SARS-CoV-2 Antibodies and Methods of Use

Abstract

The present disclosure provides antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2, and methods of making and using the same. The antibodies can be used, for example, for prevention, post-exposure prophylaxis, or treatment of SARS-CoV-2 infection. The antibodies can also be used, for example, to detect SARS-CoV-2 infection in a subject.

Description

[Technical field]

[0001] 1. CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 336,332, filed April 29, 2022, and U.S. Provisional Patent Application No. 63 / 371,454, filed August 15, 2022, each of which is incorporated by reference in its entirety herein.

[0002] 2. Reference to sequence listings submitted in electronic form The contents of the sequence listing in the sequence listing submitted electronically in an ASCII text file (File Name: 2943_218PC02_SequenceListing_ST26, Size: 89,789 bytes, and Creation Date: April 24, 2023) submitted with this application are incorporated herein by reference in their entirety.

[0003] The present disclosure relates to antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2, and methods of use thereof. [Background technology]

[0004] The coronavirus 2019 (COVID19) pandemic caused by Severe Acute Respiratory Syndrome Coronavirus Type 2 (SARS-CoV-2) has emerged. SARS-CoV-2 was first identified in Wuhan, People's Republic of China, in December 2019 and rapidly caused a global outbreak. The mortality rate of the virus is currently uncertain, but the number of cases and deaths worldwide is significant. The virus can be spread from person to person through droplets from the nose or mouth that are expelled when an infected person coughs, sneezes, or speaks. The incubation period (time from exposure to onset of symptoms) ranges from 0 to 24 days, with an average of 3 to 5 days, although infection can be transmitted through contact during this period even after recovery. Most people with SARS-CoV-2 develop symptoms within 11.5 days of exposure. Symptoms include fever, cough, and difficulty breathing. The virus has a greater impact on older patients with type 2 diabetes, heart disease, chronic obstructive pulmonary disease (COPD), and / or obesity. Most patients who contract this virus have mild symptoms, but in some, the lung infection becomes severe, leading to severe breathing difficulties or even death.

[0005] Several vaccines aimed at preventing COVID-19 have been approved, and combinations of antibodies for pre-exposure prophylaxis (Evusheld) are also available, but these are less effective against variants that have emerged since their development than against older variants. Therefore, there is an urgent need for medicines that can prevent and treat COVID-19, including cases caused by new variants of the virus. Summary of the Invention [Means for solving the problem]

[0006] Provided herein are antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, as well as compositions and combinations thereof. In some embodiments, the composition or combination comprises: (i) a first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the first antibody or antigen-binding fragment thereof comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively; and (ii) a second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the second antibody or antigen-binding fragment thereof comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 69, 70, 71, 73, 74, and 75, respectively.

[0007] In some aspects, the composition or combination comprises: (i) a first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2 and competitively inhibits binding to the spike protein of SARS-CoV-2 with an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61; and (ii) a second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2 and competitively inhibits binding to the spike protein of SARS-CoV-2 with an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 68 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 72.

[0008] In some aspects, the composition or combination comprises: (i) a first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or antigen-binding fragment thereof binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61; and (ii) a second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or antigen-binding fragment thereof binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 68 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 72.

[0009] In some embodiments, the first antibody or antigen-binding fragment thereof comprises the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, respectively, of SEQ ID NOs: 31, 32, 37, 34, 35 and 36. In some embodiments, the first antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 61.

[0010] In some embodiments, the second antibody or antigen-binding fragment thereof comprises the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of SEQ ID NOs: 69, 70, 71, 73, 74 and 74, respectively. In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 68 and a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76 and a light chain comprising the amino acid sequence of SEQ ID NO: 77.

[0011] In some embodiments, the first antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 63 and / or a VL comprising the amino acid sequence of SEQ ID NO: 61. In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 68 and / or a VL comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76 and / or a light chain comprising the amino acid sequence of SEQ ID NO: 77.

[0012] In some embodiments, the composition or combination neutralizes SARS-CoV-2 virus, and optionally the composition or combination neutralizes SARS-CoV-2 BA.2.12.1 with an IC50 of 25 ng / mL or less, or an IC50 of 20 ng / mL or less. In some embodiments, the composition or combination neutralizes SARS-CoV-2 D614G, SARS-CoV-2 alpha, SARS-CoV-2 delta+T51I+T95I, SARS-CoV-2 BA.1, SARS-CoV-2 BA.1.1, SARS-CoV-2 BA.2, SARS-CoV-2 BA.2.12.1, and / or SARS-CoV-2 BA.5 virus with an IC50 of 75 ng / mL or less, or an IC50 of 60 ng / mL or less.

[0013] In some embodiments, the first antibody or antigen-binding fragment is fully human and / or the second antibody or antigen-binding fragment is fully human. In some embodiments, the first antibody or antigen-binding fragment comprises a light chain constant region and / or the second antibody or antigen-binding fragment comprises a light chain constant region. In some embodiments, the first antibody or antigen-binding fragment comprises a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions, optionally the light chain constant region is a human IgGκ light chain constant region and / or the second antibody or antigen-binding fragment comprises a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions, optionally the light chain constant region is a human IgGκ light chain constant region. In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region. In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions, optionally where the heavy chain constant region is human IgG1, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions, optionally where the heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, the first antibody or antigen-binding fragment comprises (i) a human IgG1 heavy chain constant region and (ii) a human IgGκ light chain constant region, and / or the second antibody or antigen-binding fragment comprises (i) a human IgG1 heavy chain constant region and (ii) a human IgGκ light chain constant region.

[0014] In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region comprising a YTE mutation, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region and a light chain constant region, optionally wherein the light chain constant region is a human IgGK light chain constant region, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region comprising a YTE mutation