg of the antibody or antigen-binding fragment thereof; (ii) administering 600 mg of the antibody or antigen-binding fragment thereof; (iii) administering 1200 mg of the antibody or antigen-binding fragment thereof; (iv) administering two doses of the antibody or antigen-binding fragment thereof, optionally, each of the two doses comprises 300 mg of the antibody or antigen-binding fragment thereof, and / or the two doses are administered at 4-week intervals, 5-week intervals, 6-week intervals, 7-week intervals, 8-week intervals, 9-week intervals, 10-week intervals, 11-week intervals, 12-week intervals, 13-week intervals, 14-week intervals, 15-week intervals, or 16-week intervals, optionally at 8-16 week intervals, 12-week intervals or 8-week intervals; and / or (f) the administering reduces the viral load of SARS-CoV-2 in the subject, or the administering is a pharmaceutical composition that occurs prior to SARS-CoV-2 infection.

17. An antibody or antigen-binding fragment thereof according to any one of claims 1 to 4, which neutralizes the Omicron variant of SARS-CoV-2, optionally, the Omicron variant is selected from BA.1, BA.1.1, BA.2, BA.2.12.1, BA.3, or BA.4 / BA.5.

Images