Anti-SARS-CoV-2 Spike (S) Antibodies and Their Use in Treating COVID-19

Abstract

Infectious diseases remain a problem worldwide. The sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has infected over 640 million people worldwide. SARS-CoV-2 causes the disease COVID-19. Mutations in the SARS-CoV-2 S spike protein allow SARS-CoV-2 variants to evade neutralizing monoclonal antibodies generated from previous infections or by vaccination with SARS-CoV-2. The present invention provides antibodies that bind to the SARS-CoV-2 spike (S) protein. The present invention further relates to pharmaceutical compositions, immunotherapeutic compositions, and methods that use the aforementioned antibodies that bind to the SARS-CoV-2 spike (S) protein.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 63 / 293,451, filed December 23, 2021. The foregoing application is incorporated by reference herein in its entirety.

[0002] Reference to Electronic Sequence Listing The contents of the electronic sequence listing (NOVV_097_01WO_SeqList_ST26.xml; size: 221,021 bytes; and creation date: December 22, 2022) are incorporated herein by reference in their entirety.

[0003] Field The present disclosure relates generally to anti-sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) antibodies and fragments thereof, which are useful for treating viral infections. In particular, the anti-SARS-CoV-2 spike (S) antibodies and fragments thereof are used to treat coronavirus 19 disease (COVID-19). [Background technology]

[0004] 2. Background of the Invention Infectious diseases remain a problem worldwide. The sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has infected over 640 million people worldwide. The death toll has exceeded 6.6 million worldwide. SARS-CoV-2 coronavirus belongs to the same family of viruses as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which have killed hundreds of people in the past 17 years. SARS-CoV-2 causes the disease COVID-19. Mutations in the SARS-CoV-2 S spike protein allow SARS-CoV-2 variants to evade neutralizing monoclonal antibodies generated from previous infections with SARS-CoV-2 or by vaccination.

[0005] Therefore, it is desirable to develop broadly neutralizing antibodies to treat COVID-19. Summary of the Invention [Means for solving the problem]

[0006] Abstract Provided herein is an antibody or fragment thereof that binds to sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein. In embodiments, the light chain complementarity determining region 1 (VL CDR1) is selected from the group consisting of SEQ ID NOs: 11-14 and 76; the light chain complementarity determining region 2 (VL CDR2) is selected from the group consisting of SEQ ID NOs: 15-18 and 77; the light chain complementarity determining region 3 (VL CDR3) is selected from the group consisting of SEQ ID NOs: 19-22 and 78; the heavy chain complementarity determining region 1 (VH CDR1) is selected from the group consisting of SEQ ID NOs: 23-26 and 79; the heavy chain complementarity determining region 2 (VH CDR2) is selected from the group consisting of SEQ ID NOs: 27-30 and 80; and the heavy chain complementarity determining region 3 (VH CDR3) is selected from the group consisting of SEQ ID NOs: 31-34 and 81. In embodiments, the antibody or fragment thereof comprises: (i) a VH CDR1 set forth in SEQ ID NO: 23, a VH CDR2 set forth in SEQ ID NO: 27, and a VH CDR3 set forth in SEQ ID NO: 31; a VL CDR1 set forth in SEQ ID NO: 11, a VL CDR2 set forth in SEQ ID NO: 15, and a VL CDR3 set forth in SEQ ID NO: 19; (ii) a VH CDR1 set forth in SEQ ID NO: 24; a VH CDR2 set forth in SEQ ID NO: 28; a VH CDR3 set forth in SEQ ID NO: 32; a VL CDR1 set forth in SEQ ID NO: 12; a VL CDR2 set forth in SEQ ID NO: 16, and a VL CDR3 set forth in SEQ ID NO: 20; (iii) a VH CDR1 set forth in SEQ ID NO: 25; a VH CDR2 set forth in SEQ ID NO: 29; a VH CDR3 set forth in SEQ ID NO: 33; a VL CDR1 set forth in SEQ ID NO: 13; a VL CDR2 set forth in SEQ ID NO: 17, and a VL CDR3 set forth in SEQ ID NO: 21; (iv) a VH CDR1 set forth in SEQ ID NO: 26; a VH CDR2 set forth in SEQ ID NO: 30; a VH CDR3 set forth in SEQ ID NO: 34; a VL CDR3 set forth in SEQ ID NO: 14 CDR1; VL CDR2 set forth in SEQ ID NO: 18; and VL CDR3 set forth in SEQ ID NO: 22; or (v) VH CDR1 set forth in SEQ ID NO: 79; VH CDR2 set forth in SEQ ID NO: 80; VH CDR3 set forth in SEQ ID NO: 81; VL CDR1 set forth in SEQ ID NO: 76; VL CDR2 set forth in SEQ ID NO: 77;and a VL CDR3 as set forth in SEQ ID NO: 78. In embodiments, the amino acid sequence of the variable heavy (VH) domain comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 5-8 and 75. In embodiments, the amino acid sequence of the variable light (VL) domain comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having the amino acid sequence of any one of SEQ ID NOs: 1-4 and 74. In embodiments, an antibody comprising: (i) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5; and (ii) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1; (i) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6; and and (ii) an antibody comprising a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2; (i) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:7; and (ii) an antibody comprising a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3;Provided herein is an antibody selected from the group consisting of: (i) an antibody comprising a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:4; and (ii) an antibody comprising a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:8; and (i) an antibody comprising a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:75; and (ii) an antibody comprising a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:74. In embodiments, the antibody or fragment thereof is a monoclonal antibody, Fab, F(ab')2, Fab', scFv, or single domain antibody (sdAb). In embodiments, the antibody comprises a human IgG1 or IgG4 domain. In embodiments, the antibody or fragment thereof has a dissociation constant (K; for a SARS-CoV-2 S polypeptide or variant thereof of 50 nM or less, 10 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, or 0.001 nM or less. D). In embodiments, the antibody or fragment thereof binds to one or more CoV S polypeptides having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide set forth in any one of SEQ ID NOs: 9, 10, 35-43, 72-73, and 90-139. In embodiments, provided herein is an isolated nucleic acid molecule encoding any one of the foregoing antibodies or fragments. In embodiments, provided herein is an expression vector comprising a nucleic acid molecule encoding any one of the foregoing antibodies or fragments. In embodiments, provided herein is a host cell comprising the foregoing expression vector. In embodiments, provided herein is a pharmaceutical composition comprising an antibody or fragment provided herein and a pharma- ceutically acceptable carrier. In embodiments, provided herein is a method of treating a subject infected with SARS-CoV-2 virus or a variant thereof in need thereof, comprising administering to the subject an antibody or fragment thereof described herein. In an embodiment, the subject is 65 years of age or older. In an embodiment, the subject is immunocompromised. In an embodiment, the subject is a pregnant woman. In an embodiment, the SARS-CoV-2 variant has a PANGO lineage selected from the group consisting of B.1.1.529; BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1.

[0007] 1. An antibody or fragment thereof that binds to sudden acute respiratory syndrome coronavirus 2 (CoV) spike (S) glycoprotein, comprising: (i) a light chain complementarity determining region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 11-14 and 76; (ii) a light chain complementarity determining region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 15-18 and 77; and (iii) a light chain complementarity determining region 3 (VL CDR4) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 19-22 and 78. (iv) a heavy chain complementarity determining region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23-26 and 79; (v) a heavy chain complementarity determining region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 27-30 and 80; and (vi) a heavy chain complementarity determining region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 31-34 and 81.1. An antibody or fragment thereof that binds to sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, comprising: (i) an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polypeptide of SEQ ID NOs: 5-8 and 75. and (ii) a variable light (VL) domain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polypeptide of any one of SEQ ID NOs: 1-4 and 74. Provided herein is an antibody or fragment thereof comprising a VH CDR1 set forth in SEQ ID NO: 23, a VH CDR2 set forth in SEQ ID NO: 27, and a VH CDR3 set forth in SEQ ID NO: 31; a VL CDR1 set forth in SEQ ID NO: 11, a VL CDR2 set forth in SEQ ID NO: 15; and a VL CDR3 set forth in SEQ ID NO: 19. Provided herein is an antibody or fragment thereof comprising a VH CDR1 set forth in SEQ ID NO: 23, a VH CDR2 set forth in SEQ ID NO: 27, and a VH CDR3 set forth in SEQ ID NO: 31 ...L CDR1 set forth in SEQ ID NO: 11, a VL CDR2 set forth in SEQ ID NO: 15, and a VL CDR3 set forth in SEQ ID NO: 19. Provided herein is an antibody or fragment thereof comprising a VH CDR1 set forth in SEQ ID NO:24; a VH CDR2 set forth in SEQ ID NO:28; a VH CDR3 set forth in SEQ ID NO:32; a VL CDR1 set forth in SEQ ID NO:12; a VL CDR2 set forth in SEQ ID NO:16; and a VL CDR3 set forth in SEQ ID NO:20.Provided herein is an antibody or fragment thereof comprising a VH CDR1 set forth in SEQ ID NO:25; a VH CDR2 set forth in SEQ ID NO:29; a VH CDR3 set forth in SEQ ID NO:33; a VL CDR1 set forth in SEQ ID NO:13; a VL CDR2 set forth in SEQ ID NO:17; and a VL CDR3 set forth in SEQ ID NO:21. Provided herein is an antibody or fragment thereof comprising a VH CDR1 set forth in SEQ ID NO:26; a VH CDR2 set forth in SEQ ID NO:30; a VH CDR3 set forth in SEQ ID NO:34; a VL CDR1 set forth in SEQ ID NO:14; a VL CDR2 set forth in SEQ ID NO:18; and a VL CDR3 set forth in SEQ ID NO:22. Provided herein is an antibody or fragment thereof comprising a VH CDR1 set forth in SEQ ID NO:79; a VH CDR2 set forth in SEQ ID NO:80; a VH CDR3 set forth in SEQ ID NO:81; a VL CDR1 set forth in SEQ ID NO:76; a VL CDR2 set forth in SEQ ID NO:77; and a VL CDR3 set forth in SEQ ID NO:78. Provided herein is an antibody or fragment thereof comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:5; and (ii) a VL comprising the amino acid sequence of SEQ ID NO:1. Provided herein is an antibody or fragment thereof comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:6; and (ii) a VL comprising the amino acid sequence of SEQ ID NO:2. Provided herein is an antibody or fragment thereof comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:7; and (ii) a VL comprising the amino acid sequence of SEQ ID NO:3. Provided herein is an antibody or fragment thereof comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:8; and (ii) a VL comprising the amino acid sequence of SEQ ID NO:4. Provided herein is an antibody or fragment thereof comprising (i) a VH comprising the amino acid sequence of SEQ ID NO:75; and (ii) a VL comprising the amino acid sequence of SEQ ID NO:74. In an embodiment, the antibody or fragment thereof is a monoclonal antibody, a Fab, a F(ab')2, a Fab', a scFv, or a single domain antibody (sdAb). In an embodiment, the antibody comprises a human IgG1 or IgG4 domain.In embodiments, the antibody or fragment thereof has an equilibrium dissociation constant (K) for CoV S glycoprotein or a variant thereof of 50 nM or less, 10 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, or 0.001 nM or less. D In embodiments, the antibody or fragment thereof has a binding affinity to the CoV S glycoprotein or a variant thereof of 1.0×10 -9 Moles / liter (M) less than 1.0 x 10 -10 Less than M, 1.0×10 -11 Less than M or 1.0×10 -12The antibody or fragment thereof binds with an equilibrium dissociation constant (Kd) of less than M. In embodiments, the antibody or fragment thereof binds to one or more CoV S polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide set forth in any one of SEQ ID NOs: 9, 10, 35-43, 72-73, 90-139, and 145-147. In embodiments, the antibody or fragment thereof binds to about 2 to about 20 CoV S glycoproteins. In embodiments, the antibody or fragment thereof is a broadly neutralizing antibody. In embodiments, the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein, said epitope comprising amino acids 476, 485, 486, 487, and 489 of the CoV S glycoprotein of SEQ ID NO: 10. In embodiments, the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein, said epitope comprising amino acids 485, 486, 487, and 489 of the CoV S glycoprotein of SEQ ID NO: 10. In embodiments, the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein, said epitope comprising amino acids 378 and 385 of the CoV S glycoprotein of SEQ ID NO: 10. In embodiments, the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein, said epitope comprising amino acids 444, 445, 446, and 448 of the CoV S glycoprotein of SEQ ID NO: 10. Provided herein are isolated nucleic acid molecules encoding the antibodies or fragments thereof provided herein. Provided herein is an isolated nucleic acid molecule encoding an antibody or fragment thereof provided herein. Provided herein is an expression vector comprising a nucleic acid provided herein. Provided herein is a host cell comprising an expression vector provided herein. Provided herein is a pharmaceutical composition comprising an antibody or fragment thereof provided herein and a pharma- ceutical acceptable carrier.In embodiments, the pharmaceutical composition comprises up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 antibodies or fragments thereof provided herein. Provided herein is a method of treating a subject infected with SARS-CoV-2 virus or a variant thereof in need thereof, comprising administering to the subject an antibody or fragment thereof or a pharmaceutical composition provided herein. In embodiments, the subject is 65 years of age or older. In embodiments, the subject is immunocompromised. In embodiments, the subject is less than 2 years of age. In embodiments, the subject is a pregnant woman. In embodiments, the SARS-CoV-2 variant has a PANGO lineage selected from the group consisting of B.1.1.529;BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1. In embodiments, the SARS-CoV-2 variant has a World Health Organization designation of alpha, beta, gamma, delta, epsilon, iota, kappa, zeta, mu, or omicron. [Brief description of the drawings]

[0008] [Figure 1A-1] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1A-2]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1A-3] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1A-4] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1A-5]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1A-6] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1B-1] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1B-2]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1B-3] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1B-4] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1B-5]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1B-6] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1C-1] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1C-2]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1C-3] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1C-4] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1C-5]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1C-6] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1D-1] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1D-2]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1D-3] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1D-4] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1D-5]1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42). [Figure 1D-6] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to the SARS-CoV-2 S protein for SARS-CoV-2 S polypeptides associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), the SARS-CoV-2 gamma strain (SEQ ID NO: 38), the SARS-CoV-2 beta strain (SEQ ID NO: 36), the SARS-CoV-2 delta strain (SEQ ID NO: 37), the SARS-CoV-2 alpha strain (SEQ ID NO: 39), and the SARS-CoV-2 Omicron strain (SEQ ID NO: 42).

[0009] [Figure 1E-1] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1E-2] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1E-3]Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1E-4] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1E-5] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1E-6] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1E-7] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1E-8] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-1]Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-2] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-3] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-4] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-5] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-6] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-7]Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1F-8] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-1] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-2] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-3] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-4] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-5]Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-6] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-7] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1G-8] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-1] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-2] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-3]Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-4] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-5] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-6] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-7] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1H-8] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-1]Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-2] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-3] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-4] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-5] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-6] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-7]Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. [Figure 1I-8] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain.

[0010] [Figure 2A] Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2B] Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2C]Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2D] Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2E] Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2F]Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2G] Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2H] Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35). [Figure 2I]Figures 2A-2E show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various recombinant SARS-CoV-2 S proteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various SARS-CoV-2 S proteins associated with the SARS-CoV-2 S omicron strain. Figure 2I further shows the EC50 for binding of 199.9 to the SARS-CoV-2 S protein derived from the SARS-CoV-2 parental strain (SEQ ID NO: 35).

[0011] [Figure 3A] Figures 3A-3D show the crystal structure of the SARS-CoV-2 S glycoprotein (Protein Databank ID: 6XCN). The key residues for binding of 35.13 (Figure 3A), 425.6 (Figure 3B), 239.12 (Figure 3C), and 322.3 (Figure 3D) Fabs are shown as spheres. The structures to the right of each figure show the key residues in the SARS-CoV-2 S receptor binding domain (RBD) for binding of each Fab (Protein Databank ID: 6Z2M). [Figure 3B] Figures 3A-3D show the crystal structure of the SARS-CoV-2 S glycoprotein (Protein Databank ID: 6XCN). The key residues for binding of 35.13 (Figure 3A), 425.6 (Figure 3B), 239.12 (Figure 3C), and 322.3 (Figure 3D) Fabs are shown as spheres. The structures to the right of each figure show the key residues in the SARS-CoV-2 S receptor binding domain (RBD) for binding of each Fab (Protein Databank ID: 6Z2M). [Figure 3C]Figures 3A-3D show the crystal structure of the SARS-CoV-2 S glycoprotein (Protein Databank ID: 6XCN). The key residues for binding of 35.13 (Figure 3A), 425.6 (Figure 3B), 239.12 (Figure 3C), and 322.3 (Figure 3D) Fabs are shown as spheres. The structures to the right of each figure show the key residues in the SARS-CoV-2 S receptor binding domain (RBD) for binding of each Fab (Protein Databank ID: 6Z2M). [Figure 3D] Figures 3A-3D show the crystal structure of the SARS-CoV-2 S glycoprotein (Protein Databank ID: 6XCN). The key residues for binding of 35.13 (Figure 3A), 425.6 (Figure 3B), 239.12 (Figure 3C), and 322.3 (Figure 3D) Fabs are shown as spheres. The structures to the right of each figure show the key residues in the SARS-CoV-2 S receptor binding domain (RBD) for binding of each Fab (Protein Databank ID: 6Z2M).

[0012] [Figure 4A] Figures 4A-4C show the minimum sample dilutions (Neut99) of 35.13 (Figure 4A), 425.6 (Figure 4B), and 322.3 (Figure 4C) required to neutralize greater than 99% of the concentrations of SARS-CoV-2 tested. [Figure 4B] Figures 4A-4C show the minimum sample dilutions (Neut99) of 35.13 (Figure 4A), 425.6 (Figure 4B), and 322.3 (Figure 4C) required to neutralize greater than 99% of the concentrations of SARS-CoV-2 tested. [Figure 4C] Figures 4A-4C show the minimum sample dilutions (Neut99) of 35.13 (Figure 4A), 425.6 (Figure 4B), and 322.3 (Figure 4C) required to neutralize greater than 99% of the concentrations of SARS-CoV-2 tested.

[0013] [Figure 5A] Figures 5A-5C show hACE2 receptor inhibition by antibodies 35.13 (Figure 5A), 425.6 (Figure 5B), and 322.3 (Figure 5C). [Figure 5B] Figures 5A-5C show hACE2 receptor inhibition by antibodies 35.13 (Figure 5A), 425.6 (Figure 5B), and 322.3 (Figure 5C). [Figure 5C] Figures 5A-5C show hACE2 receptor inhibition by antibodies 35.13 (Figure 5A), 425.6 (Figure 5B), and 322.3 (Figure 5C).

[0014] [Figure 6A] Figures 6A-6B show pseudovirus neutralization by antibodies 35.13 (Figure 6A) and 425.6 (Figure 6B). [Figure 6B] Figures 6A-6B show pseudovirus neutralization by antibodies 35.13 (Figure 6A) and 425.6 (Figure 6B).

[0015] [Figure 7] FIG. 7 shows an alignment of the SARS-CoV-2 S glycoproteins from the ancestral, beta, delta, gamma, BA.1, BA.2, BA.5, and BQ.1.1 SARS-CoV-2 viruses. The important amino acids for binding of 322.3 to the SARS-CoV-2 S glycoprotein (K378 and T385) are boxed. The important amino acids for binding of 425.6 to the SARS-CoV-2 S glycoprotein (K444, V445, G446, and N448) are boxed. The important amino acids for binding of 425.6 to the SARS-CoV-2 S glycoprotein (K444, V445, G446, and N448) are boxed. The numbering of the important amino acids is relative to the SARS-CoV-2 S glycoprotein in SEQ ID NO: 10. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] Detailed Description of the Invention definition As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. So, for example, reference to "a protein" can refer to one protein or a mixture of such proteins, and reference to "the method" includes reference to equivalent steps and / or methods known to those skilled in the art, and so forth.

[0017] As used herein, the term "adjuvant" refers to a compound that, when used in combination with an immunogen, increases or otherwise alters or modifies the immune response induced against the immunogen. Modification of immune response can include enhancing or broadening the specificity of either or both antibody and cellular immune responses. As used herein, the term "about" or "approximately," when preceding a numerical value, indicates a range of plus or minus 10% of the value. For example, "about 100" includes 90 and 110.

[0018] As used herein, the terms "immunogen," "antigen," and "epitope" refer to substances such as proteins, including glycoproteins, and peptides, that are capable of eliciting an immune response.

[0019] As used herein, "substantially" refers to the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) such that the substance forms a majority percentage of the sample in which it is contained. For example, in a sample, a substantially purified component constitutes 85% of the sample, preferably 85%-90%, more preferably at least 95%-99.5%, and most preferably at least 99%. When a component is substantially replaced, the amount remaining in the sample is less than or equal to about 0.5% to about 10%, preferably less than about 0.5% to about 1.0%.

[0020] The terms "treat," "treatment," and "treating," as used herein, refer to an approach for obtaining beneficial or desired results, e.g., clinical results. For purposes of this disclosure, beneficial or desired results may include inhibiting or suppressing the onset or progression of an infection or disease; ameliorating or reducing the occurrence of symptoms of an infection or disease; or a combination thereof.

[0021] "Prevention," as used herein, is used interchangeably with "prophylaxis" and can mean preventing an infection or disease altogether, or preventing the onset of a symptom of that infection or disease; delaying the onset of an infection or disease or a symptom thereof; or reducing the severity of a subsequently occurring infection or disease or a symptom thereof.

[0022] As used herein, an "effective dose" or "effective amount" refers to an amount of antibody sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. An effective dose or amount may be determined, for example, by measuring the amount of neutralizing secretory and / or serum antibodies, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent (ELISA), or microneutralization assays.

[0023] As used herein, the term "subject" includes humans and other animals. Typically, the subject is a human. For example, the subject may be an adult, a teenager, a child (2-14 years old), an infant (birth-2 years), or a newborn (up to 2 months old). In certain embodiments, the subject is up to 4 months old, or up to 6 months old. In embodiments, the adult is about 65 years old or older, or about 60 years old or older. In embodiments, the subject is a pregnant woman, or a woman who intends to become pregnant. In other embodiments, the subject is not a human; for example, a non-human primate; for example, a baboon, chimpanzee, gorilla, or macaque. In certain embodiments, the subject may be a pet, for example, a dog or cat.

[0024] In an embodiment, the subject is immunocompromised. In an embodiment, the immunocompromised subject is administered a medicament that causes immunosuppression. Non-limiting examples of medicaments that cause immunosuppression include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressants (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, tumor necrosis factor (TNF) blockers (e.g., etanercept, adalimumab, infliximab). In an embodiment, the immunocompromised subject is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In an embodiment, the virus is a respiratory virus, e.g., respiratory syncytial virus, influenza, parainfluenza, adenovirus, or picornavirus. In an embodiment, the immunocompromised subject has acquired immune deficiency syndrome (AIDS). In an embodiment, the immunocompromised subject is a person living with human immunodeficiency virus (HIV). In an embodiment, the immunocompromised subject is immunocompromised due to a treatment regimen designed to prevent inflammation or prevent rejection of the graft. In an embodiment, the immunocompromised subject is a subject who has received a graft. In an embodiment, the immunocompromised subject has undergone radiation therapy or splenectomy.In embodiments, the immunocompromised subject is a patient with a condition that is associated with cancer, an autoimmune disease, tuberculosis, a substance use disorder (e.g., alcohol, opioid, or cocaine use disorder), stroke or cerebrovascular disease, solid organ or blood stem cell transplant, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), autoimmune polyglandular syndrome type 1 (APS-1), B-cell expansion with NF-κB and T-cell anergy (BENTA) disease, caspase 8 deficiency state (CEDS), chronic granulomatous disease (CGD), common variable immunodeficiency (CVID), congenital neutropenic syndrome, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) deficiency, DOCK8 deficiency, GATA2 deficiency, glycosylation disorders with immune deficiency, hyperimmunoglobulin E syndrome, or a combination of these. The subject has been diagnosed with: hyperimmunoglobulin M syndrome (HIES), diabetes mellitus, type 1 diabetes, type 2 diabetes, interferon gamma deficiency, interleukin 12 deficiency, interleukin 23 deficiency, leukocyte adhesion deficiency, lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency, PI3 kinase disease, PLCG2-associated antibody deficiency and immune dysregulation (PLAID), severe combined immunodeficiency (SCID), STAT3 dominant negative disease, STAT3 gain of function disease, warts, hypogammaglobulinemia, infection, and myeloid cell pool (WHIM) syndrome, Wiskott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disease (XLP), uremia, malnutrition, or XMEN disease. In an embodiment, the immunocompromised subject is a current or former tobacco smoker. In embodiments, the immunocompromised subject has a B cell deficiency, a T cell deficiency, a macrophage deficiency, a cytokine deficiency, a phagocyte deficiency, impaired phagocyte function, a complement deficiency, or a combination thereof.

[0025] In embodiments, the subject is overweight or obese. In embodiments, an overweight subject is > 25 kg / m 2 and <30 kg / m 2 In embodiments, an obese subject has a body mass index (BMI) of ≥ 30 kg / m 2In an embodiment, the subject has a mental health condition. In an embodiment, the mental health condition is depression, schizophrenia, or anxiety.

[0026] As used herein, the term "pharmaceutical acceptable" means approved by a regulatory agency of the U.S. Federal or state government, or listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other generally recognized pharmacopoeias, for use in mammals, and more specifically, in humans. These compositions may be useful as vaccine and / or antigen compositions for inducing a protective immune response in vertebrates.

[0027] As used herein, the term "modification," when it refers to a SARS-CoV-2 spike (S) polypeptide, refers to a mutation, deletion, or addition of one or more amino acids of a CoV S polypeptide. The location of the modification within a CoV S polypeptide can be determined based on alignment of the sequence of the polypeptide to SEQ ID NO: 10 (a CoV S polypeptide containing a signal peptide) or SEQ ID NO: 9 (a mature CoV S polypeptide lacking the signal peptide).

[0028] The term SARS-CoV-2 "variant," used interchangeably herein with "heterologous SARS-CoV-2 strain," refers to a SARS-CoV-2 virus that includes a CoV S polypeptide that has one or more modifications when compared to the SARS-CoV S polypeptide having the amino acid sequence of SEQ ID NO:9. For example, a SARS-CoV-2 variant may have at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, or at least about 35 modifications compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:9. at least 1, and at most 2, at most 3, at most 4, at most 5, at most 6, at most 7, at most 8, at most 9, at most 10, at most 11, at most 12, at most 13, at most 14, at most 15, at most 16, at most 17, at most 18, at most 19, at most 20, at most 21, at most 22, at most 23, at most 24, at most 25, at most 26, at most 27, at most 29, at most 30, at most 31, at most 32, at most 33, at most 34, at most 35, at most 36, at most 37, at most 38, at most 39, at most 40, at most 41, at most 42, at most 43, at most 44, at most 45, at most 46, at most 47, at most 48, at most 49, at most 50, at most 51, at most 52, at most 53, at most 54, at most 55, at most 56, at most 57, at most 58, at most 59, at most 60, at most 61, at most 62, at most 63, at most 64, at most 65, at most 66, at most 67, at most 68, at most 69, at most 70, at most 71, at most 72, at most 73, at most 74, at most 75, at most 76, at most 77, at most 78, at most 79, at most 80, at most 81, at most 82, at most 83, at most 84, at most 85, at most 86, at most 87, at most up to 8, up to 29, up to 30, up to 31, up to 32, up to 33, up to 34, up to 35 modifications, up to 40 modifications, up to 45 modifications, up to 50 modifications, up to 55 modifications, up to 60 modifications, up to 65 modifications, up to 70 modifications, up to 75 modifications, up to 80 modifications, up to 85 modifications, up to 90 modifications, up to 95 modifications, or up to 100 modifications.In embodiments, a SARS-CoV-2 variant may have between about 2 and about 35 modifications, between about 5 and about 10 modifications, between about 5 and about 20 modifications, between about 10 and about 20 modifications, between about 15 and about 25 modifications, between about 20 and about 30 modifications, between about 20 and about 40 modifications, between about 25 and about 45 modifications, between about 25 and about 100 modifications, between about 25 and about 45 modifications, or between about 35 and about 100 modifications, when compared to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:9.

[0029] In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 9. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having between about 70% and about 99.9% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 9. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having between about 70% and about 99.5% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 9. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having between about 90% and about 99.9% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:9 or SEQ ID NO:10. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having between about 90% and about 99.8% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:9. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having between about 95% and about 99.9% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:9. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having between about 95% and about 99.8% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:9. In embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus comprising a CoV S polypeptide having between about 95% to about 99% identity to a CoV S polypeptide having the amino acid sequence of SEQ ID NO:9.In embodiments, the heterologous SARS-CoV-2 strain has a World Health Organization designation of alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu, or omicron. In embodiments, the heterologous SARS-CoV-2 strain has a PANGO lineage selected from the group consisting of B.1.1.529; BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1. The following reference describes the nomenclature of Pango strains and is incorporated by reference in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0030] In embodiments, the heterologous SARS-CoV-2 strain has an Omicron World Health Organization tag. In embodiments, the heterologous SARS-CoV-2 strain with an Omicron World Health Organization tag has at least 35 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 9. In embodiments, the heterologous SARS-CoV-2 strain with an Omicron World Health Organization tag has 35-55, 35-65, 35-75, 35-85, 35-95, or 35-105 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 9. In embodiments, the modifications are T6I, T6R, A14S, A54V, V70A, T82I, G129D, H133Q, K134E, W139R, E143G, F144L, Q170E, I197V, L199I, V200E, V200G, G239V, G244S, G326D, G326H, R333T, L355I, S358 F, S358L, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, V432P, G433S, L439 R, L439Q, N447K, S464N, T465K, E471A, F473V, F473S, F477S, Q480R, G483S, Q485R, N488Y , Y492H, T534K, T591I, D601G, G626V, H642Y, N645S, N666K, P668H, S691L, N751K, D783Y, N843K, Q941H, N956K, L968F, D1186N, a deletion of amino acid 11, a deletion of amino acid 12, a deletion of amino acid 13, a deletion of amino acid 56, a deletion of amino acid 57, a deletion of amino acid 130, a deletion of amino acid 131, a deletion of amino acid 132, a deletion of amino acid 144, a deletion of amino acid 145, a deletion of amino acid 198, and an insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215, and combinations thereof.

[0031] In embodiments, the variant CoV S polypeptide comprises a combination of modifications selected from the group consisting of: (i)A54V, T82I, G129D, L199I, G326D, S358L, S360P, S362F, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N 751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215; (ii) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (iii) T6R, A14S, T82I, G129D, E143G, L199I, G326D, S358L, S360P, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N751K, D783Y, N843K, Q941H, N956K, L968F, a deletion of amino acid 144, a deletion of amino acid 145, a deletion of amino acid 198, and an insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215; (iv) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, K404N, N427K, L439Q, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, S691L, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (v) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (vi) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, D601G, H642Y, N645S, N666K, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (vii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, G626V, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (viii) V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (ix) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (x) T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, N447K, S464N, T465K, E471A, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (xi)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, L439R, N447K, S 464N, T465K, E471A, F473S, Q485R, N488Y, Y492H, T591I, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, D1186N, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (xii) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N645S, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xiii) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xiv)T6I, A14S, V70A, G129D, H133Q, Q170E, V200E, G239V, G326H, R333T, L355I, S 358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S46 4N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, and deletion of amino acid 131; (xv)T6I, A14S, G129D, H133Q, Q170E, V200E, G326H, R333T, L355I, S358F, S360P, S36 2F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F 473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131; (xvi) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xvii) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; and (xviii) T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131; (xix) a deletion of amino acid 56, a deletion of amino acid 57, and a deletion of amino acid 131, N488Y, A557D, D601G, P668H or P668R, T703I, S969A, and D1105H; (xx) D67A, K404N, E471K, N488Y, D601G, and A688V; (xxi) D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V; (xxii) D67A, D202G, deletion of one, two, or three amino acids within amino acids 228 to 230, K404N, E471K, N488Y, D601G, and A688V; (xxiii) D67A, L229H, R233I, N488Y, K404N, E471K, D601G, and A688V; (xxiv) L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F; (xxv) W139C and L439; (xxvi) deletion of amino acid 144, deletion of amino acid 145, T6R, E143G, L439R, T465K, D601G, P668R, and D937N; (xxvii) deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, L439R, T465K, D601G, P668R, and D937N; (xxviii) deletion of amino acid 144, deletion of amino acid 145, T6R, T82I, G129D, Y132H, E143G, A209V, K404N L439R, T465K, D601G, P668R, and D937N; (xxix) deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R, and D937N; (xxx) deletion of amino acid 144, deletion of amino acid 145, T6R, W51H, H53W, G129D, E143G, D200V, L201R, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R, and D937N; (xxxi) deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, K404N, L439R, T465K, E471Q, D601G, P668R, and D937N; (xxxii) Q39R, A54V, E471K; D601G, Q664H, F875L, and deletion of 1, 2, 3, or 4 of amino acids 56, 57, 131, 132; (xxxiii) T82I, D240G, E471K, D601G, and A688V; (xxxiv) L439R, E471Q, D601G, P668R, and Q1058H; (xxxv) G62V, T63I, R233N, L439Q, F477S, D601G, T846N, and deletion of 1, 2, 3, 4, 5, or 6 of amino acids 234 to 240; (xxxvi) T82I, Y131S, Y132N, R333K, E471K, N488Y, D601G, P668H, and D937N; and (xxxvii)G129D, G326D, S360P, S362F, K404N, N427K, T465K, E471A or E471K, Q480K or Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, and N953K; Here, the amino acids of the CoV S glycoprotein are numbered relative to the polypeptide having the amino acid sequence of SEQ ID NO:9.

[0032] As used herein, the terms "antibody" and "antibodies" (immunoglobulin) encompass monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFvs), single-chain antibodies, single domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments exhibiting the desired biological activity, disulfide-linked Fvs (sdFvs), and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to an antibody of the invention), intrabodies, and epitope-binding fragments of any of the above. In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen-binding site. The immunoglobulin molecule can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

[0033] Native antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, but the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by several constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end, with the constant domain of the light chain aligned with the first constant domain of the heavy chain and the light chain variable domain aligned with the variable domain of the heavy chain. Light chains are classified as either lambda or kappa chains based on the amino acid sequence of the light chain constant region. The variable domain of a kappa light chain may also be referred to herein as VK. The term "variable region" may also be used to describe the variable domain of a heavy or light chain. Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains. Such antibodies may be from any animal, including, but not limited to, human, monkey, pig, horse, rabbit, dog, cat, mouse, etc.

[0034] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody to its particular antigen. However, the variability is not uniformly distributed throughout the variable domains of antibodies. It is concentrated in segments called complementarity determining regions (CDRs) in both the light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FWs). Native heavy and light chain variable domains each contain four FW regions that predominantly adopt a β-sheet configuration, connected by three CDRs, which form loop connections and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held together in close proximity by the FW regions and, together with the CDRs from the other chain, contribute to the formation of the antigen-binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domains are generally not directly involved in antigen binding, but may influence antigen binding affinity and exhibit various effector functions, such as participation of the antibody in ADCC, CDC, and / or apoptosis.

[0035] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are involved in its binding to antigen. The hypervariable region is defined as amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., residues 24-34 (VL CDR1), 50-56 (VL CDR2), and 89-97 (VL CDR3) of the light chain variable domain, and residues 31-35 (VH CDR1), 50-65 (VH CDR2), and 95-102 (VH CDR3) of the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and / or residues from the "hypervariable loops" (e.g., residues 26-32 (VL CDR1), 50-52 (VL CDR2), and 91-96 (VL CDR3) of the light chain variable domain, and residues 26-32 (VH CDR1), 53-55 (VH CDR3) of the heavy chain variable domain). "Framework" or "FW" residues are those variable domain residues which flank the CDRs. FW residues are present in chimeric, humanized, human, domain antibodies, diabodies, vaccibodies, linear antibodies, and bispecific antibodies.

[0036] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies that make up the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized by hybridoma cells uncontaminated by other immunoglobulin-producing cells. Alternative production methods are known to those skilled in the art, for example, monoclonal antibodies can be produced by cells stably or transiently transfected with heavy and light chain genes encoding the monoclonal antibody.

[0037] The modifier "monoclonal" refers to the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and should not be construed as requiring the antibody to be engineered by any particular method. The term "monoclonal" is used herein to refer to an antibody derived from a clonal population of cells, including any eukaryotic, prokaryotic, or phage clone, and not the method by which the antibody was engineered. For example, the monoclonal antibodies used in accordance with the present invention may be produced by the hybridoma method first described, for example, by Kohler et al., Nature, 256:495 (1975), or by any recombinant DNA method (see, for example, U.S. Patent No. 4,816,567), including isolation from a phage antibody library using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991). These methods can be used to produce monoclonal mammalian, chimeric, humanized, human, domain antibodies, diabodies, vaccibodies, linear antibodies, and bispecific antibodies.

[0038] The term "chimeric" antibody includes antibodies in which at least one portion of the heavy and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and at least one other portion of the chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey, e.g., a baboon, a rhesus monkey, or a cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780).

[0039] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibodies) in which native CDR residues have been replaced by residues from the corresponding CDRs of a non-human species (donor antibody), e.g., mouse, rat, rabbit, or non-human primate, with the desired specificity, affinity, and capacity. In some instances, residues in the FW regions of the human immunoglobulin have been replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are not found in either the recipient or donor antibody. These modifications are made to further refine antibody performance. In general, the heavy or light chain of a humanized antibody will comprise substantially all of at least one or more variable domains, with all or substantially all of the CDRs corresponding to those of a non-human immunoglobulin, and all or substantially all of the FWs being of human immunoglobulin sequences. In certain embodiments, a humanized antibody will comprise at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0040] A "human antibody" may be an antibody derived from a human or from a transgenic organism that has been "engineered" to produce specific human antibodies in response to antigen challenge, and may be produced by any method known in the art. In one particular technique, elements of human heavy and light chain loci are introduced into a strain of organism derived from an embryonic stem cell line that contains targeted disruptions of endogenous heavy and light chain loci. The transgenic organism is capable of synthesizing human antibodies specific to human antigens, and the organism can be used to produce hybridomas that secrete human antibodies. A human antibody may also be an antibody in which the heavy and light chains are encoded by nucleotide sequences derived from one or more sources of human DNA. Fully human antibodies may also be constructed by genetic or chromosomal transfection methods, and phage display technology, or in vitro activated B cells, all of which are known in the art.

[0041] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells and subsequently cause lysis of the target cells. In one embodiment, such cells are human cells. Without wishing to be limited to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs). NK cells, the primary cells for mediating ADCC, express FcγRIII, whereas monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To evaluate the ADCC activity of a molecule, in vitro ADCC assays can be carried out, such as those described in U.S. Patent No. 5,500,362 or 5,821,337.Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells.Alternatively, or in addition, the ADCC activity of a molecule of interest can be evaluated in vivo, for example, in an animal model, such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998).

[0042] "Complement-dependent cytotoxicity" or "CDC" refers to the ability of a molecule to initiate complement activation and lyse a target in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule (e.g., an antibody) complexed with a cognate antigen. To assess complement activation, a CDC assay may be performed, for example, as described in Gazzano-Santaro et al., J. Immunol. Methods, 202:163 (1996).

[0043] An "effector cell" is a white blood cell that expresses one or more FcRs and performs effector function. The cell expresses at least FcγRI, FcγRII, FcγRII and / or FcγRIV and performs ADCC effector function. Examples of human white blood cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils.

[0044] The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody. In one embodiment, the FcR is a native sequence human FcR. In certain embodiments, the FcR also binds IgG antibodies (gamma receptors), including allelic variants and alternatively spliced ​​forms of these receptors, and includes receptors of the FcγRI, FcγRII, FcγRII, and FcγRIV subclasses. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See Daeron, Annu. Rev. Immunol., 15:203-234 (1997). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

[0045] "Fv" is the minimum antibody fragment that contains a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent or covalent association. In this arrangement, the three CDRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen binding specificity to the antibody. However, even a single variable domain (or half of an Fv that contains only three CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

[0046] The "affinity" of an antibody for an epitope used in the treatments described herein is a term well understood in the art and refers to the degree, or strength, of binding of an antibody to an epitope. Affinity can be measured and / or expressed in several ways known in the art, including, but not limited to, equilibrium dissociation constant (KD or Kd), apparent equilibrium dissociation constant (KD' or Kd'), and IC50 (amount required to produce 50% inhibition in a competitive assay). For the purposes of the present invention, affinity is understood to be the average affinity for a given population of antibodies binding to an epitope. The KD' values ​​reported herein in mg IgG per mL or mg / mL refer to mg Ig per mL of serum, although plasma can be used. When antibody affinity is used as a basis for administration of or selection for the treatment methods described herein, antibody affinity can be measured before and / or during treatment, and the resulting values ​​can be used by clinicians in assessing whether a human patient is a suitable candidate for treatment.

[0047] As used herein, the term "avidity" is a measure of the overall binding strength (i.e., both antibody arms) of an antibody binding to an antigen. Avidity depends on three factors: (i) the affinity of the antibody for the epitope on the antigen; (ii) the valency of both the antibody and the antigen; and (iii) the structural arrangement of the interacting moieties. Antibody avidity can be determined by any means known in the art, for example, but not limited to, by measuring the dissociation of antigen-antibody binding in antigen excess, by modification of indirect fluorescent antibody as described in Gray et al., J. Virol. Meth., 44:11-24. (1993).

[0048] As used herein, the term "neutralizing antibody" refers to an antibody that reduces the ability of a pathogen to initiate or persist an infection in a host. A neutralizing anti-CoV S glycoprotein antibody is an antibody that reduces the ability of the SARS-CoV-2 virus or a variant thereof to initiate or persist an infection in a host.

[0049] "Epitope" is a term well understood in the art and means any chemical moiety that exhibits specific binding to an antibody. An "antigen" is a moiety or molecule that contains an epitope and therefore also specifically binds to an antibody.

[0050] The term "antibody half-life" as used herein refers to a pharmacokinetic property of an antibody that is a measure of the average survival time of antibody molecules after their administration. Antibody half-life can be expressed as the time required to eliminate 50 percent of a known amount of immunoglobulin from a patient's body or a specific compartment thereof, for example, as measured in serum or plasma, i.e., circulating half-life, or as measured in other tissues. Half-life can vary depending on the immunoglobulin or class of immunoglobulin. In general, an increase in antibody half-life results in an increase in the mean residence time (MRT) in the circulation for the administered antibody.

[0051] The term "isotype" refers to the classification of the heavy or light chain constant region of an antibody. The constant domain of an antibody is not involved in binding to an antigen, but exhibits various effector functions. Depending on the amino acid sequence of the heavy chain constant region, a given human antibody or immunoglobulin can be assigned to one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further divided into subclasses (isotypes), e.g., IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma 3), and IgG4 (gamma 4), as well as IgA1 and IgA2. The heavy chain constant regions corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The structures and three-dimensional configurations of the different classes of immunoglobulins are well known. Of the various human immunoglobulin classes, only human IgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. Human IgG1 and IgG3 are known to mediate ADCC in humans. Human light chain constant regions can be classified into two major classes, kappa and lambda.

[0052] As used herein, the term "immunogenic" means that a compound is capable of eliciting an immune response (stimulating the production of specific antibodies and / or the proliferation of specific T cells).

[0053] As used herein, the term "broadly neutralizing antibody" refers to an antibody or fragment thereof that binds to the SARS-CoV-2 S glycoprotein of two or more heterologous SARS-CoV-2 strains. In embodiments, a broadly neutralizing antibody binds to the SARS-CoV-2 S glycoprotein of at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, or at least 20 heterologous SARS-CoV-2 strains. In embodiments, the broadly neutralizing antibody binds to the SARS-CoV-2 S glycoprotein of at least 2, and up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, up to 10, up to 11, up to 12, up to 13, up to 14, up to 15, up to 16, up to 17, up to 18, up to 19, or up to 20 heterologous SARS-CoV-2 strains. In embodiments, the broadly neutralizing antibody binds to the SARS-CoV-2 S glycoprotein of between 2 and 10 heterologous SARS-CoV-2 strains.

[0054] Antibodies that bind to the SARS-CoV-2 spike polypeptide

[0055] The present invention relates to antibodies that bind to the SARS-CoV-2 spike polypeptide and variants thereof (anti-CoV S glycoprotein antibodies), and compositions comprising such antibodies. The SARS-CoV-2 spike polypeptide ("CoV S glycoprotein") may comprise the following amino acid sequence:

[0056] [ka]

[0057] In embodiments, the CoV S glycoprotein comprises an N-terminal signal peptide, and the protein has the amino acid sequence of SEQ ID NO: 10. The signal peptide is underlined.

[0058] [ka]

[0059] The CoV S glycoprotein (SEQ ID NO:9) is divided into an S1 subunit (amino acids 1-672 of SEQ ID NO:9) and an S2 subunit (amino acids 673-1260 of SEQ ID NO:9). The S1 subunit is further divided into an N-terminal domain (NTD, amino acids 1-318 of SEQ ID NO:9), a receptor binding domain (RBD, amino acids 318-514 of SEQ ID NO:9), subdomains 1 and 2 (SD1 / 2, amino acids 529-668 of SEQ ID NO:9), and a furin cleavage site (amino acids 669-672 of SEQ ID NO:2). The S2 subunit contains an HR1 domain (amino acids 889-971 of SEQ ID NO:9), an HR2 domain (amino acids 1150-1200 of SEQ ID NO:2), a transmembrane domain (TM, amino acids 1201-1224 of SEQ ID NO:2), and a cytoplasmic domain (CD, amino acids 1225-1260 of SEQ ID NO:9). In embodiments, the anti-CoV S glycoprotein antibody binds to the S1 subunit, the S2 subunit, the NTD, the RBD, the furin cleavage site, the HR1 domain, the TM domain, the CD, or a combination thereof of the SARS-CoV 2 S glycoprotein.

[0060] In embodiments, the CoV S glycoprotein has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 modifications.

[0061] Exemplary modifications to the CoV S glycoprotein are shown in the table below. [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6] [Table 6-7] [Table 6-8] [Table 6-9] [Table 6-10]

[0062] In embodiments, the CoV S glycoprotein has a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 9, 10, 35-43, 72, 73, 90-139, and 145-147.

[0063] In embodiments, anti-CoV S glycoprotein antibodies may mediate antigen-dependent cell-mediated cytotoxicity (ADCC). In embodiments, the invention is directed to anti-CoV S glycoprotein antibodies of the IgG1, IgG2, IgG3, IgG4, or IgG5 isotype. In embodiments, the antibodies mediate human ADCC, CDC, and / or apoptosis.

[0064] In one embodiment, the anti-CoV S glycoprotein antibody comprises a variable heavy chain (VH) and a variable light chain (VL). In an embodiment, the anti-CoV S glycoprotein antibody comprises a VL having an amino acid sequence of any one of SEQ ID NOs: 1-4 and 74, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-4 and 74. In an embodiment, the anti-CoV S glycoprotein antibody comprises a VH having an amino acid sequence of any one of SEQ ID NOs: 5-8 and 75, or an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 5-8 and 75. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL of SEQ ID NO: 1, or a VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and a VH of SEQ ID NO: 5, or a VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL of SEQ ID NO: 2, or a VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, and a VH of SEQ ID NO: 6, or a VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.In embodiments, the anti-CoV S glycoprotein antibody comprises a VL of SEQ ID NO: 3, or a VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3, and a VH of SEQ ID NO: 7, or a VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL of SEQ ID NO: 4, or a VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4, and a VH of SEQ ID NO: 8, or a VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL of SEQ ID NO:75, or a VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:75, and a VH of SEQ ID NO:74, or a VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO:74.

[0065] In an embodiment, the VL of SEQ ID NO: 1-4 comprises an N-terminal leader sequence. Up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids of the N-terminal leader sequence of any one of SEQ ID NO: 1-4 and 74 may be removed. In an embodiment, an antibody comprising a VL without an N-terminal leader sequence is provided herein. In an embodiment, the VH of any one of SEQ ID NO: 5-8 and 75 comprises an N-terminal leader sequence. Up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids of the N-terminal leader sequence of any one of SEQ ID NO: 5-8 and 75 may be removed. In an embodiment, an antibody comprising a VH without an N-terminal leader sequence is provided herein. In embodiments, the antibodies described herein comprise up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids of the N-terminal leader sequence of the VH or VL.

[0066] In embodiments, the VL and VH are selected from Table 1 below. The solid underlined amino acids are the N-terminal leader sequences of the VL and VH. The bolded amino acids are the CDRs of each VL and VH. The framework regions are dotted underlined. [Table 1-1] [Table 1-2]

[0067] In an embodiment, the anti-CoV S glycoprotein antibody comprises a variable heavy chain complementarity determining region 1 (VH CDR1) having an amino acid sequence of any one of SEQ ID NOs: 23-26 and 79. In an embodiment, the anti-CoV S glycoprotein antibody comprises a variable heavy chain complementarity determining region 2 (VH CDR2) having an amino acid sequence of any one of SEQ ID NOs: 27-30 and 80. In an embodiment, the anti-CoV S glycoprotein antibody comprises a variable heavy chain complementarity determining region 3 (VH CDR3) having an amino acid sequence of any one of SEQ ID NOs: 31-34 and 81. In an embodiment, the anti-CoV S glycoprotein antibody comprises a variable light chain complementarity determining region 1 (VL CDR1) having an amino acid sequence of any one of SEQ ID NOs: 11-14 and 76. In an embodiment, the anti-CoV S glycoprotein antibody comprises a variable light chain complementarity determining region 2 (VL CDR2) having an amino acid sequence of any one of SEQ ID NOs: 15-18 and 77. In embodiments, the anti-CoV S glycoprotein antibody comprises a variable light chain complementarity determining region 3 (VL CDR3) having the amino acid sequence of any one of SEQ ID NOs: 19-22 and 78.

[0068] In embodiments, provided herein is an anti-CoV S glycoprotein antibody comprising a VL CDR1 selected from the group consisting of SEQ ID NOs: 11-14, and 76; a VL CDR2 selected from the group consisting of SEQ ID NOs: 15-18, and 77; a VL CDR3 selected from the group consisting of SEQ ID NOs: 19-22, and 78; a VH CDR1 selected from the group consisting of SEQ ID NOs: 23-26, and 79; a VH CDR2 selected from the group consisting of SEQ ID NOs: 27-30, and 80; and a VH CDR3 selected from the group consisting of SEQ ID NOs: 31-34, and 81.

[0069] In embodiments, the VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are independently selected from Table 2. [Table 2-1] [Table 2-2]

[0070] In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 23, a VH CDR2 of SEQ ID NO: 27, and a VH CDR3 of SEQ ID NO: 31. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL CDR1 of SEQ ID NO: 11, a VL CDR2 of SEQ ID NO: 15, and a VL CDR3 of SEQ ID NO: 19. In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 23, a VH CDR2 of SEQ ID NO: 27, and a VH CDR3 of SEQ ID NO: 31, as well as a VL CDR1 of SEQ ID NO: 11, a VL CDR2 of SEQ ID NO: 15, and a VL CDR3 of SEQ ID NO: 19.

[0071] In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 24, a VH CDR2 of SEQ ID NO: 28, and a VH CDR3 of SEQ ID NO: 32. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 16, and a VL CDR3 of SEQ ID NO: 20. In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 24, a VH CDR2 of SEQ ID NO: 28, and a VH CDR3 of SEQ ID NO: 32, as well as a VL CDR1 of SEQ ID NO: 12, a VL CDR2 of SEQ ID NO: 16, and a VL CDR3 of SEQ ID NO: 20.

[0072] In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 25, a VH CDR2 of SEQ ID NO: 29, and a VH CDR3 of SEQ ID NO: 33. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL CDR1 of SEQ ID NO: 13, a VL CDR2 of SEQ ID NO: 17, and a VL CDR3 of SEQ ID NO: 21. In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 25, a VH CDR2 of SEQ ID NO: 29, and a VH CDR3 of SEQ ID NO: 33, as well as a VL CDR1 of SEQ ID NO: 13, a VL CDR2 of SEQ ID NO: 17, and a VL CDR3 of SEQ ID NO: 21.

[0073] In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 26, a VH CDR2 of SEQ ID NO: 30, and a VH CDR3 of SEQ ID NO: 34. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL CDR1 of SEQ ID NO: 14, a VL CDR2 of SEQ ID NO: 18, and a VL CDR3 of SEQ ID NO: 22. In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 26, a VH CDR2 of SEQ ID NO: 30, and a VH CDR3 of SEQ ID NO: 34, as well as a VL CDR1 of SEQ ID NO: 14, a VL CDR2 of SEQ ID NO: 18, and a VL CDR3 of SEQ ID NO: 22.

[0074] In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO: 80, and a VH CDR3 of SEQ ID NO: 81. In embodiments, the anti-CoV S glycoprotein antibody comprises a VL CDR1 of SEQ ID NO: 76, a VL CDR2 of SEQ ID NO: 77, and a VL CDR3 of SEQ ID NO: 78. In embodiments, the anti-CoV S glycoprotein antibody comprises a VH CDR1 of SEQ ID NO: 79, a VH CDR2 of SEQ ID NO: 80, and a VH CDR3 of SEQ ID NO: 81, as well as a VL CDR1 of SEQ ID NO: 76, a VL CDR2 of SEQ ID NO: 77, and a VL CDR3 of SEQ ID NO: 78.

[0075] The present invention encompasses antibodies that bind to SARS-CoV 2 S glycoprotein, comprising derivatives of a VH domain, VH CDR1, VH CDR2, VH CDR3, VK domain, VK CDR1, VK CDR2, or VK CDR3 described herein that are capable of binding to SARS-CoV 2 S glycoprotein or a variant thereof. In embodiments, the anti-CoV S glycoprotein antibody binds to the CoV S glycoprotein of a SARS-CoV-2 strain having a PANGO lineage selected from the group consisting of: B.1.1.529; BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1.

[0076] Modifications (e.g., additions, deletions, and / or substitutions) can be introduced into the nucleotide sequence encoding the antibody using standard techniques known to those of skill in the art, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which are routinely used to create amino acid substitutions. In another embodiment, the VH and / or VK CDR derivatives may have conservative amino acid substitutions (e.g., as described above) made at one or more predicted non-essential amino acid residues (i.e., amino acid residues that are not important for the antibody to specifically bind to the SARS-CoV-2 S glycoprotein). Mutations can also be randomly introduced along all or part of the VH and / or VL CDR coding sequence, for example, by saturation mutagenesis, and the resulting mutations can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.

[0077] The present invention further encompasses antibodies that bind to the SARS-CoV-2 S glycoprotein, wherein the antibody or antibody fragment comprises one or more CDRs, the CDRs comprising an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence of one or more CDRs described herein. The percent identity of two amino acid sequences can be determined by any method known to one of skill in the art, including, but not limited to, BLAST protein searches.

[0078] Framework regions of anti-CoV S glycoprotein antibodies

[0079] In embodiments, the anti-CoV S glycoprotein antibody comprises a VL and a VH each containing four framework regions (FW1, FW2, FW3, and FW4). In embodiments, FW1, FW2, FW3, and FW4 of the VL are independently selected from Table 4. In embodiments, FW1, FW2, FW3, and FW4 of the VH are independently selected from Table 5. [Table 4-1] [Table 4-2] [Table 5-1] [Table 5-2]

[0080] The Kabat numbering is based on the seminal work of Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Publication No. 91-3242 (published in a three-volume set by the National Institutes of Health, National Technical Information Service) (hereinafter "Kabat"). Kabat provides a multiple sequence alignment of immunoglobulin chains from antibody isotypes of multiple species. The aligned sequences are numbered according to a single numbering system, the Kabat numbering system. The Kabat sequences have been updated since their publication in 1991 and are available as an electronic sequence database (latest downloadable version 1997). Any immunoglobulin sequence can be numbered according to Kabat by aligning it with the Kabat reference sequence. Thus, the Kabat numbering system provides a uniform system for numbering immunoglobulin chains. Unless otherwise indicated, all immunoglobulin amino acid sequences described herein are numbered according to the Kabat numbering system. Similarly, every single amino acid position referred to herein is numbered according to the Kabat numbering system.

[0081] In another embodiment, the anti-CoV S glycoprotein antibody of the invention has a nucleotide sequence of at least 10 2 M -1 , at least 5 × 10 2 M -1 , at least 10 3 M -1 , at least 5 × 10 3 M -1 , at least 10 4 M -1 , at least 5 × 10 4 M -1 , at least 10 5 M -1 , at least 5 × 10 5 M -1 , at least 10 6M -1 , at least 5 × 10 6 M -1 , at least 10 7 M -1 , at least 5 × 10 7 M -1 , at least 10 8 M -1 , at least 5 × 10 8 M -1 , at least 10 9 M -1 , at least 5 × 10 9 M -1 , at least 10 10 M -1 , at least 5 × 10 10 M -1 , at least 10 11 M -1 At least 5×10 11 M -1 , at least 10 12 M -1 , at least 5 × 10 12 M -1 , at least 10 13 M -1 At least 5×10 13 M -1 , at least 10 14 M -1 , at least 5 × 10 14 M -1 , at least 10 15 M -1 , or at least 5 × 10 15 M -1 Affinity constant or K a (k on / k off In embodiments, the anti-CoV S glycoprotein antibodies of the invention may have a 5×10 antibody titer, as assessed using the methods described herein or known to those of skill in the art (e.g., BIAcore assay, ELISA). -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×10 -4 Less than M, 10 -4 Less than M, 5×10 -5 Less than M, 10 -5Less than M, 5×10 -6 Less than M, 10 -6 Less than M, 5×10 -7 Less than M, 10 -7 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -9 Less than M, 10 -9 Less than M, 5×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, 10 -11 Less than M, 5×10 -12 Less than M, 10 -12 Less than M, 5×10 -13 Less than M, 10 -13 Less than M, 5×10 -14 Less than M, 10 -14 Less than M, 5×10 -15 Less than M or 10 -15 Dissociation constant or K less than M d (k off / k on ).

[0082] The present invention further provides a polynucleotide comprising a nucleotide sequence encoding an anti-CoV S glycoprotein antibody or fragment thereof described herein. The present invention also encompasses polynucleotides, e.g., as defined herein, that hybridize under stringent or less stringent hybridization conditions to a polynucleotide encoding an anti-CoV S glycoprotein antibody.

[0083] Stringent hybridization conditions include, but are not limited to, hybridization to filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by one or more washes in 0.2×SSC / 0.1% SDS at about 50-65° C., highly stringent conditions, such as hybridization to filter-bound DNA in 6×SSC at about 45° C., followed by one or more washes in 0.1×SSC / 0.2% SDS at about 60° C., or any other stringent hybridization conditions known to those of skill in the art (see, e.g., Ausubel, FM et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY at pages 6.3.1 to 6.3.6 and 2.10.3).

[0084] Polynucleotide can be obtained and the nucleotide sequence of polynucleotide can be determined by any method known in the art.For example, if the nucleotide sequence of antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (for example, as described in Kutmeier et al., BioTechniques 17:242 (1994)), which briefly includes the synthesis of overlapping oligonucleotides that contain the part of the sequence encoding the antibody, the annealing and ligation of the oligonucleotides, and then the amplification of the ligated oligonucleotides by PCR.

[0085] Polynucleotides encoding antibodies may also be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, nucleic acid encoding an immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library or a cDNA library generated from any tissue or cell expressing the antibody, e.g., a hybridoma cell selected to express the antibody, or nucleic acid isolated therefrom, preferably polyA+RNA) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific for a particular gene sequence, e.g., to identify a cDNA clone from a cDNA library encoding the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.

[0086] The invention also provides polynucleotide sequences encoding the VH and VL framework regions and CDRs of the antibodies described herein, as well as expression vectors for their efficient expression in mammalian cells.

[0087] In one embodiment, the anti-CoV S glycoprotein antibodies described herein mediate antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and / or apoptosis. In one embodiment, the anti-CoV S glycoprotein antibodies of the invention mediate antibody-dependent cellular cytotoxicity (ADCC) and / or apoptosis. In one embodiment, the anti-CoV S glycoprotein antibodies of the invention have enhanced antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the anti-CoV S glycoprotein antibodies of the invention comprise a variant Fc region that mediates enhanced antibody-dependent cellular cytotoxicity (ADCC). In one embodiment, the anti-CoV S glycoprotein antibodies of the invention comprise an Fc region having a complex N-glycoside-linked glycan linked to Asn297 that is not linked to N-acetylglucosamine at the end where the fucose is reduced, said Fc region mediating enhanced antibody-dependent cellular cytotoxicity (ADCC).

[0088] Production of humanized anti-CoV S glycoprotein antibodies Humanized antibodies described herein may be modified by any method known in the art, including, but not limited to, CDR-grafting (see, e.g., European Patent No. EP 239,400; International Publication No. WO 91 / 09967; and U.S. Pat. Nos. 5,225,539, 5,530,101, and 5,585,089, each of which is incorporated by reference in its entirety), veneering or resurfacing (see, e.g., European Patent Nos. EP 592,106 and EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al., 1994, Proc. Natl. Acad. Sci., 1997). 91:969-973, each of which is incorporated herein by reference in its entirety), chain shuffling (see, e.g., U.S. Pat. No. 5,565,332, which is incorporated herein by reference in its entirety), as well as other techniques such as those described in, e.g., U.S. Pat. No. 6,407,213, U.S. Pat. No. 5,766,886, International Publication No. WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55 (23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994), each of which is incorporated herein by reference in its entirety. Often, FW residues in the FW region are substituted with corresponding residues from the CDR donor antibody to alter, preferably improve, antigen binding. These FW substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and FW residues to identify FW residues important for antigen binding, and by sequence comparison to identify unusual FW residues at particular positions (see, e.g., Queen et al., U.S. Pat. No. 5,585,089; and Riechmann et al., 1988, Nature, 332:323, which are incorporated herein by reference in their entirety).

[0089] A humanized anti-CoV S glycoprotein antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Thus, a humanized antibody comprises one or more CDRs from a non-human immunoglobulin molecule and a framework region from a human. Antibody humanization is well known in the art and can be performed essentially according to the method of Winter and coworkers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1989)). (1988)), by replacing rodent CDRs or CDR sequences with the corresponding sequences of a human antibody, i.e., CDR grafting (EP 239,400; PCT Publication WO 91 / 09967; and U.S. Pat. Nos. 4,816,567; 6,331,415; 5,225,539; 5,530,101; 5,585,089; 6,548,640, the contents of which are incorporated herein by reference in their entireties). In such humanized chimeric antibodies, substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FW residues are replaced by residues from analogous sites in rodent antibodies.Humanization of anti-CoV S glycoprotein antibodies can also be achieved by veneering or resurfacing (EP 592,106; EP 519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., Protein Engineering, 7(6):805-814 (1994); and Roguska et al., Proc. Natl. Acad. Sci., 91:969-973 (1994)) or chain shuffling (U.S. Patent No. 5,565,332), the contents of which are incorporated herein by reference in their entireties.

[0090] The selection of both light and heavy human variable domains used in the production of humanized antibodies is to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is most closely related to that of the rodent is then screened for the presence of specific residues that may be important for antigen binding, proper structure formation and / or stability of the intended humanized mAb (Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987), the contents of which are incorporated herein by reference in their entirety). The resulting FW sequence that matches the desired criteria is then used as the human donor FW region for the humanized antibody.

[0091] Another method uses a specific FW derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same FW may be used for several different humanized anti-CoV S glycoprotein antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993), the contents of which are incorporated herein by reference in their entirety).

[0092] Anti-CoV S glycoprotein antibodies can be humanized with high affinity for SARS-CoV-2 S glycoprotein and with retention of other desirable biological properties. According to one aspect of the invention, humanized antibodies are prepared by a process of analysis of parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and are familiar to those skilled in the art. Computer programs are available that illustrate and display the structures of the predicted three-dimensional configurations of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind to the SARS-CoV-2 S glycoprotein. In this way, FW residues can be selected and combined from the recipient and import sequences such that the desired antibody characteristics, e.g., affinity for the SARS-CoV-2 S glycoprotein, are achieved. In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0093] A "humanized" antibody may retain a similar antigen specificity as the original antibody, i.e., in the present invention, the ability to bind to the SARS-CoV-2 S glycoprotein. However, using certain methods of humanization, the binding affinity and / or specificity of an antibody to the SARS-CoV-2 S glycoprotein may be altered using the method of "directed evolution" as described by Wu et al., J. Mol. Biol, 294:151 (1999), the contents of which are incorporated herein by reference in their entirety.

[0094] The humanized anti-CoV S glycoprotein antibodies described herein can be constructed by selection of separate human framework regions for grafting of the 239.12, 322.3, 425.6, and 35.13 CDRs, as described herein.

[0095] Monoclonal anti-CoV S glycoprotein antibodies Monoclonal anti-CoV S glycoprotein antibodies exhibit binding specificity for SARS-CoV-2 antigens and may mediate human ADCC, CDC and / or apoptosis mechanisms. Such antibodies can be made using a wide variety of techniques known in the art, including the use of hybridoma, recombination, and phage display technologies, or a combination thereof. The antibodies are highly specific and directed to a single antigenic site. Engineered anti-CoV S glycoprotein antibodies can be produced by any means known in the art, including, but not limited to, the techniques described below and improvements thereto. Large-scale, high-yield production typically involves culturing host cells that produce the engineered anti-CoV S glycoprotein antibodies and recovering the anti-CoV S glycoprotein antibodies from the host cell culture.

[0096] Hybridoma Technology Monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al., in Monoclonal Antibodies and T Cell Hybridomas, 563-681 (Elsevier, NY, 1981), which are incorporated herein by reference in their entireties. For example, in the hybridoma method, a mouse or other suitable host animal, such as a hamster or macaque, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Lymphocytes may also be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0097] The hybridoma cells thus prepared are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridoma typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells.

[0098] A specific embodiment uses myeloma cells that fuse efficiently, support stable and high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. Among these, myeloma cell lines are mouse myeloma lines, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from the American Type Culture Collection, Rockville, MD, USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001 (1984);Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0099] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the SARS-CoV-2 S glycoprotein. The binding specificity of the monoclonal antibodies produced by the hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0100] After hybridoma cells producing antibodies of the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI 1640 medium. In addition, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0101] The monoclonal antibodies secreted by the subclones are preferably separated from the culture medium or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0102] Recombinant DNA techniques DNA encoding the anti-CoV S glycoprotein antibodies described herein is easily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to genes encoding the heavy and light chains of the anti-CoV S glycoprotein antibodies). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, to obtain the synthesis of anti-CoV S glycoprotein antibodies in the recombinant host cells.

[0103] In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. H and V LThe DNA sequence encoding the domain is amplified from an animal cDNA library (e.g., a human or mouse cDNA library of the affected tissue). H and V L The DNA encoding the domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages, including fd and M13, and V II and V LThe domain is usually recombinantly fused to either gene III or gene VIII of the phage. Phage expressing an antigen-binding domain that binds to a particular antigen can be selected or identified with the antigen, for example, using labeled antigen, or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to generate the antibodies of the invention include those described in Brinkman et al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J. Immunol. Methods, 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton et al., 1994, Advances in Immunology, 57:191-280; International Application No. PCT / GB91 / O1. 134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, Nos. 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108, each of which is incorporated herein by reference in its entirety.

[0104] After phage selection, as described in the above references, antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen-binding fragments, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, as described below. Techniques for recombinantly producing Fab, Fab', and F(ab')2 fragments can also be used, using methods known in the art, such as those disclosed in PCT Publication No. WO92 / 22324; Mullinax et al., 1992, BioTechniques, 12(6):864-869; Sawai et al., 1995, AJRI, 34:26-34; and Better et al., 1988, Science, 240:1041-1043 (the above references are incorporated by reference in their entirety).

[0105] Antibodies may be isolated from antibody phage libraries made using techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991). Marks et al., J. Mol. Biol., 222:581-597 (1991) describe the isolation of mouse and human antibodies, respectively, using phage libraries. Chain shuffling can be used in the production of high affinity (nM range) human antibodies (Marks et al., Bio / Technology, 10:779-783 (1992)), and can also be used in combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of anti-CoV S glycoprotein antibodies.

[0106] To generate a full antibody, a PCR primer containing the VH or VL nucleotide sequence, a restriction site, and a flanking sequence for protecting the restriction site can be used to amplify the VH or VL sequence in the scFv clone. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, such as the human gamma 4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region, such as the human kappa or lambda constant region. The vector for expressing the VH or VL domain can include an EF-la promoter, a secretion signal, a cloning site for the variable domain, a constant domain, and a selection marker such as neomycin. The VH and VL domains can also be cloned into one vector expressing the necessary constant region. The heavy chain conversion vector and the light chain conversion vector are then co-transfected into a cell line using techniques known to those skilled in the art to generate a stable or transient cell line expressing a full-length antibody, such as an IgG.

[0107] The DNA may also be modified, for example, by substituting the coding sequence with human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0108] Chimeric antibodies The anti-CoV S glycoprotein antibodies herein specifically include chimeric antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical or homologous to the corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and another portion of the chain is identical or homologous to the corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey, e.g., a baboon, a rhesus monkey, or a cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780).

[0109] In embodiments, the K of the anti-CoV S glycoprotein antibodies described herein or against the SARS-CoV-2 S glycoprotein are D can be 50nM or less, 10nM or less, 1nM or less, 0.5nM or less, 0.1nM or less, 0.05nM or less, 0.01nM or less, or 0.001nM or less. Methods and reagents suitable for determining such binding characteristics of the antibody of the present invention, or its modified / mutated derivatives, are known in the art and / or commercially available (see above, and for example, U.S. Patent No. 6,849,425, U.S. Patent No. 6,632,926, U.S. Patent No. 6,294,391, and U.S. Patent No. 6,143,574, each of which is incorporated herein by reference in its entirety). Also, instruments and software designed for such kinetic analysis are commercially available (e.g., Biacore® A100 and Biacore® 2000 instruments; Biacore International AB, Uppsala, Sweden).

[0110] Identity or similarity with respect to sequences is defined herein as the percentage of amino acid residues in a candidate sequence that are identical (i.e., the same residues) or similar (i.e., amino acid residues from the same group based on common side chain properties, see below) to an anti-CoV S glycoprotein antibody, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Neither N-terminal, C-terminal or internal extensions, deletions, nor insertions into the antibody sequence outside the variable domain shall be construed as affecting sequence identity or similarity.

[0111] Methods for comparing the identity of two or more sequences are well known in the art. Identity percentage is calculated using the online tool CLUSTALW2. The following default parameters can be used for CLUSTALW2 pairwise alignment: protein weight matrix=Gonnet; gap open=10; gap widening=0.1. Unless otherwise stated, the identity percentage herein is calculated using the CLUSTALW2 tool.

[0112] To generate an altered antibody, one or more amino acid alterations (e.g., substitutions) are introduced into one or more of the hypervariable regions of the species-dependent antibody. One or more alterations (e.g., substitutions) of framework region residues can also be introduced into the anti-CoV S glycoprotein antibody, where these result in improved binding affinity of the antibody mutant to the antigen from the second mammalian species. Examples of framework region residues to modify include those that non-covalently bind directly to the antigen (Amit et al., Science, 233:747-753 (1986)); those that interact with / affect the conformation of the CDRs (Chothia et al., J. Mol. Biol., 196:901-917(1987)); and / or those that modify the V domain of the CDRs (Chothia et al., J. Mol. Biol., 196:901-917(1987)). L- V HIn certain embodiments, modification of one or more of such framework region residues results in an enhancement of the binding affinity of the antibody to an antigen from a second mammalian species. For example, from about one to about five framework residues may be altered in this embodiment of the invention. Sometimes, this may be sufficient to yield an antibody mutant suitable for use in preclinical trials, even when no hypervariable region residues have been altered. Usually, however, the altered antibody will include additional hypervariable region modifications.

[0113] The hypervariable region residues that are altered may be varied randomly, particularly if the starting binding affinity of the anti-CoV S glycoprotein antibody for an antigen from a second mammalian species is such that such randomly produced altered antibodies can be readily screened.

[0114] One useful procedure for generating such altered antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells, Science, 244:1081-1085 (1989)). In this, one or more of the hypervariable region residues are replaced by alanine or polyalanine residues to affect amino acid interactions with an antigen from a second mammalian species. Those hypervariable region residues which demonstrate functional sensitivity to the substitution are then improved by introducing additional or other mutations at or for the sites of substitution. Thus, while the site for introducing amino acid sequence variation is predefined, the nature of the mutation per se need not be predefined. Ala mutants produced in this manner are screened for their biological activity as described herein.

[0115] Another procedure for generating such altered antibodies involves affinity maturation using phage display (Hawkins et al., J. Mol. Biol., 254:889-896 (1992) and Lowman et al., Biochemistry, 30(45):10832-10837 (1991)). Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene 111 product of M13 packaged within each particle. The phage-displayed mutants are then screened for their biological activity (e.g., binding affinity) as disclosed herein.

[0116] Mutations in the antibody sequence may include substitutions, deletions including internal deletions, additions including additions resulting in fusion proteins, or conservative substitutions of amino acid residues within and / or adjacent to the amino acid sequence, which results in a "silent" change in that the change produces a functionally equivalent anti-CoV S glycoprotein antibody. Conservative amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residues involved. For example, non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine, polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine, positively charged (basic) amino acids include arginine, lysine, and histidine, and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. In addition, glycine and proline are residues that can affect chain orientation. Non-conservative substitutions involve replacing a member of one of these classes with a member of another class. Furthermore, non-classical amino acids or chemical amino acid analogs can be introduced as substitutions or additions to the antibody sequence, if desired. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids, such as, in general, β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and amino acid analogs.

[0117] In another embodiment, the sites selected for modification are affinity matured using phage display (see above).

[0118] Any technique for mutagenesis known in the art can be used to alter individual nucleotides in the DNA sequence for the purposes of making amino acid substitutions in the antibody sequence or to create / remove restriction sites to facilitate further manipulation. Such techniques include, but are not limited to, chemical mutagenesis, in vitro site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Hutchinson, C. et al., J. Biol. Chem., 253:6551 (1978)), oligonucleotide-directed mutagenesis (Smith, Ann. Rev. Genet., 19:423-463 (1985); Hill et al., Methods Enzymol., 155:558-568 (1987)), PCR-based overlap extension (Ho et al., Gene, 77:51-59 (1989)), PCR-based megaprimer mutagenesis (Sarkar et al., Biotechniques, 8:404-407 (1990)), and the like. The modification can be confirmed by double-stranded dideoxy DNA sequencing.

[0119] In certain embodiments of the invention, anti-CoV S glycoprotein antibodies can be modified to produce fusion proteins, i.e., antibodies or fragments thereof fused to a heterologous protein, polypeptide or peptide.

[0120] Additional fusion proteins may be generated by the techniques of gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be used to alter the activity of anti-CoV S glycoprotein antibodies (e.g., antibodies or fragments thereof with higher affinity and lower dissociation rates). See generally U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, as well as Patten et al., 1997, Curr. Opinion Biotechnol., 8:724-33; Harayama, 1998, Trends Biotechnol. 16(2):76-82; Hansson et al., 1999, J. Mol. Biol., 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308- 313 (each of these patents and publications is incorporated herein by reference in its entirety). The antibody may further be a binding domain immunoglobulin fusion protein as described in U.S. Patent Application Publication No. 20030118592, U.S. Patent Application Publication No. 200330133939, and PCT Publication No. WO02 / 056910, all to Ledbetter et al., which are incorporated by reference in their entireties.

[0121] Domain Antibodies The anti-CoV S glycoprotein antibodies of the compositions and methods of the invention are domain antibodies, e.g., heavy (V) domain antibodies of human antibodies. H ) chain or light (V LThe domain antibody may be an antibody that contains a small functional binding unit of an antibody corresponding to the variable region of the IgG1 chain. Examples of domain antibodies include, but are not limited to, those available from Domantis Limited (Cambridge, UK) and Domantis Inc. (Cambridge, MA, USA) that are specific for therapeutic targets (see, for example, WO04 / 058821; WO04 / 003019; U.S. Patent Nos. 6,291,158; 6,582,915; 6,696,245; and 6,593,081).

[0122] Diamond Body In certain embodiments of the invention, the anti-CoV S glycoprotein antibody is a "diabody." The term "diabody" refers to a small antibody fragment with two antigen-binding sites, which comprises a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) (V H -V L (By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain, creating two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0123] linear antibody In certain embodiments of the invention, the anti-CoV S glycoprotein antibody is a linear antibody. A linear antibody comprises a pair of tandem Fd segments (V H -C H1 -V H -C H1 ) pairs. Linear antibodies can be bispecific or monospecific. See Zapata et al., Protein Eng., 8(10):1057-1062 (1995).

[0124] antibody fragment An "antibody fragment" includes a portion of a full-length antibody, generally the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab ’ , F(ab ’ ) 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0125] Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed above. Fab'-SH fragments can also be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Other techniques for the production of antibody fragments will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See, for example, WO93 / 16185. In certain embodiments, the antibody is not a Fab fragment.

[0126] bispecific antibody Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes.

[0127] Methods for making bispecific antibodies are known in the art (e.g., Millstein et al., Nature, 305:537-539 (1983); Traunecker et al., EMBO J., 10:3655-3659 (1991); Suresh et al., Methods in Enzymology, 121:210 (1986); Kostelny et al., J. Immunol., 148(5):1547-1553 (1992); Hollinger et al., Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993); Gruber et al., J. Immunol., 152:5368 (1994)). (1994); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,81; 95,731,168; 4,676,980; and 4,676,980, WO94 / 04690; WO91 / 00360; WO92 / 200373; WO93 / 17715; WO92 / 08802; and EP 03089).

[0128] In one embodiment, when the anti-CoV S glycoprotein antibodies of the compositions and methods of the invention are bispecific, the anti-CoV S glycoprotein antibodies may be human or humanized and may have specificity for an epitope on the SARS-CoV-2 S glycoprotein and T cells, or may be capable of binding to human effector cells, such as, for example, monocytes / macrophages and / or natural killer cells, that result in cell death.

[0129] In one embodiment, the anti-CoV S glycoprotein antibody of the invention is a bispecific antibody capable of specifically binding to a first and a second antigen, wherein said first antigen is SARS-CoV-2 S glycoprotein and said second antigen is an Fc gamma receptor selected from the group consisting of FcγRI, FcγRIIA, FcγRIIB, FcγRIIIA and / or FcγRIV. In a further embodiment, the anti-CoV S glycoprotein antibody of the invention is a bispecific antibody capable of specifically binding to SARS-CoV-2 and FcγRIIB. In another embodiment, the anti-CoV S glycoprotein antibody of the invention is a bispecific antibody capable of specifically binding to SARS-CoV-2 S glycoprotein and human FcγRIIB.

[0130] Variant Fc Regions The present invention provides anti-CoV S glycoprotein antibodies having a variant Fc domain, which is a non-naturally occurring Fc region, e.g., an Fc region that includes one or more non-naturally occurring amino acid residues. Also encompassed by the variant Fc region of the present invention are Fc regions that include amino acid deletions, additions, and / or modifications.

[0131] Fc region, as used herein, will be understood to include the polypeptides that include the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the N-terminal flexible hinge to these domains. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes the immunoglobulin domains Cgamma2 and Cgamma3 (Cγ2 and Cγ3), and the hinge between Cgamma1 (Cγ1) and Cgamma2 (Cγ2). Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined as comprising residues C226 or P230 to its carboxyl terminus, where the numbering is according to the EU index as in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). "EU index as shown in Kabat" refers to the residue numbering of the human IgG1 EU antibody as described in Kabat et al., supra. Fc may refer to this region in isolation or in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein may be an antibody, an Fc fusion, or any protein or protein domain that comprises an Fc region, including, but not limited to, a protein that comprises a variant Fc region that is a non-naturally occurring variant of Fc. Note: Polymorphisms have been observed at several Fc positions, including but not limited to Kabat 270, 272, 312, 315, 356, and 358, so slight differences may exist between the presented and prior art sequences.

[0132] The present invention encompasses anti-CoV S glycoprotein antibodies having variant Fc domains. Variant Fc domains may have altered binding properties for Fc ligands (e.g., Fc receptors, C1q) compared to comparable molecules (e.g., proteins having the same amino acid sequence except for having a wild-type Fc region). Examples of binding properties include, but are not limited to, binding specificity, equilibrium dissociation constant (K D ), dissociation and association rates (k off and k on ), binding affinity and / or avidity. D Binding molecules (e.g., Fc variant proteins such as antibodies) with high K D It is generally understood that a binding molecule having a K D Those skilled in the art can determine which kinetic parameters are most important for a given antibody application.

[0133] The affinity and binding properties of an Fc domain for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art for determining Fc-FcγR interactions, i.e., specific binding of an Fc region to an FcγR, including, but not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA), or radioimmunoassay (RIA)), or kinetics (e.g., BIACORE® analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labels in one or more of the components being examined and / or may employ a variety of detection methods, including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), focusing on antibody-immunogen interactions.

[0134] In one embodiment, the anti-CoV S glycoprotein antibody with the variant Fc domain has enhanced binding to one or more Fc ligands compared to a comparable molecule. In another embodiment, the anti-CoV S glycoprotein antibody with the variant Fc domain has an affinity for an Fc ligand that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold greater than that of a comparable molecule. In a specific embodiment, the anti-CoV S glycoprotein antibody with the variant Fc domain has enhanced binding to an Fc receptor. In another specific embodiment, the anti-CoV S glycoprotein antibody with the variant Fc domain has enhanced binding to the Fc receptor FcγRIIIA. In further specific embodiments, anti-CoV S glycoprotein antibodies with variant Fc domains have enhanced binding to the Fc receptor FcγRIIB. In yet another specific embodiment, anti-CoV S glycoprotein antibodies with variant Fc domains have enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, anti-CoV S glycoprotein antibodies with variant Fc domains have enhanced binding to C1q compared to comparable molecules.

[0135] In one embodiment, an anti-CoV S glycoprotein antibody of the invention comprises a variant Fc domain, wherein said variant Fc domain has enhanced binding affinity to Fc gamma receptor IIB compared to an equivalent non-variant Fc domain. In a further embodiment, an anti-CoV S glycoprotein antibody of the invention comprises a variant Fc domain, wherein said variant Fc domain has an affinity for Fc gamma receptor IIB that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold greater than that of the equivalent non-variant Fc domain.

[0136] The serum half-life of a protein comprising an Fc region can be increased by increasing the binding affinity of the Fc region to FcRn, hi one embodiment, an antibody comprising a variant Fc domain has an enhanced serum half-life compared to a comparable molecule.

[0137] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the formation of cytotoxicity in which secreted Ig bound to Fc receptors (FcR) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. Specific high-affinity IgG antibodies against the surface of the target cells "arm" the cytotoxic cells and are absolutely necessary for such killing. Lysis of the target cells is extracellular, requires direct cell-to-cell contact, and does not involve complement.

[0138] The ability of the antibody comprising the variant Fc domain to mediate the lysis of target cells by ADCC can be assayed.To evaluate ADCC activity, the Fc variant protein of interest is added to target cells in combination with immune effector cells, which can be activated by antigen-antibody complexes to cause cytolysis of target cells.Cytolysis is generally detected by the release of label (e.g., radioactive substrate, fluorescent dye, or natural intracellular protein) from lysed cells.Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361; Wilkinson et al., 2001, J Immunol Methods 258:183-191; Patel et al., 1995 J Tmmunol Methods 184:29-38. ADCC activity of the Fc variant protein of interest may also be assessed in vivo, for example in animal models such as those disclosed in Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-656.

[0139] In one embodiment, the antibody with the variant Fc domain has enhanced ADCC activity compared to the comparable molecule. In a specific embodiment, the antibody with the variant Fc domain has at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold greater ADCC activity than the comparable molecule. In another specific embodiment, the antibody with the variant Fc domain has enhanced binding to the Fc receptor FcγRIIIA and enhanced ADCC activity compared to the comparable molecule. In other embodiments, the antibody with the variant Fc domain has enhanced ADCC activity and enhanced serum half-life compared to the comparable molecule.

[0140] In one embodiment, the antibody with the variant Fc domain has reduced ADCC activity compared to the comparable molecule. In a specific embodiment, the Fc variant protein has at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold less ADCC activity than the comparable molecule. In another specific embodiment, the antibody with the variant Fc domain has reduced binding to the Fc receptor FcγRIIIA and reduced ADCC activity compared to the comparable molecule. In another embodiment, the antibody with the variant Fc domain has reduced ADCC activity and enhanced serum half-life compared to the comparable molecule.

[0141] "Complement dependent cytotoxicity" and "CDC" refer to the lysis of a target cell in the presence of complement. The complement activation pathway is initiated by the binding of the first component of the complement system (C1q) to a molecule, e.g., an antibody, complexed with a cognate antigen. To assess complement activation, a CDC assay, e.g., as described in Gazzano-Santoro et al., 1996, J. Immunol. Methods, 202:163, may be performed. In one embodiment, an antibody with a variant Fc domain has enhanced CDC activity compared to a comparable molecule. In specific embodiments, the Fc variant protein has CDC activity that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold greater than that of a comparable molecule. In other embodiments, an antibody with a variant Fc domain has enhanced CDC activity and enhanced serum half-life compared to a comparable molecule.

[0142] In one embodiment, an antibody having a variant Fc domain has reduced binding to one or more Fc ligands compared to a comparable molecule. In another embodiment, an antibody having a variant Fc domain has an affinity for an Fc ligand that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower than that of a comparable molecule. In a specific embodiment, an antibody having a variant Fc domain has reduced binding to an Fc receptor. In another specific embodiment, an antibody having a variant Fc domain has reduced binding to the Fc receptor FcγRIIIA. In further specific embodiments, antibodies having variant Fc domains described herein have an affinity for the Fc receptor FcγRIIIA that is at least about 5-fold lower than that of a comparable molecule, and wherein said antibodies having variant Fc domains have an affinity for the Fc receptor FcγRIIB that is within about 2-fold of that of a comparable molecule. In yet another specific embodiment, the Fc variant proteins have reduced binding to the Fc receptor FcRn. In yet another specific embodiment, antibodies having variant Fc domains have reduced binding to C1q compared to a comparable molecule.

[0143] In one embodiment, the invention provides an antibody having a variant Fc domain, wherein the Fc region is selected from the group consisting of 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 347, 251, 252, 254, 25 , 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443. Optionally, the Fc region may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752; WO04 / 074455; WO04 / 099249; WO04 / 063351; WO05 / 070963; WO05 / 040217, WO05 / 092925 and WO06 / 020114).

[0144] In one embodiment, the invention provides a formulation, wherein the Fc region is 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 343, 344, 345, 347, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, Formulations are provided that comprise a non-naturally occurring amino acid residue at one or more positions selected from the group consisting of 2, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440 and 443. Optionally, the Fc region may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752; WO04 / 074455; WO04 / 099249; WO04 / 063351; WO05 / 070963; WO05 / 040217, WO05 / 092925 and WO06 / 020114).

[0145] In a specific embodiment, the invention relates to an antibody having a variant Fc domain, wherein the Fc region is one of the following: 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235C, 235D, 235D, 235E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235C, 235D, 235E, 234N, 234Q, 234T, 234H, 234 P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 2 39F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 2621, 262A, 262T, 262 E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 2 65F, 265V, 2651, 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E , 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 29 8H, 2981, 298T, 298F, 2991, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 3051, 313F, 316D, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328 1, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 3 30H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 3311, 331C, 331Y, 331H, 331R, 331N,The antibodies include at least one non-naturally occurring amino acid residue selected from the group consisting of 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W. Optionally, the Fc region may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752 and WO05 / 040217).

[0146] In a particular embodiment, the invention relates to an antibody having a variant Fc domain, wherein the Fc region is selected from the group consisting of 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241L, 241Y, 241E ... R, 243W, 243L 243Y, 243R, 243Q, 244H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 2621, 262A, 262T, 262 E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 264M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 2 65F, 265V, 2651, 265L, 265H, 265T, 2661, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E , 280A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H, 269G, 297S, 297D, 297E, 29 8H, 2981, 298T, 298F, 2991, 299L, 299A, 299S, 299V, 299H, 299F, 299E, 3051, 313F, 316D, 325Q, 325L, 3251, 325D, 325E, 325A, 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 328Q, 328F, 328 1, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T, 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 3 30H, 331G, 331A, 331L, 331M, 331F, 331W, 331K, 331Q, 331E, 331S, 331V, 3311, 331C, 331Y, 331H, 331R, 331N,The antibodies include at least one non-naturally occurring amino acid residue selected from the group consisting of 331D, 3311, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y and 434W. Optionally, the Fc region may include non-naturally occurring amino acid residues at additional and / or alternative positions known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; PCT Patent Publications WO01 / 58957; WO02 / 06919; WO04 / 016750; WO04 / 029207; WO04 / 035752 and WO05 / 040217).

[0147] In another embodiment, the invention provides an antibody having a variant Fc domain, wherein the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330, and 332, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L, and 332E, as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may further comprise an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat, and at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

[0148] In another embodiment, the invention provides an antibody having a variant Fc domain, wherein the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 234, 235 and 331, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y and 331S, as numbered by the EU index as set forth in Kabat. In a further specific embodiment, the Fc variant of the invention comprises the non-naturally occurring amino acid residues 234F, 235F and 331S, as numbered by the EU index as set forth in Kabat. In another specific embodiment, the Fc domain of the invention comprises the non-naturally occurring amino acid residues 234F, 235Y and 331S, as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may further comprise additional non-naturally occurring amino acids at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat, and at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

[0149] In another embodiment, the invention provides an antibody having a variant Fc domain, wherein the Fc region comprises at least a non-naturally occurring amino acid at one or more positions selected from the group consisting of 239, 330, and 332, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant protein preparation, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L, and 332E, as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may further comprise an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant protein preparation, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 239D, 330L and 332E, as numbered by the EU index as set forth in Kabat, and at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

[0150] In another embodiment, the invention provides an antibody having a variant Fc domain, wherein the Fc region comprises at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 234, 235, and 331, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant protein preparation, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat. Optionally, the Fc region may further comprise an additional non-naturally occurring amino acid at one or more positions selected from the group consisting of 252, 254, and 256, as numbered by the EU index as set forth in Kabat. In a specific embodiment, the invention provides an Fc variant protein preparation, wherein the Fc region comprises at least one non-naturally occurring amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S, as numbered by the EU index as set forth in Kabat, and at least one non-naturally occurring amino acid at one or more positions selected from the group consisting of 252Y, 254T and 256E, as numbered by the EU index as set forth in Kabat.

[0151] In one embodiment, the Fc variant of the present invention is described in Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faseb J 9:115-119; Jefferis et al, 1996, Immunol Lett 54:101-104; Lund et al, 1996, J Immunol 157:4963-4969; Armour et al., 1999, Eur J Immunol 29:2613-2624; Idusogie et al, 2000, J Immunol 164:4178-4184; Reddy et al, 2000, J Immunol 164:1925-1933; Xu et al., 2000, Cell Immunol 200:16-26; Idusogie et al, 2001, J Immunol 166:2571-2575; Shields et al., 2001, J Biol Chem 276:6591-6604; Jefferis et al, 2002, Immunol Lett 82:57-65; Presta et al., 2002, Biochem Soc Trans 30:487-490; U.S. Patent No. 5,624,821; U.S. Patent No. 5,885,573; U.S. Patent No. 5,677,425; U.S. Patent No. 6,165,745; U.S. Patent No. 6,277,375; U.S. Patent No. 5,86 No. 9,046; No. 6,121,022; No. 5,624,821; No. 5,648,260; No. 6,528,624; No. 6,194,551; No. 6,737,056; Nos. 6,821,505; 6,277,375; U.S. Patent Application Publication No. 2004 / 0002587 and PCT Publications WO94 / 29351; WO99 / 58572; WO00 / 42072; WO02 / 060919; WO04 / 029207; WO04 / 099249; WO04 / 063351. Fc regions containing deletions, additions, and / or modifications are also encompassed by the present invention. Other modifications / substitutions / additions / deletions of the Fc domain will be readily apparent to one of skill in the art.

[0152] Methods for creating non-naturally occurring Fc regions are known in the art. For example, amino acid substitutions and / or deletions can be created by mutagenesis methods, including but not limited to site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCR mutagenesis (Higuchi, in "PCR Protocols: A Guide to Methods and Applications", Academic Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably, site-directed mutagenesis is performed by overlap extension PCR (Higuchi, in "PCR Technology: Principles and Applications for DNA Amplification", Stockton Press, New York, pp. 61-70 (1989)). The technique of overlap extension PCR (Higuchi, supra) can also be used to introduce any desired mutation into the target sequence (starting DNA). For example, the first round of overlap extension PCR involves amplifying the target sequence with an external primer (primer 1) and an internal mutagenesis primer (primer 3), and separately with a second external primer (primer 4) and an internal primer (primer 2), resulting in two PCR segments (segments A and B). The internal mutagenesis primer (primer 3) is designed to contain a mismatch to the target sequence that defines the desired mutation. In the second round of PCR, the products of the first round of PCR (segments A and B) are amplified by PCR using two external primers (primers 1 and 4). The resulting full-length PCR segment (segment C) is digested with a restriction enzyme, and the resulting restriction fragment is cloned into an appropriate vector.As a first step in mutagenesis, the starting DNA (e.g., encoding an Fc fusion protein, antibody, or simply an Fc region) is operably cloned into a mutagenesis vector. Primers are designed to reflect the desired amino acid substitutions. Other methods useful for creating variant Fc regions are known in the art (e.g., U.S. Pat. Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624 ... (see U.S. Patent Application Publication Nos. 6,194,551; 6,737,056; 6,821,505; 6,277,375; U.S. Patent Application Publication No. 2004 / 0002587 and PCT Publication Nos. WO94 / 29351; WO99 / 58572; WO00 / 42072; WO02 / 060919; WO04 / 029207; WO04 / 099249; WO04 / 063351).

[0153] In some embodiments, an antibody having a variant Fc domain comprises one or more engineered glycoforms, i.e., carbohydrate compositions that are covalently attached to a molecule comprising an Fc region. Engineered glycoforms can be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms may be generated by any method known to those skilled in the art, for example, by using engineered or variant expression strains, by co-expression with one or more enzymes, e.g., DI N-acetylglucosaminyltransferase III (GnTI11), by expressing a molecule comprising an Fc region in a different organism or in a cell line derived from a different organism, or by modifying the carbohydrate after the molecule comprising an Fc region is expressed. Methods for generating engineered glycoforms are known in the art and include, but are not limited to, Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473); U.S. Patent No. 6,602,684; U.S. Patent Application No. 10 / 277,370; U.S. Patent Application No. 10 / 113,929; PCT WO00 / 61739A1; PCT WO01 / 292246A1; PCT WO02 / 311140A1; PCT Examples include those described in WO02 / 30954A1; Potillegent™ technology (Biowa, Inc. Princeton, NJ); GlycoMAb™ glycosylation engineering technology (GLYCART biotechnology AG, Zurich, Switzerland). See, e.g., WO00061739; EA01229125; US 20030115614; Okazaki et al., 2004, JMB, 336: 1239-49.

[0154] Antibody glycosylation

[0155] In yet another embodiment, the glycosylation of the antibody utilized according to the invention is modified. For example, an aglycosylated antibody can be generated (i.e., the antibody lacks glycosylation). Glycosylation can be altered, for example, to increase the affinity of the antibody for the target antigen. Such carbohydrate modification can be accomplished, for example, by altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made, which result in the elimination of one or more variable region framework glycosylation sites, thereby eliminating glycosylation at that site. Such aglycosylation can increase the affinity of the antibody for the antigen. Such an approach is described in further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861. One or more amino acid substitutions can also be made to result in the elimination of a glycosylation site present in the Fc region (e.g., asparagine 297 of IgG). Furthermore, aglycosylated antibodies may be produced in bacterial cells that lack the necessary glycosylation machinery.

[0156] Antibodies with altered glycosylation types can also be made, for example, hypofucosylated antibodies with reduced amounts of fucosyl residues or antibodies with increased bisecting GlcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells to express the recombinant antibodies of the present invention, thereby producing antibodies with altered glycosylation. See, e.g., Shields, RL et al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1, as well as U.S. Pat. No. US 6,946,292; European Patent No. EP 1,176,195; PCT Publication No. WO 03 / 035835; WO 99 / 54342, each of which is incorporated by reference herein in its entirety.

[0157] Effector function operation It may be desirable to modify the anti-CoV S glycoprotein antibodies of the present invention with respect to effector function. For example, cysteine ​​residues may be introduced into the Fc region, thereby allowing the formation of interchain disulfide bonds in this region. Homodimeric antibodies thus generated may have improved internalization capability and / or increased complement-mediated cell killing and / or antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, B., J. Immunol., 148:2918-2922 (1992). Homodimeric antibodies with enhanced antitumor activity may also be prepared using heterobifunctional cross-linkers, as described in Wolff et al., Cancer Research, 53:2560-2565 (1993). Antibodies with dual Fc regions may also be engineered, thereby enhancing complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design, 3:219-230 (1989).

[0158] Other methods of engineering the Fc region of an antibody to alter effector function are known in the art (e.g., U.S. Patent Application Publication No. 20040185045 and PCT Publication No. WO2004 / 016750, both to Koenig et al., which describe altering the Fc region to enhance binding affinity to FcγRIIB compared to binding affinity to FcγRIIA; see also PCT Publication No. WO99 / 58572 to Armour et al., WO99 / 51642 to Idusogie et al., and US 6,395,272 to Deo et al., the disclosures of which are incorporated herein in their entireties). Methods of modifying an Fc region to decrease binding affinity to FcγRIIB are also known in the art (e.g., U.S. Patent Application Publication No. 20010036459 and PCT Publication No. WO01 / 79299, both to Ravetch et al., the disclosures of which are incorporated herein in their entireties). Engineered antibodies with variant Fc regions that have enhanced binding affinity for FcγRIIIA and / or FcγRIIA compared to the wild-type Fc region have also been described (e.g., PCT Publication WO 2004 / 063351 to Stavenhagen et al., the disclosure of which is incorporated herein in its entirety).

[0159] In vitro assays known in the art can be used to determine whether the anti-CoV S glycoprotein antibodies used in the compositions and methods of the invention are capable of mediating ADCC, such as that described herein.

[0160] Anti-CoV S glycoprotein antibody production / production Once a desired anti-CoV S glycoprotein antibody has been engineered, the anti-CoV S glycoprotein antibody can be produced on a commercial scale using methods well known in the art for large-scale production of antibodies For example, this can be accomplished using recombinant expression systems such as, but not limited to, those described below.

[0161] Recombinant Expression Systems Recombinant expression of an antibody or variant thereof generally requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once a polynucleotide encoding an antibody molecule, or an antibody heavy or light chain, or a portion thereof, is obtained, a vector for the production of the antibody molecule can be produced by recombinant DNA technology using techniques well known in the art. See, for example, U.S. Patent No. 6,331,415, which is incorporated herein by reference in its entirety. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody-encoding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing an antibody-encoding sequence and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Thus, the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule, an antibody heavy or light chain, an antibody heavy or light chain variable domain, or a portion thereof, or a heavy or light chain CDR, operably linked to a promoter. Such vectors may contain nucleotide sequences encoding the constant region of an antibody molecule (see, e.g., International Publication Nos. WO 86 / 05807 and WO 89 / 01036, and U.S. Pat. No. 5,122,464), and the variable domain of an antibody can be cloned into such a vector for expression of the entire heavy chain, the entire light chain, or both the entire heavy and light chains.

[0162] In another embodiment, anti-CoV S glycoprotein antibodies can be produced using targeted homologous recombination to produce all or a portion of the anti-CoV S glycoprotein antibody (see U.S. Pat. Nos. 6,063,630, 6,187,305, and 6,692,737). In certain embodiments, anti-CoV S glycoprotein antibodies can be produced using random recombination techniques to produce all or a portion of the anti-CoV S glycoprotein antibody (see U.S. Pat. Nos. 6,361,972, 6,524,818, 6,541,221, and 6,623,958). Anti-CoV S glycoprotein antibodies can also be produced in cells expressing the antibody from the genomic sequence of the cell containing the modified immunoglobulin locus using Cre-mediated site-specific homologous recombination (see U.S. Pat. No. 6,091,001). The host cell line may be derived from human or non-human species, including, but not limited to, mouse, and Chinese hamster. If production of human or humanized antibodies is desired, the host cell line must be a human cell line. These methods may be advantageously used to engineer stable cell lines which continually express the antibody molecule.

[0163] Once the expression vector has been transferred into the host cell by conventional techniques, the transfected cells are then cultured by conventional techniques to produce the antibody. Thus, the present invention includes a host cell containing a polynucleotide encoding the antibody of the present invention or a fragment thereof, or a heavy or light chain thereof, or a portion thereof, or a single chain antibody of the present invention, operably linked to a heterologous promoter. In certain embodiments, for the expression of a double-chain antibody, vectors encoding both the heavy and light chains may be co-expressed in the host cell for the expression of the entire immunoglobulin molecule, as detailed below.

[0164] A variety of host-expression vector systems may be utilized to express anti-CoV S glycoprotein antibodies or portions thereof that can be used in engineering and generating anti-CoV S glycoprotein antibodies (see, e.g., U.S. Pat. No. 5,807,715). For example, mammalian cells such as Chinese hamster ovary cells (CHO) in conjunction with vectors such as the major intermediate-early gene promoter element from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., Gene, 45:101 (1986); and Cockett et al., Bio / Technology, 8:2 (1990)). In addition, a host cell line may be selected that modulates the expression of the inserted antibody sequences or modifies and processes the antibody gene product in the specific manner desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell line or host system can be selected to ensure the correct modification and processing of the expressed antibody or its part.For this purpose, eukaryotic host cells can be used that have the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of gene product.Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O and T47D, NSO (a mouse myeloma cell line that does not endogenously produce any functional immunoglobulin chains), CRL7030 and HsS78Bst cells.

[0165] In bacterial systems, several expression vectors may be advantageously selected depending on the intended use for the antibody molecule being expressed. For example, a vector directing the expression of high levels of a fusion protein product that is easily purified for the generation of pharmaceutical compositions containing anti-CoV S glycoprotein antibodies may be desirable if large amounts of such antibodies are to be produced. Such vectors include, but are not limited to, the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791 (1983)); pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J. Biol. Chem., 24:5503-5509 (1989)), in which antibody coding sequences may be individually ligated into the vector in frame with the lac Z coding region such that a fusion protein is produced. pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST). Generally, such fusion proteins are soluble and can be easily purified from lysed cells by adsorption and binding to a glutathione agarose affinity matrix followed by elution in the presence of free glutathione. pGEX vectors are designed to introduce athrombin and / or factor Xa protease cleavage sites into the expressed polypeptide so that the cloned target gene product can be released from the GST moiety.

[0166] In an insect system, Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes. The virus grows in Sporoclopera frugiperda cells. The antibody coding sequence can be cloned individually into non-essential regions (e.g., the polyhedrin gene) of the virus and placed under the control of an AcNPV promoter (e.g., the polyhedrin promoter).

[0167] In mammalian host cells, several virus-based expression systems can be utilized. In the case where adenovirus is used as an expression vector, the sequence encoding the antibody of interest can be ligated to the adenovirus transcription / translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene can then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into non-essential regions of the viral genome (e.g., regions E1 or E3) results in recombinant viruses that are viable and capable of expressing antibody molecules in infected hosts (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). Specific initiation signals may also be required for efficient translation of the inserted antibody-encoding sequence. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must generally be in frame with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression can be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see, e.g., Bittner et al., Methods in Enzymol., 153:51-544(1987)).

[0168] Stable expression can be used for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express antibody molecules may be created. Host cells can be transformed with appropriately engineered vectors containing expression control elements (e.g., promoters, enhancers, transcription terminators, polyadenylation sites, etc.), and selectable marker genes. After introduction of the foreign DNA, cells may be grown in rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells that have stably integrated the plasmid into their chromosomes to grow and form foci that can then be cloned and expanded into cell lines. Plasmids encoding anti-CoV S glycoprotein antibodies can be used to introduce the gene / cDNA into any cell line suitable for production in culture.

[0169] Several selection systems can be used, including, but not limited to, herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes, respectively. - , hgprt -or aprr cells. Anti-metabolite resistance can also be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981)), gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)), neo, which confers resistance to the aminoglycoside G-418 (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-2 15 (1993)), and hygro, which confers resistance to hygromycin (Santerre et al., Gene, 30:147 (1984)).The desired recombinant clones can be selected by routine application of methods generally known in the field of recombinant DNA technology, such as those described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kricgler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); Colberre-Garapin et al., 1981, J. Mol. Biol., 150:1, which are incorporated herein by reference in their entireties.

[0170] The expression level of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. Academic Press, New York (1987)). If the marker in the vector system expressing the antibody is amplifiable, increasing the level of inhibitor present in the host cell culture will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, the production of the antibody will also increase (Crouse et al., Mol. Cell. Biol., 3:257 (1983)). The antibody expression level can be amplified by the use of recombinant methods and tools known to those skilled in the art of recombinant protein production, including techniques that remodel the surrounding chromatin and enhance transgene expression in the form of an active artificial transcription domain.

[0171] The host cell may be co-transfected with two expression vectors, the first encoding a polypeptide derived from the heavy chain and the second encoding a polypeptide derived from the light chain. The two vectors may contain the same or different selectable markers. A single vector capable of encoding and expressing both heavy and light chain polypeptides may also be used. In such a situation, the light chain must be located 5' to the heavy chain to avoid excess non-toxic heavy chain (Proudfoot, Nature 322:562-65 (1986); and Kohler, 1980, Proc. Natl. Acad. Sci. USA, 77:2197 (1980)). The coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.

[0172] Once an antibody molecule has been produced by recombinant expression, it may be purified by any method known in the art for the purification of immunoglobulin molecules, such as, for example, by chromatography (e.g., ion exchange, affinity, particularly affinity for specific antigens to Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins. Additionally, the antibodies of the present invention or fragments thereof may be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0173] Antibody purification and isolation When using recombinant techniques, antibodies can be produced intracellularly, in the periplasmic space, or directly secreted into the medium. If antibodies are produced intracellularly, as a first step, particulate debris, either host cells or lysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al., Bio / Technology, 10:163-167 (1992) describes a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation. If antibody mutants are secreted into the medium, the supernatant of such expression systems is generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

[0174] The antibody composition prepared from the cells can be purified, for example, using hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography, either alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain present in the antibody mutant. Protein A can be used to purify antibodies based on human gamma l, gamma 2, or gamma 4 heavy chains (Lindmark et al., J. Immunol. Methods, 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human gamma 3 (Guss et al., EMBO J., 5:15671575 (1986)). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as porous glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. If the antibody contains a CH3 domain, Bakerbond ABX resin (JT Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification, such as fractionation on ion exchange columns, ethanol precipitation, reversed-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on anion or cation exchange resins (such as polyaspartic acid columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0175] After any preliminary purification steps, the mixture containing the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5 and 4.5, optionally performed at a low salt concentration (e.g., about 0-0.25 M salt).

[0176] Therapeutic anti-CoV S glycoprotein antibody The anti-CoV S glycoprotein antibodies used in the compositions and methods of the invention may be human or humanized antibodies that can treat COVID-19 or neutralize the SARS-CoV-2 virus or its variants. In certain embodiments, the anti-CoV S glycoprotein antibodies may be chimeric or murine antibodies. In certain embodiments, the anti-CoV S glycoprotein antibodies may be monoclonal human, humanized, or chimeric antibodies. The anti-CoV S glycoprotein antibodies used in the compositions and methods of the invention may be human or humanized antibodies of IgG1 or IgG3 human isotype, or any IgG1 or IgG3 allele found in the human population. In other embodiments, the anti-CoV S glycoprotein antibodies used in the compositions and methods of the invention may be human or humanized antibodies of IgG2 or IgG4 human isotype, or any IgG2 or IgG4 allele found in the human population.

[0177] In certain embodiments, the antibody is an isotype-switched variant of a known antibody, such as those described above (eg, to an IgG1 or IgG3 human isotype).

[0178] The anti-CoV S glycoprotein antibodies used in the compositions and methods of the disclosure may be naked antibodies, immunoconjugates or fusion proteins.

[0179] Screening of antibodies for SARS-CoV-2 S glycoprotein binding Binding assays can be used to identify antibodies that bind to the SARS-CoV-2 S glycoprotein. Binding assays may be performed either as direct binding assays or as competitive binding assays. Binding can be detected using standard ELISA or standard flow cytometry assays. In direct binding assays, candidate antibodies are tested for binding to the SARS-CoV-2 S glycoprotein. Competitive binding assays, on the other hand, evaluate the ability of a candidate antibody to compete with known anti-CoV S glycoprotein antibodies or other compounds for binding to the SARS-CoV-2 S glycoprotein.

[0180] In a direct binding assay, the SARS-CoV-2 S glycoprotein is contacted with a candidate antibody under conditions that allow binding of the candidate antibody to the SARS-CoV-2 S glycoprotein. Binding can be performed in solution or on a solid surface. The candidate antibody may be pre-labeled for detection. Any detectable compound can be used for labeling, including but not limited to luminescent, fluorescent or radioisotope or groups containing same, or nonisotopic labels, e.g., enzymes or dyes. After a period of incubation sufficient for binding, the reaction is exposed to conditions and manipulations that remove excess or non-specifically bound antibody. Typically, this involves washing with an appropriate buffer. Finally, the presence of a complex between the candidate antibody and the SARS-CoV-2 S glycoprotein is detected.

[0181] In a competitive binding assay, a candidate antibody is evaluated for its ability to inhibit or displace the binding of a known anti-CoV S glycoprotein antibody (or other compound) to the SARS-CoV-2 S glycoprotein. A labeled, known binder of SARS-CoV-2 S glycoprotein may be mixed with the candidate antibody and placed under conditions under which an interaction between them would normally occur, with and without the addition of the candidate antibody. The amount of labeled, known binder of SARS-CoV-2 glycoprotein that binds to the SARS-CoV-2 glycoprotein may be compared to the amount bound in the presence or absence of the candidate antibody.

[0182] In one embodiment, the binding assay is performed with one or more components immobilized on a solid surface to facilitate the formation and detection of antibody-antigen complexes. In various embodiments, the solid support can be, but is not limited to, polyvinylidene fluoride, polycarbonate, polystyrene, polypropylene, polyethylene, glass, nitrocellulose, dextran, nylon, polyacrylamide, and agarose. The support structure can include beads, membranes, microparticles, the interior surface of a reaction vessel, such as a microtiter plate, test tube, or other reaction vessel. Immobilization of the SARS-CoV-2 S glycoprotein or fragments thereof, or other components, can be achieved by covalent or non-covalent attachment. In one embodiment, attachment can be indirect, i.e., through an attached antibody. In another embodiment, the SARS-CoV-2 S glycoprotein and negative control are tagged with an epitope, such as glutathione S-transferase (GST), such that attachment to the solid surface can be mediated by a commercially available antibody, such as anti-GST (Santa Cruz Biotechnology).

[0183] For example, such affinity binding assays can be performed using SARS-CoV-2 S glycoprotein immobilized on a solid support. Typically, the non-immobilized component of the binding reaction, in this case the candidate anti-CoV S glycoprotein antibody, is labeled to allow detection. A variety of labeling methods are available, such as luminescent, chromophore, fluorescent or radioisotope or groups containing same, and nonisotopic labels, such as enzymes or dyes, can be used. In one embodiment, the candidate anti-CoV S glycoprotein antibody is labeled with a fluorophore, such as fluorescein isothiocyanate (FITC, available from Sigma Chemicals, St. Louis). Such affinity binding assays can be performed using SARS-CoV-2 S glycoprotein immobilized on a solid surface. The anti-CoV S glycoprotein antibody is then incubated with the antigen, and specific binding of the antibody is detected by methods known in the art, including, but not limited to, BiaCore Analyses, ELISA, FMET and R1A methods.

[0184] Finally, the label remaining on the solid surface can be detected by any detection method known in the art, for example, if the candidate anti-CoV S glycoprotein antibody is labeled with a fluorophore, a fluorometer can be used to detect the complex.

[0185] SARS-CoV-2 S glycoprotein can be added to the binding assay in the form of intact cells expressing SARS-CoV-2 S glycoprotein or isolated membranes containing human SARS-CoV-2 S glycoprotein. Thus, direct binding to SARS-CoV-2 glycoprotein may be assayed in intact cells in culture or animal models in the presence and absence of candidate anti-CoV S glycoprotein antibodies. Labeled candidate anti-CoV S glycoprotein antibodies may be mixed with cells expressing SARS-CoV-2 S glycoprotein and candidate anti-CoV S glycoprotein antibodies may be added. Isolated membranes may be used to identify candidate anti-CoV S glycoprotein antibodies that interact with SARS-CoV-2 S glycoprotein. For example, in a typical experiment using isolated membranes, cells may be genetically engineered to express SARS-CoV-2 S glycoprotein. Membranes may be harvested by standard techniques and used in in vitro binding assays. A labeled candidate anti-CoV S glycoprotein antibody (e.g., a fluorescently labeled antibody) is bound to the membrane and assayed for specific activity, with specific binding being determined by comparison to a binding assay performed in the presence of an excess of unlabeled (non-radioactive) candidate anti-CoV S glycoprotein antibody. Polypeptides corresponding to one or more regions of the SARS-CoV-2 S glycoprotein (e.g., the RBD), or fusion proteins containing one or more regions of the SARS-CoV-2 S glycoprotein, can also be used in non-cell-based assay systems to identify antibodies that bind to portions of the SARS-CoV-2 S glycoprotein. In non-cell-based assays, recombinantly expressed human SARS-CoV-2 S glycoprotein is attached to a solid substrate, e.g., a test tube, a microliter well, or a column, by means well known to those of skill in the art (see Ausubel et al., supra). Test antibodies are then assayed for their ability to bind to the SARS-CoV-2 S glycoprotein.

[0186] The binding reaction may also be carried out in solution. In this assay, the labeled component is allowed to interact with its binding partner in solution. If the size difference between the labeled component and its binding partner allows such separation, separation can be achieved by passing the product of the binding reaction through an ultrafilter whose pores allow the passage of the unbound labeled component, but not its binding partner or the labeled component bound to its partner. Separation can also be achieved using any reagent that allows the capture of the binding partner of the labeled component from the solution, such as an antibody against the binding partner.

[0187] In one embodiment, for example, a phage library can be screened by passing phage from a successive phage display library through a column containing the SARS-CoV-2 S glycoprotein or a portion thereof (e.g., the RBD of the SARS-CoV-2 S glycoprotein), or a derivative, analog, fragment, or domain thereof, linked to a solid phase, such as plastic beads. By varying the stringency of the wash buffer, it is possible to enrich for phage expressing peptides with high affinity for the SARS-CoV-2 S glycoprotein. Phage isolated from the column can be cloned and affinity can be measured directly. Knowing which antibodies and their amino acid sequences confer the strongest binding to the SARS-CoV-2 S glycoprotein, computer models can be used to identify molecular contacts between the SARS-CoV-2 S glycoprotein and candidate antibodies.

[0188] In another specific embodiment, the solid support is a membrane containing SARS-CoV-2 S glycoprotein attached to a microtiter dish. Candidate antibodies can be bound to cells expressing the library antibodies, for example, cultured under conditions that allow expression of the library members in the microliter dish. Library members that bind to SARS-CoV-2 are harvested. Such methods are generally described, for example, in Parmley and Smith, 1988, Gene, 73:305-318; Fowlkes et al., 1992, BioTechniques, 13:422-427; PCT Publication No. WO94 / 18318, and the references cited herein above. Antibodies identified as binding to SARS-CoV-2 S glycoprotein can be of any of the antibody types or modifications described above.

[0189] Screening antibodies for human ADCC effector function Human IgG class antibodies, which have functional characteristics such as a long half-life in serum and the ability to mediate various effector functions, are used in certain embodiments of the present invention (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)). Human IgG class antibodies are further classified into the following four subclasses: IgG1, IgG2, IgG3 and IgG4. Numerous studies have been conducted on ADCC and CDC as effector functions of IgG class antibodies, and it has been reported that among human IgG class antibodies, the IgG1 subclass has the highest ADCC and CDC activities in humans (Chemical Immunology, 65, 88 (1997)).

[0190] The expression of ADCC and CDC activity of human IgG1 subclass antibodies generally involves binding of the Fc region of the antibody to a receptor for the antibody (hereinafter referred to as "FcγR") present on the surface of an effector cell, such as a killer cell, a natural killer cell, or an activated macrophage. Various complement components can bind. It has been suggested that several amino acid residues in the hinge region and the second domain of the C region (hereinafter referred to as "Cγ2 domain") of the antibody are important for binding (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995), Chemical Immunology, 65, 88 (1997)), as well as the carbohydrate chain in the Cγ2 domain (Chemical Immunology, 65, 88 (1997)).

[0191] Anti-CoV S glycoprotein antibodies can be modified with respect to effector functions, for example to enhance the ADCC and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions in the Fc region of the antibody. Cysteine ​​residues can also be introduced into the Fc region, allowing the formation of interchain disulfide bonds in this region. In this way, homodimeric antibodies can be created that may have improved internalization capabilities and / or increased complement-mediated cell killing and ADCC (Caron et al., J. Exp. Med., 176:1191-1195 (1992) and Shopes, J. Immunol., 148:2918-2922 (1992)). Heterobifunctional cross-linkers can also be used to create homodimeric antibodies with enhanced anti-tumor activity (Wolff et al., Cancer Research, 53:2560-2565 (1993)). Antibodies can also be engineered to have two or more Fc regions that provide enhanced complement lysis and ADCC capabilities (Stevenson et al., Anti-Cancer Drug Design, (3)219-230 (1989)).

[0192] Other methods of engineering the Fc region of an antibody to alter effector function are known in the art (e.g., U.S. Patent Application Publication No. 20040185045 and PCT Publication No. WO2004 / 016750, both to Koenig et al., which describe altering an Fc region to enhance binding affinity to FcγRIIB compared to binding affinity to FcγRIIA; see also PCT Publication No. WO99 / 58572 to Armour et al., WO99 / 51642 to Idusogic et al., and US6,395,272 to Dco et al., the disclosures of which are incorporated herein in their entireties). Methods of modifying an Fc region to decrease binding affinity to FcγRIIB are also known in the art (e.g., U.S. Patent Application Publication No. 20010036459 and PCT Publication No. WO01 / 79299, both to Ravetch et al., the disclosures of which are incorporated herein in their entireties). Engineered antibodies with variant Fc regions that have enhanced binding affinity for FcγRIIIA and / or FcγRIIA compared to the wild-type Fc region have also been described (e.g., PCT Publication WO 2004 / 063351 to Stavenhagen et al.; the disclosure of which is incorporated herein in its entirety).

[0193] At least four different types of FcγR have been found, called FcγRI (CD64), FcγRII (CD32), FcγRIII (CD16), and FcγRIV, respectively. In humans, FcγRII and FcγRIII are further classified into FcγRIIa and FcγRIIb, and FcγRIIIa and FcγRIIIb, respectively. FcγR is a membrane protein belonging to the immunoglobulin superfamily, FcγRII, FcγRIII, and FcγRIV have an a chain with an extracellular region containing two immunoglobulin-like domains, and FcγRI has an a chain with an extracellular region containing three immunoglobulin-like domains as a constituent component, and the a chain is involved in IgG binding activity. In addition, FcγRI and FcγRIII have a gamma or zeta chain as a constituent component that has signaling function associated with the alpha chain (Annu. Rev. Immunol., 18, 709 (2000); Annu. Rev. Immunol., 19, 275 (2001)). FcγRIV is described by Bruhns et al., Clin. Invest. Med., (Canada) 27:3D (2004).

[0194] To assess the ADCC activity of an anti-CoV S glycoprotein antibody of interest, an in vitro ADCC assay such as that described in U.S. Pat. No. 5,500,362 or 5,821,337 can be used. The assay may also be performed using commercially available kits, such as CytoTox 96® (Promega). Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, and NK cell lines. NK cell lines expressing transgenic Fc receptors (e.g., CD16) and related signaling polypeptides (e.g., FCεRI-γ) can also serve as effector cells (see, e.g., Campbell, WO 2006 / 023148 ​​A2). For example, the ability of any particular antibody to mediate lysis of target cells by complement activation and / or ADCC can be assayed. The cells of interest are grown and labeled in vitro, and the antibody is added to the cell culture in combination with immune cells that can be activated by antigen-antibody complexes, i.e., effector cells involved in ADCC responses. The antibody can also be tested for complement activation. In either case, cytolysis of the target cells is detected by the release of label from the lysed cells. The extent of target cytolysis can also be determined by detecting the release of cytoplasmic proteins (e.g., LDH) into the supernatant. In practice, antibodies can be screened using the patient's own serum as a source of complement and / or immune cells. Antibodies that can mediate human ADCC in in vitro tests can then be used therapeutically in that particular patient. The ADCC activity of the molecule of interest can also be evaluated in vivo, for example in animal models such as those disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998). Additionally, techniques for modulating (ie, increasing or decreasing) the level of ADCC and, optionally, CDC activity of an antibody are well known in the art.See, for example, U.S. Patent No. 6,194,551. The antibodies of the present invention may be capable of, or may be modified to, induce ADCC and / or CDC. Assays for determining ADCC function may be performed using human effector cells to assess human ADCC function. Such assays may also include those intended to screen for antibodies that induce, mediate, enhance, or block cell death by necrotic and / or apoptotic mechanisms. Such methods, including assays utilizing viability dyes, methods to detect and analyze caspases, and assays that measure DNA breakage, may be used to assess the apoptotic activity of cells cultured in vitro with the anti-CoV S glycoprotein antibody of interest.

[0195] For example, annexin V or TdT-mediated dUTP nick end labeling (TUNEL) assay can be performed to detect apoptotic activity as described in Decker et al., Blood (USA) 103:2718-2725 (2004). TUNEL assay involves culturing cells of interest with fluorescein-labeled dUTP for incorporation into DNA strand breaks. The cells are then processed for analysis by flow cytometry. Annexin V assay detects the appearance of phosphatidylserine (PS) on the outside of the cell membrane of apoptotic cells using fluorescein-conjugated annexin V, which specifically recognizes exposed PS molecules. In conjunction, viability dyes such as propidium iodide can be used to exclude late apoptotic cells. Cells are stained with labeled annexin V and analyzed by flow cytometry.

[0196] neutralizing antibody In embodiments, the anti-CoV S glycoprotein antibodies described herein are neutralizing antibodies. In embodiments, the anti-CoV S glycoprotein antibodies neutralize the SARS-CoV-2 virus or variants thereof.

[0197] Anti-CoV S glycoprotein antibody conjugates According to certain aspects of the invention, compounds may be conjugated to anti-CoV S glycoprotein antibodies for use in the compositions and methods of the invention. In certain embodiments, these conjugates may be made as fusion proteins.

[0198] Covalent modifications of anti-CoV S glycoprotein antibodies are included within the scope of the present invention. They may be produced by chemical synthesis or by enzymatic or chemical cleavage of the antibody, if applicable. Other types of covalent modifications of anti-CoV S glycoprotein antibodies are introduced into the molecule by reacting targeted amino acid residues of the antibody with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0199] Cysteinyl residues are most commonly reacted with a-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Similarly, iodine reagents can be used. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetylphosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoic acid, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0200] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5 to 7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful, and the reaction can be performed in 0.1 M sodium cacodylate at pH 6.0.

[0201] Lysyl and amino terminal residues are reacted with succinic anhydride or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing α- and / or ε-amino containing residues include imidoesters such as methylpicolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, 0-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylic acid.

[0202] Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues generally requires that the reaction be carried out under alkaline conditions due to the high pKa of the guanidine functional group. Furthermore, these reagents can react with the ε-amino group of lysine and the epsilon-amino group of arginine.

[0203] The specific modification of tyrosyl residues may be made, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosyl residues can be 125 I or 131 I to prepare labeled proteins for use in radioimmunoassay.

[0204] Carboxyl side chains (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R--N=C=N--R'), where R and R' are different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide (carbodiimidc) or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0205] Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively, which are deamidated under neutral or basic conditions, and the deamidated forms of these residues are within the scope of the invention.

[0206] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (TE Creighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.

[0207] Another type of covalent modification involves chemical or enzymatic coupling of glycosides to antibodies. These procedures are advantageous in that they do not require production of the antibody in a host cell that has glycosylation capabilities for N- or O-linked glycosylation. Depending on the coupling mode used, the sugar can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups such as those of cysteine, (d) free hydroxyl groups such as those of serine, threonine, or hydroxyproline, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO87 / 05330 published September 11, 1987 and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0208] Pharmaceutical Compositions The present invention also relates to compositions comprising anti-CoV S glycoprotein antibodies and methods of using said compositions for the treatment of COVID-19 in human subjects.

[0209] The present invention relates to pharmaceutical compositions comprising anti-CoV S glycoprotein antibodies of IgG1 or IgG3 human isotype. The present invention also relates to pharmaceutical compositions comprising anti-CoV S glycoprotein antibodies of IgG2 or IgG4 human isotype capable of mediating human ADCC. In certain embodiments, the present invention also relates to pharmaceutical compositions comprising monoclonal human, humanized, or chimeric anti-CoV S glycoprotein antibodies, which may be produced by means known in the art.

[0210] In other specific embodiments, anti-CoV S glycoprotein antibodies can mediate ADCC, complement-dependent cytotoxicity, or apoptosis.

[0211] Antibody Half-Life In embodiments, the half-life of the anti-CoV S glycoprotein antibodies described herein is from about 1 hour to about 60 days. For example, the half-life of an anti-CoV S glycoprotein antibody may be up to about 1 hour, up to about 2 hours, up to about 3 hours, up to about 4 hours, up to about 5 hours, up to about 6 hours, up to about 7 hours, up to about 8 hours, up to about 9 hours, up to about 10 hours, up to about 11 hours, up to about 12 hours, up to about 13 hours, up to about 14 hours, up to about 15 hours, up to about 16 hours, up to about 17 hours, up to about 18 hours, up to about 19 hours, up to about 21 hours, up to about 22 hours, up to about 23 hours, up to about 24 hours, up to about 25 hours, up to about 26 hours, up to about 27 hours, up to about 28 hours, up to about 29 hours, up to about 30 hours, up to about 31 hours, up to about 32 hours, up to about 33 hours, up to about 34 hours, up to about 35 hours, up to about 36 hours, up to about 37 hours, up to about 38 hours, up to about 39 hours, up to about 40 hours, up to about 41 hours, up to about 42 hours, up to about 43 hours, up to about 44 hours, up to about 45 hours, up to about 46 hours, up to about 47 hours, up to about 48 hours, up to about 49 hours, up to about 50 hours, up to about 51 hours, up to about 52 hours, up to about 53 hours, up to about 54 hours, up to about 55 hours, up to about 56 hours, up to about 57 hours, up to about 58 hours, up to about 59 hours, up to about 6 9 hours, up to about 20 hours, up to about 21 hours, up to about 22 hours, up to about 23 hours, up to about 24 hours, up to about 2 days, up to about 3 days, up to about 4 days, up to about 5 days, up to about 6 days, up to about 7 days, up to about 8 days, up to about 9 days, up to about 10 days, up to about 11 days, up to about 12 days, up to about 13 days, up to about 14 days, up to about 15 days, up to about 16 days, up to about 17 days, up to about 18 days, up to about 19 days, up to about 20 days, up to about 21 days, up to about 22 days, up to about 23 days, up to about 24 days, up to about 25 days, up to about 26 days, up to about 27 days, up to about 28 days, up to about 29 days, up to about 30 days, up to about 31 days, up to about 32 days, up to about 33 days, up to about 34 days, up to about 35 days, up to about 36 days, up to about 37 days, up to about 38 days, up to about 39 days, The half-life of the antibody of the compositions and methods of the present invention can be extended by methods known in the art. Such extension can then reduce the amount and / or frequency of administration of the antibody composition. Antibodies with improved in vivo half-lives and methods for preparing them are disclosed in US Pat. No. 6,277,375; and International Publication Nos. WO 98 / 23289 and WO 97 / 3461.

[0212] The serum circulation of anti-CoV S glycoprotein antibodies in vivo can also be extended by attaching inert polymer molecules, such as high molecular weight polyethylene glycol (PEG), with or without multifunctional linkers to the antibody, either by site-specific conjugation of PEG to the N- or C-terminus of the antibody, or via the epsilon-amino groups present on the lysyl residues. Linear or branched polymer derivatization that results in minimal loss of biological activity is used. The degree of conjugation can be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion chromatography or by ion exchange chromatography. PEG-derivatized antibodies can be tested for binding activity and for in vivo efficacy using methods known to those skilled in the art, for example, by immunoassays described herein.

[0213] In addition, the antibody of the composition and method of the present invention can be conjugated to albumin to make the antibody more stable in vivo or have a longer half-life in vivo.Techniques are well known in the art, see, for example, International Publication Nos. WO93 / 15199, WO93 / 15200 and WO01 / 77137; and European Patent No. EP413,622, all of which are incorporated herein by reference.

[0214] Pharmaceutical Formulation, Administration, and Dosing The pharmaceutical formulations of the invention contain anti-CoV S glycoprotein antibodies as an active ingredient. The formulations contain an effective amount of naked antibody, immunoconjugate, or fusion protein to produce the desired response, in units of weight or volume suitable for administration to a human patient, and are preferably sterile.

[0215] The anti-CoV S glycoprotein antibody composition may be formulated with a pharma- ceutically acceptable carrier. The term "pharmaceutically acceptable" means one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and other therapeutic agents as appropriate. Such pharma- ceutical acceptable preparations may also routinely contain compatible solid or liquid fillers, diluents, or encapsulating substances suitable for administration to humans. When used in medicines, salts must be pharma- ceutical acceptable, although pharma- ceutical unacceptable salts may be conveniently used to prepare pharma- ceutical acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharma- ceutical acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, and the like. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth metal salts, such as sodium, potassium or calcium salts. The term "carrier" refers to a natural or synthetic organic or inorganic component with which an active ingredient is combined to facilitate application. The components of the pharmaceutical composition can also be mixed with the antibody of the present invention, and with each other, in a manner such that there is no interaction that substantially imparts the desired pharmaceutical effectiveness.

[0216] According to certain aspects of the invention, anti-CoV S glycoprotein antibody compositions can be prepared for storage in the form of lyophilized formulations or aqueous solutions by mixing antibodies or immunoconjugates having the desired degree of purity with physiologically acceptable carriers, excipients or stabilizers as required (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed and include, but are not limited to, buffers, such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens, such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin. , gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN®, PLURONICS™, or polyethylene glycol (PEG).

[0217] The anti-CoV S glycoprotein antibody compositions may also contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

[0218] The anti-CoV S glycoprotein antibody composition may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier which constitutes one or more accessory ingredients. In general, the anti-CoV S glycoprotein antibody composition is prepared by uniformly and intimately bringing into association the active compound with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

[0219] Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation of the anti-CoV S glycoprotein antibody, which is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be used are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conveniently used as a solvent or suspending medium. For this purpose, any non-irritating solid oil may be used, including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, may be used in the preparation of injectables. Suitable carrier formulations for oral, subcutaneous, intravenous, intramuscular, etc. administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0220] The active ingredient may also be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, for example, in microcapsules prepared by coacervation techniques, or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules, and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0221] The formulations to be used for in vivo administration are typically sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0222] Sustained release preparations may be prepared. Suitable examples of sustained release preparations include semipermeable matrices of solid hydrophobic polymers containing anti-CoV S glycoprotein antibodies, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate) or poly(vinyl alcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid are capable of releasing molecules for over 100 days, while certain hydrogels release proteins for shorter periods of time. If encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular SS bond formation by thio-disulfide exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions. In certain embodiments, the pharmaceutically acceptable carriers used in the compositions of the present invention do not affect human ADCC or CDC.

[0223] The anti-CoV S glycoprotein antibodies disclosed herein may also be formulated as immunoliposomes. "Liposomes" are small vesicles composed of various types of lipids, phospholipids, and / or surfactants, which are useful for delivery of drugs (e.g., the anti-CoV S glycoprotein antibodies disclosed herein) to humans. The components of the liposome are usually arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibodies of the present invention are prepared by methods known in the art, for example, as described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Pat. No. 5,013,556. Particularly useful liposomes can be made by reverse phase evaporation using a lipid composition comprising phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are extruded through filters of defined pore size to obtain liposomes with desired diameter. The antibody of the present invention can be conjugated to liposomes via disulfide exchange reaction as described in Martin et al., J. Biol. Chem., 257:286-288 (1982). Therapeutic agents can also be contained within liposomes. See Gabizon et al., J. National Cancer Inst., (19)1484 (1989).

[0224] In certain embodiments, the pharmaceutical compositions of the invention are stable at 4° C. In certain embodiments, the pharmaceutical compositions of the invention are stable at room temperature.

[0225] Administration of the compositions of the invention to a human patient may be by any route, including, but not limited to, intravenous, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via aerosol), buccal (e.g., sublingual), topical (i.e., both cutaneous and mucosal surfaces, including airway surfaces), intrathecal, intraarticular, intrapleural, intracerebral, intraarterial, intraperitoneal, oral, intralymphatic, intranasal, rectal or intravaginal administration, by perfusion through a regional catheter, or by direct intralesional injection. In one embodiment, the compositions of the invention are administered by intravenous push or intravenous infusion, given over a predefined period of time (e.g., 0.5 to 2 hours). The compositions of the invention may be delivered by peristaltic means or in the form of a depot, although the most suitable route in any given case will depend on factors such as the species, age, sex and general condition of the subject, the nature and severity of the condition being treated, and / or the nature of the particular composition being administered (i.e., dosage, formulation), as is well known in the art.

[0226] In embodiments, the dose of the composition comprising anti-CoV S glycoprotein antibodies is measured in mg / kg of the patient's body weight. In other embodiments, the dose of the composition comprising anti-CoV S glycoprotein antibodies is measured in mg / kg of the patient's lean body mass (i.e., body weight minus body fat content). In yet other embodiments, the dose of the composition comprising anti-CoV S glycoprotein antibodies is measured in mg / m of the patient's body surface area. 2 In yet other embodiments, the dose of the composition comprising the anti-CoV S glycoprotein antibody is measured in units of mg per dose administered to a patient. Any measurement of dose may be used in conjunction with the compositions and methods of the invention, and dosage units may be converted by standard means in the art.

[0227] It will be clear to those skilled in the art that dosage can be selected based on several factors, including age, sex, species and condition of subject.For example, the effective amount of the composition of the present invention can be extrapolated from the dose-response curve derived from in vitro test system or animal model (e.g., cotton rat or monkey) test system.Models and methods for evaluating the efficacy of antibody are known in the art (Wooldridge et al., Blood, 89(8): 2994-2998 (1997)), which is incorporated herein by reference in its entirety).

[0228] Examples of dosing regimens that can be used in the methods of the invention include, but are not limited to, daily, three times a week (intermittent), weekly, every 14 days, monthly, every 6-8 weeks, every 2 months, every 6 months, or yearly.

[0229] In embodiments, the dose of anti-CoV S glycoprotein antibody ranges from 10 mg to about 2 g. For example, the dose of anti-CoV S glycoprotein antibody can be about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 1000 mg, about 1500 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 1000 mg, about 150 ... The amount of the active ingredient may be about 0 mg, about 900 mg, about 950 mg, about 1 g, about 1.05 g, about 1.1 g, about 1.15 g, about 1.2 g, about 1.25 g, about 1.3 g, about 1.35 g, about 1.4 g, about 1.45 g, about 1.5 g, about 1.55 g, about 1.6 g, about 1.65 g, about 1.7 g, about 1.75 g, about 1.8 g, about 1.85 g, about 1.9 g, about 1.95 g, about 2 g, or any range or subrange therebetween.

[0230] In embodiments, the disclosure provides a method for treating a subject infected with a SARS-CoV-2 virus or a variant thereof, comprising administering a composition comprising an anti-CoV S glycoprotein antibody described herein, wherein the SARS-CoV-2 variant has a PANGO lineage selected from the group consisting of B.1.1.529; BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1.

[0231] Toxicity testing The tolerability, toxicity and / or efficacy of compositions and / or treatment regimens of the invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., to determine the LD50 (the dose lethal to 50% of the population), the ED50 (the dose therapeutically effective in 50% of the population), and the IC50 (the dose effective to achieve 50% inhibition).

[0232] Data obtained from cell culture assays and animal studies can be used in formulating a dosage range of compositions and / or treatment regimens for use in humans. The dosage of such agents can be within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage can vary within this range depending on the dosage form used and the route of administration utilized. For any therapy used in the methods of the present invention, the therapeutically effective dose can be estimated by an appropriate animal model. Depending on the species of the animal model, the dose can be adjusted for human use according to art-accepted formulas provided, for example, by Freireich et al., Quantitative comparison of toxicity of anticancer agents in mouse, rat, monkey, dog, and human, Cancer Chemotherapy Reports, NCI 1966 40:219-244. Data obtained from cell culture assays can be useful for predicting potential toxicity. Animal studies can be used to formulate a particular dose that achieves a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound that achieves half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Plasma drug levels can be measured, for example, by high performance liquid chromatography, ELISA, or by cell-based assays. EXAMPLES

[0233] Example 1 Discovery of antibodies 239.12; 322.3; 425.6; and 35.13

[0234] Using hybridoma technology, we generated five antibodies that bind to the SARS-CoV-2 S polypeptide from the SARS-CoV-2 virus.

[0235] Mice were injected with CoV S glycoproteins BV2373, BV2438, BV2465, or BV2540, which comprise the amino acid sequences of SEQ ID NOs: 35, 36, 37, and 119, respectively. The amino acid sequences of BV2373, BV2438, BV2465, and BV2540 are provided in Table A. [Table A-1] [Table A-2] [Table A-3] [Table A-4]

[0236] Antibody-producing B cells from the mice were fused with immortalized B cells to generate hybridomas, which were screened for antibodies that bind to the CoV S glycoprotein.

[0237] Antibodies 239.12 and 322.3 were identified from hybridomas produced by mice immunized with BV2373. Antibody 425.6 was identified from hybridomas produced by mice immunized with BV2438. Antibody 35.13 was identified from hybridomas produced by mice immunized with BV2465. Antibody 35.13 was identified from hybridomas produced by mice immunized with BV2540.

[0238] The VH and VL sequences of 239.12, 322.3, 425.6, 35.13, and 199.9 are identified in Table B.

[0239] [Table B-1] [Table B-2] Example 2 Characterization of the binding of antibodies 239.12; 322.3; 425.6; 35.13, and 199.9 to the SARS-CoV-2 S glycoprotein

[0240] OBJECTIVE:The ability of antibodies 239.12, 322.3, 425.6, 35.13, and 199.9 to bind to CoV S glycoproteins from the parent SARS-CoV-2 virus (SARS-CoV-2 virus with SARS-CoV-2 S glycoprotein of SEQ ID NO: 9) and multiple SARS-CoV-2 variants was investigated. The ability of the aforementioned antibodies to inhibit the interaction between hACE2 and SARS-CoV-2 S glycoprotein was also investigated. Finally, the ability of the aforementioned antibodies to neutralize live SARS-CoV-2 virus and pseudovirus was investigated.

[0241] Methods - Recombinant CoV S glycoprotein production: The gene encoding the CoV S glycoprotein was codon-optimized for expression in Spodoptera frugiperda (Sf9) cells and synthetically generated from the full-length CoV S glycoprotein gene sequence. The CoV S glycoprotein contains an inactive furin cleavage site QQAQ (SEQ ID NO: 144) and two proline point mutations were introduced at K986P and V987P, where the amino acids are numbered according to SEQ ID NO: 10. Table J contains the amino acid sequences of the SARS-CoV-2 S glycoprotein used herein.

[0242] Methods - Antibody production: Antibodies were produced according to the method described in Example 1.

[0243] Methods - Biolayer Interferometry: Binding kinetics of CoV S glycoprotein to brMAbs captured on biosensors (i.e., 239.12; 322.3; 425.6; 35.13; and 199.9) were performed using an Octet QK 384 instrument (ForteBio, Fremont, CA). Briefly, to measure binding to CoV S glycoprotein (i.e., SARS-CoV-2 rS), each brMAb was coupled to an anti-mouse Fc biosensor at 2 μg / mL for 600 seconds, followed by a wash step where a baseline measurement was taken. The amino acid sequence of the CoV S glycoprotein is found in Table J. The association of CoV S glycoprotein was then measured for 600 seconds, followed by a 600 second dissociation step. Binding kinetics were analyzed using Octet software HT10.0. To measure the binding kinetics of brMAb to spiked RBD-His, RBD-His (2 μg / mL) was coupled to a Ni-NTA biosensor. After a baseline measurement, antibody association was measured for 600 seconds, followed by dissociation for 600 seconds. Binding kinetics were analyzed using Octet software HT10.0.

[0244] Methods - ELISA: 96-well microtiter plates were coated with 1.0 μg / mL of SARS-CoV-2 S protein. After blocking of non-specific binding, serial dilutions of monoclonal antibodies were added and antibody binding was measured using horseradish peroxidase (HRP)-conjugated anti-mouse. Substrate turnover was measured at OD450nm. EC50 values ​​were calculated by 4-parameter curve fitting.

[0245] Methods - hACE2 receptor inhibition: The ability of antibodies to block the interaction between human angiotensin-converting enzyme 2 (hACE2) receptor and CoV S glycoprotein was assessed by ELISA. Briefly, 96-well plates were coated with 1.0 μg / mL CoV S glycoprotein overnight at 4°C. Plates were washed with PBS-T and non-specific binding was blocked with TBS Startblock blocking buffer. Serum or mAb solutions were added in two-fold serial dilutions starting with a 1:20 dilution to coat the wells for 1 hour at room temperature. After washing, 30 ng / mL histidine-tagged hACE2 was added to the wells for 1 hour at room temperature. HRP-conjugated anti-histidine IgG was added and incubated for 1 hour, followed by the addition of TMB substrate. Plates were read at OD450nm using a SpectraMax Plus plate reader and data was analyzed with SoftMax Pro software. For each sample, the % inhibition of each dilution was calculated in the SoftMax Pro program using the following equation: 100-[(mean result / control value in positive control) x 100].

[0246] Plots of % inhibition versus serum dilution were generated and curve fitting was performed by four-parameter logistic (4PL) curve fitting to the data. The 50% inhibition of binding of hACE2 to CoV S glycoprotein (IC 50 Serum antibody titers or antibody concentrations at 100-200 ng / mL were determined in the SoftMax Pro program. Individual animal hACE2 receptor inhibitory titers, group geometric mean titers, and 95% CI were plotted using GraphPad Prism 7.05 software. For titers below the LOD of the assay, titers <20 (starting dilution) were reported and a value of "10" was assigned to the sample to calculate the group mean titer.

[0247] Methods - Live SARS-CoV-2 Neutralization Assay: Handling of live SARS-CoV-2 was performed in the Animal Biosafety Level 3 facility at the University of Maryland, School of Medicine (Baltimore, MD), the selective agent. Vero / TMPRSS2 cells were maintained in complete medium consisting of DMEM (Quality Biological) supplemented with 10% (v / v) fetal bovine serum (heat inactivated, Sigma-Aldrich), 1% (v / v) penicillin / streptomycin, and 1% (v / v) L-glutamine (final concentration of 2 mM, Gibco). Stock viruses for SARS-CoV-2 isolates were prepared in Vero / TMPRSS2 cells and sequence confirmed. Monoclonal antibodies were run in duplicate for a final starting concentration of 10 μg / mL, followed by 1:2 serial dilutions to obtain a dilution series of 12 with each well containing 100 μL. Lower sample concentrations were processed as necessary. All dilutions were performed in DMEM (Quality Biological) supplemented with 10% (v / v) fetal bovine serum (heat inactivated, Sigma), 1% (v / v) penicillin / streptomycin (Gemini Bio-products), and 1% (v / v) L-glutamine (final concentration of 2 mM, Gibco). The dilution plates were then transported to a BSL-3 laboratory and 100 μL of diluted SARS-CoV-2 inoculum was added to each well to result in a multiplicity of infection (MOI) of 0.01 upon transfer to the titration plate. Untreated virus-only controls and mock-infected controls were included on all plates. The sample / virus mixtures were then incubated at 37°C (5.0% CO2) for 1 h before 100 μL was transferred to a clear 96-well titer plate with confluent Vero / TMPRSS2 cells. The titer plates were incubated at 37 °C (5.0% CO2) for 48–72 h (depending on the variant) followed by visual CPE determination for each sample dilution. The first sample dilution showing CPE was determined as the minimum sample dilution required to neutralize >99% of the concentration of SARS-CoV-2 tested (Neutralization). 99 ) was reported.

[0248] Methods - Pseudovirus Neutralization Assay: SARS-CoV-2 pseudoviruses were generated using a lentiviral platform. Briefly, backbone and helper plasmids containing the CoV S glycoprotein were obtained. An Omicron variant of pcDNA3.1 was synthesized by GenScript using a gene encoding the CoV S glycoprotein sequence from the EPICoV database, followed by codon optimization and deletion of the cytoplasmic tail for the prototype (SARS-CoV-2 virus encoding spike glycoprotein of SEQ ID NO: 9) pseudovirus. HEK293T cells were cultured at 1 × 10 in 6-well tissue culture plates. 6 Cells were seeded at 1000 x 10 cells / well, incubated overnight at 37°C, and transfected using LIPOFECTAMINE™ 3000 with a plasmid encoding a lentiviral backbone expressing a marker protein (luciferase or Zs green), a plasmid expressing CoV S glycoprotein, and a plasmid expressing other HIV proteins for virion formation. 72 hours after transfection, the supernatant was collected and filtered through a 0.45 μM filter to obtain pseudovirus stocks. Aliquots of the pseudovirus stocks were stored at -80°C.

[0249] Pseudovirus neutralization assays were then performed using a HEK293T cell line stably expressing hACE2. Solutions containing the antibodies described herein were serially diluted 2-fold in HEK293T cell culture medium (DMEM + 10% FBS + 1% penicillin + streptomycin + glutamine, no puromycin) and 50 μL was added to each well in a 96-well tissue culture plate. 50 microliters of SARS-CoV-2 pseudovirus stock (corresponding to 3-7% GFP) was then added to each well, followed by incubation at 37°C for 1 hour. 2.5 × 10 IgG in 100 μL of HEK293T medium containing puromycin were then added to each well. 4Single cells of HEK293T / hACE2 cells were added to the wells, followed by incubation at 37°C for 72 hours. After incubation, the medium was carefully removed using a pipette, and 50 μL of trypsin was added to dislodge the cells. Manual agitation using a pipette was used to dislodge the cells, and 4% paraformaldehyde prepared in PBS was added to each well. Viral replication was determined by measuring fluorescence at 488-510 nm using a Guava flow cytometer and InCyte software (Luminex). Data was analyzed, neutralization curves were generated for each sample in GraphPad Prism, and 50% neutralization titers (EC50) were calculated by 4-parameter curve fitting.

[0250] Results - Biolayer Interferometry: Table C shows the binding kinetics of antibodies 239.12, 322.3, 425.6, and 35.13 to the RBD of CoV S glycoprotein relative to the CoV S glycoprotein from the Omicron strain of SARS-CoV-2.

[0251] [Table C]

[0252] Table D shows the binding kinetic parameters for each antibody evaluated. As shown in Table D, each antibody bound to multiple CoV S glycoproteins. Specifically, 239.12 bound to CoV S glycoproteins associated with SARS-CoV-2 parental, gamma, delta, and alpha strains. 322.3 binds to CoV S glycoproteins associated with SARS-CoV-2 parental, gamma, beta, delta, alpha, and multiple Omicron strains. 35.13 binds to CoV S glycoproteins associated with SARS-CoV-2 parental, gamma, beta, delta, alpha, and multiple Omicron strains. 425.6 binds to CoV S glycoproteins associated with SARS-CoV-2 parental, gamma, beta, delta, alpha, and multiple Omicron strains.

[0253] 1A-1D show binding curves of 239.12 (FIG. 1A), 322.3 (FIG. 1B), 425.6 (FIG. 1C), and 35.13 (FIG. 1D) to CoV S glycoproteins associated with the SARS-CoV-2 parental strain (SEQ ID NO: 35), SARS-CoV-2 gamma strain (SEQ ID NO: 38), SARS-CoV-2 beta strain (SEQ ID NO: 36), SARS-CoV-2 delta strain (SEQ ID NO: 37), SARS-CoV-2 alpha strain (SEQ ID NO: 39), and SARS-CoV-2 Omicron strain (SEQ ID NO: 42).

[0254] Figures 1E-1I show the binding curves of 239.12 (Figure 1E), 322.3 (Figure 1F), 425.6 (Figure 1G), 35.13 (Figure 1H), and 199.9 (Figure 1I) to various CoV S glycoproteins related to the SARS-CoV-2 S omicron strain. [Table D-1] [Table D-2] [Table D-3] [Table D-4] [Table D-5] [Table D-6]

[0255] Results - ELISA: Figures 2A-2D show the EC50s for binding of 239.12 (Figure 2A), 322.3 (Figure 2B), 425.6 (Figure 2C), 35.13 (Figure 2D) to various CoV S glycoproteins. Figures 2E-2I show the EC50s for binding of 239.12 (Figure 2E), 322.3 (Figure 2F), 425.6 (Figure 2G), 35.13 (Figure 2H), and 199.9 (Figure 2I) to various CoV S glycoproteins associated with the SARS-CoV-2 S Omicron strain. Table E shows the EC50 for binding of 35.13 to CoV S glycoprotein. Table F1 shows the EC50 (ng / mL) for antibody binding to CoV S glycoproteins from the Omicron strain (239.12, 322.3, 426.7, and 35.13). Table F2 shows the EC50 (ng / mL) for binding of 199.9 to CoV S glycoprotein. [Table E] [Table F1] [Table F2-1] [Table F2-2]

[0256] Results - hACE2 receptor inhibition: Table G shows the concentration of 35.13 that inhibits 50% of the interaction between hACE2 and CoV S glycoprotein (IC50). Table H1 shows the concentration of antibody that inhibits 50% of the interaction between hACE2 and omicron-related CoV S glycoprotein (IC50). Table H2 shows the concentration of 199.9 that inhibits 50% of the interaction between hACE2 and CoV S glycoprotein (IC50). Figures 5A-5C show hACE2 receptor inhibition of 35.13 (Figure 5A), 425.6 (Figure 5B), and 322.3 (Figure 5C). [Table G] [Table H1] [Table H2-4]

[0257] Results - Live SARS-CoV-2 Neutralization Assay: SARS-CoV-2 Neutralization Assay: 35.13 neutralized the parent SARS-CoV-2, and variant strains up to Omicron BA.4. 35.13 did not neutralize BA.4.6 or BA.5 in this assay. 425.6 showed potent neutralizing activity against all variants tested, except against Omicron BQ.1.1. Figures 4A-4C show the minimum sample dilutions (Neut99) of 35.13 (Figure 4A), 425.6 (Figure 4B), and 322.3 (Figure 4C) required to neutralize >99% of the concentrations of SARS-CoV-2 tested.

[0258] Table I shows the concentration at which 50% of the SARS-CoV-2 virus is neutralized by 425.6. "ND" means that the data point has not yet been collected. [Table I]

[0259] Results - Pseudovirus Neutralization: Figures 6A-6B show pseudovirus neutralization by antibodies 35.13 (Figure 6A) and 425.6 (Figure 6B). [Table J-1] [Table J-2] [Table J-3] [Table J-4] [Table J-5]

Table J-6

Table J-7

Table J-8

Table J-9

Table J-10

Table J-11

Table J-12

Table J-13

Table J-14

Table J-15

Table J-16

Table J-17

Table J-18

Table J-19

Table J-20

Table J-21

Table J-22

Table J-23

Table J-24

Table J-25

Table J-26

Table J-27

Table J-28

Table J-29

Table J-30

Table J-31

Table J-32

Table J-33

Table J-34

Table J-35

Table J-36

Table J-37

Table J-38

Table J-39

[0260] Objective: Alanine scanning mutagenesis was performed to identify the epitopes of the 239.12, 322.3, 425.6, and 35.13 antibodies.

[0261] Methods: Shotgun mutagenesis epitope mapping services were provided by Integral Molecular (Philadelphia, PA) using a SARS-CoV-2 (Wuhan-Hu-1 strain) S protein RBD shotgun mutagenesis mutation library generated using a full-length expression construct for the S protein. 184 residues of the RBD were individually mutated to alanine and alanine residues to serine. The mutant library was placed in a 384-well microplate, transiently transfected into HEK293T cells, and allowed to express for 22 hours. Cells were then incubated with antibody at concentrations previously determined using an independent binding titration curve in cells expressing wild-type spike. Cells were fixed in 4% (v / v) paraformaldehyde (Electron Microscopy Sciences) and permeabilized with 0.2% (w / v) saponin (Sigma-Aldrich) in PBS with calcium and magnesium (PBS++) before incubation with brMAb diluted in PBS++, 10% normal goat serum (Sigma), and 0.1% saponin. Antibodies were detected using 3.75 μg / mL Alexa-Fluor-488-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories) in 10% normal goat serum with 0.1% saponin. Cells were washed three times with PBS++ / 0.1% saponin followed by two washes with PBS and mean cell fluorescence was detected using a high-throughput Intellicyte iQue flow cytometer (Sartorius). Antibody reactivity for each mutant S protein clone was calculated relative to wild-type S protein reactivity by subtracting the signal from mock-transfected controls and normalizing to the signal from wild-type S transfected controls. Mutations within clones were identified as important for the mAb epitope if they did not support reactivity of the test mAb but did support reactivity of other SARS-CoV-2 antibodies.This counterscreening strategy facilitates the elimination of S protein mutants that are locally misfolded or have expression defects. The amino acid sequence of Novavax recombinant spike (rS) protein was aligned in Clustal Omega (ebi.ac.uk / Tools / msa / clustalo). Any point mutations from the ancestral sequence were noted and amino acids important for brMAb binding were highlighted. Residues involved in hACE2 binding and class 1-4 antibody binding were noted as described in Barnes, CO, Jette, CA, Abernathy, ME et al. SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Nature 588, 682-687 (2020). Note that the Barnes antibody classes were defined using human mAbs and that the mAbs described in this work were generated in mice.

[0262] Results: Table K shows the binding reactivity of 322.3, 239.12, and 425.6 Fabs to mutant SARS-CoV-2 S RBD compared to binding to wild-type SARS-CoV-2 S RBD. The underlined amino acids were determined to be important for binding. The amino acids in Table K are numbered with respect to the SARS-CoV-2 S protein of SEQ ID NO: 10. [Table K]

[0263] The amino acids important for binding of 35.13 Fab to SARS-CoV-2 glycoprotein (residues 476, 485, 486, 487, 489 of SEQ ID NO: 10) are shown in Table L. Further structural data indicated that amino acids 485, 486, 487, and 489 were particularly important for binding of 35.13 to SARS-CoV-2 S glycoprotein. In addition, structural data confirmed that amino acids 378 and 385 were important for binding of 322.3 to SARS-CoV-2 S glycoprotein, and that amino acids 444, 445, 446, and 448 were important for binding of 425.6 to SARS-CoV-2 S glycoprotein. [Table L]

[0264] Figures 3A-3D show the crystal structure of the SARS-CoV-2 S glycoprotein (Protein Databank ID: 6XCN). The critical residues for binding to the 35.13 (Figure 3A), 425.6 (Figure 3B), 239.12 (Figure 3C), and 322.3 (Figure 3D) Fabs are shown as spheres. The structures to the right of each figure show the critical residues in the SARS-CoV-2 S receptor binding domain (RBD) for binding to each Fab (Protein Databank ID: 6Z2M). Figure 7 shows the alignment of the SARS-CoV-2 S glycoproteins from the ancestral, beta, delta, gamma, BA.1, BA.2, BA.5, and BQ.1.1 SARS-CoV-2 viruses. The critical amino acids (K378 and T385) for binding of 322.3 to the SARS-CoV-2 S glycoprotein are boxed. The important amino acids for binding of 425.6 to the SARS-CoV-2 S glycoprotein are boxed (K444, V445, G446, and N448). The important amino acids for binding of 425.6 to the SARS-CoV-2 S glycoprotein are boxed (K444, V445, G446, and N448). The numbering of the important amino acids is relative to the SARS-CoV-2 S glycoprotein in SEQ ID NO: 10. Numbered embodiments 1. An antibody or fragment thereof that binds to the sudden acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein. 2. The antibody or fragment thereof according to embodiment 1, wherein light chain complementarity determining region 1 (VL CDR1) is selected from the group consisting of SEQ ID NOs: 11 to 14; light chain complementarity determining region 2 (VL CDR2) is selected from the group consisting of SEQ ID NOs: 15 to 18; light chain complementarity determining region 3 (VL CDR3) is selected from the group consisting of SEQ ID NOs: 19 to 22; heavy chain complementarity determining region 1 (VH CDR1) is selected from the group consisting of SEQ ID NOs: 23 to 26; heavy chain complementarity determining region 2 (VH CDR2) is selected from the group consisting of SEQ ID NOs: 27 to 30; and heavy chain complementarity determining region 3 (VH CDR3) is selected from the group consisting of SEQ ID NOs: 31 to 34. 3. (i) a VH CDR1 set forth in SEQ ID NO: 23, a VH CDR2 set forth in SEQ ID NO: 27, and a VH CDR3 set forth in SEQ ID NO: 31; a VL CDR1 set forth in SEQ ID NO: 11, a VL CDR2 set forth in SEQ ID NO: 15; and a VL CDR3 set forth in SEQ ID NO: 19; (ii) a VH CDR1 set forth in SEQ ID NO: 24; a VH CDR2 set forth in SEQ ID NO: 28; a VH CDR3 set forth in SEQ ID NO: 32; a VL CDR1 set forth in SEQ ID NO: 12; a VL CDR2 set forth in SEQ ID NO: 16; and a VL CDR3 set forth in SEQ ID NO: 20; (iii) the VH CDR1 set forth in SEQ ID NO: 25; the VH CDR2 set forth in SEQ ID NO: 29; the VH CDR3 set forth in SEQ ID NO: 33; the VL CDR1 set forth in SEQ ID NO: 13; the VL CDR2 set forth in SEQ ID NO: 17; and the VL CDR3 set forth in SEQ ID NO: 21; or (iv) VH CDR1 set forth in SEQ ID NO: 26; VH CDR2 set forth in SEQ ID NO: 30; VH CDR3 set forth in SEQ ID NO: 34; VL CDR1 set forth in SEQ ID NO: 14; VL CDR2 set forth in SEQ ID NO: 18; and VL CDR3 set forth in SEQ ID NO: 22 3. The antibody or fragment thereof of embodiment 1 or 2, comprising: 4. An antibody or fragment thereof according to any one of embodiments 1 to 3, wherein the amino acid sequence of the variable heavy (VH) domain comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 5 to 8. 5. An antibody or fragment thereof according to any one of embodiments 1 to 4, wherein the amino acid sequence of the variable light (VL) domain comprises or consists of an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide having an amino acid sequence of any one of SEQ ID NOs: 1 to 4. 6.Antibodies (i) an antibody comprising a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:5; and (ii) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:1; (i) an antibody comprising a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:6; and (ii) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:2; (i) an antibody comprising a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:7; and (ii) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:3; and (i) a VH comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:4; and (ii) a VL comprising an amino acid sequence having at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to SEQ ID NO:8. 6. The antibody or fragment thereof according to any one of embodiments 1 to 5, which is selected from the group consisting of: 7. The antibody or fragment thereof according to any one of embodiments 1 to 6, wherein the antibody or fragment thereof is a monoclonal antibody, Fab, F(ab')2, Fab', scFv, or a single domain antibody (sdAb). 8. The antibody or fragment thereof according to any one of embodiments 1 to 7, wherein the antibody comprises a human IgG1 or IgG4 domain. 9. The antibody or fragment thereof according to any one of embodiments 1 to 8, wherein the antibody or fragment thereof has a dissociation constant (KD) for a SARS-CoV-2 S polypeptide or variant thereof of 50 nM or less, 10 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, or 0.001 nM or less. 10. The antibody or fragment thereof according to any one of embodiments 1 to 9, wherein the antibody or fragment thereof binds to one or more CoV S polypeptides having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polypeptides set forth in SEQ ID NOs: 9, 10, 35-43, and 72-73. 11. An isolated nucleic acid molecule encoding an antibody or fragment thereof according to any one of embodiments 1 to 10. 12. An expression vector comprising a nucleic acid segment encoding the antibody or fragment thereof according to any one of embodiments 1 to 10. 13. A host cell comprising an expression vector according to embodiment 12. 14. A pharmaceutical composition comprising an antibody according to any one of embodiments 1 to 10 and a pharma- ceutically acceptable carrier. 15. A method for treating a subject infected with the SARS-CoV-2 virus or a variant thereof in need thereof, comprising administering to the subject an antibody or fragment thereof according to any one of embodiments 1 to 10, or a pharmaceutical composition according to embodiment 14. 16. The method of embodiment 15, wherein the subject is 65 years of age or older. 17. The method of embodiment 15, wherein the subject is immunocompromised. 18. The method of embodiment 15, wherein the subject is a pregnant woman. 19. The method of embodiment 15, wherein the SARS-CoV-2 variant is selected from the group consisting of: B.1.1.7 SARS-CoV-2 strain; B.1.351 SARS-CoV-2 strain; P.1 SARS-CoV-2 strain; Cal.20C SARS-CoV-2 strain; B.1.617.2 SARS-CoV-2 strain; B.1.525 SARS-CoV-2 strain; B.1.526 SARS-CoV-2 strain; B.1.617.1 SARS-CoV-2 strain; C.37 SARS-CoV-2 strain; B.1.621 SARS-CoV-2 strain; and B.1.1.529 SARS-CoV-2 strain.

[0265] Incorporation by Reference All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entirety for all purposes. However, the mention of any references, articles, publications, patents, patent publications, and patent applications cited herein is not, and should not be construed as, any form of admission or suggestion that they constitute valid prior art or form part of the common general knowledge in any country throughout the world. The following patent documents are incorporated by reference in their entirety for all purposes: International Application No. PCT / US2022 / 080700, filed November 30, 2022; International Publication No. WO 2021 / 154812; and International Publication No. WO 2022 / 203963.

Claims

1. An antibody or fragment thereof that binds to severe acute respiratory syndrome coronavirus 2 (CoV) spike (S) glycoprotein, (i) a variable light chain complementarity determining region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 11-14 and 76; (ii) a variable light chain complementarity-determining region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 15-18 and 77; (iii) a variable light chain complementarity-determining region 3 (VL CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 19-22 and 78; (iv) a variable heavy chain complementarity determining region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23-26 and 79; (v) a variable heavy chain complementarity determining region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 27-30 and 80; and (vi) a variable heavy chain complementarity-determining region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 31-34 and 81. An antibody or fragment thereof comprising:

2. An antibody or fragment thereof that binds to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein, (i) a variable heavy (VH) domain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of SEQ ID NOs: 5-8 and 75; and (ii) a variable light (VL) domain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polypeptide of any one of SEQ ID NOs: 1-4 and 74. An antibody or fragment thereof comprising:

3. (i) a variable heavy (VH) domain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide of SEQ ID NOs: 5-8 and 75; and (ii) a variable light (VL) domain comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to the polypeptide of any one of SEQ ID NOs: 1-4 and 74. The antibody or fragment thereof of claim 1, comprising:

4. (i) a variable light chain complementarity determining region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 11-14 and 76; (ii) a variable light chain complementarity-determining region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 15-18 and 77; (iii) a variable light chain complementarity-determining region 3 (VL CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 19-22 and 78; (iv) a variable heavy chain complementarity determining region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 23-26 and 79; (v) a variable heavy chain complementarity determining region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 27-30 and 80; and (vi) a variable heavy chain complementarity-determining region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity to a sequence selected from the group consisting of SEQ ID NOs: 31-34 and 81. The antibody or fragment thereof of claim 2, comprising:

5. The antibody or fragment thereof according to claim 4, wherein the antibody or fragment thereof is selected from the group consisting of VH CDR1s set forth in SEQ ID NOs: 23 to 26, 79, VH CDR2s set forth in SEQ ID NOs: 27 to 30, 80, and VH CDR3s set forth in SEQ ID NOs: 31 to 34, 81; VL CDR1s set forth in SEQ ID NOs: 11 to 14, 76, VL CDR2s set forth in SEQ ID NOs: 15 to 18, 77; and VL CDR3s set forth in SEQ ID NOs: 19 to 22, 78.

6. (i) a VH comprising the amino acid sequence of SEQ ID NOs: 5-8, 75; and (ii) VL comprising the amino acid sequence of SEQ ID NOs: 1 to 4, 74 The antibody or fragment thereof according to claim 4, selected from the group consisting of:

7. Monoclonal antibodies, Fab, F(ab') ２ 5. The antibody or fragment thereof of claim 4, which is a Fab', scFv, or single domain antibody (sdAb).

8. The antibody or fragment thereof of claim 4, wherein the antibody comprises a human IgG1 or IgG4 domain.

9. an equilibrium dissociation constant (K) for the CoV S glycoprotein or a variant thereof of 50 nM or less, 10 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, or 0.001 nM or less; Ｄ 5. The antibody or fragment thereof according to claim 4, having the following structure:

10. 1.0 × 10 to CoV S glycoprotein or its variants －９ moles / liter (M) less than 1.0 x 10 －１０ Less than M, 1.0 x 10 －１１ Less than M or 1.0 x 10 －１２ The antibody or fragment thereof of claim 4, which binds with an equilibrium dissociation constant (Kd) of less than M.

11. 5. The antibody or fragment thereof of claim 4, which binds to one or more CoV S polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity to a polypeptide set forth in any one of SEQ ID NOs: 9, 10, 35-43, 72-73, 90-139, and 145-147.

12. The antibody or fragment thereof of claim 4, wherein the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein, and the epitope comprises amino acids 476, 485, 486, 487, and 489 of the CoV S glycoprotein of SEQ ID NO:

10.

13. The antibody or fragment thereof of claim 4, wherein the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein, and the epitope comprises amino acids 378 and 385 of the CoV S glycoprotein of SEQ ID NO:

10.

14. The antibody or fragment thereof of claim 4, wherein the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein, and the epitope comprises amino acids 444, 445, 446, and 448 of the CoV S glycoprotein of SEQ ID NO:

10.

15. A pharmaceutical composition comprising the antibody or fragment thereof of claim 4 and a pharmaceutically acceptable carrier.

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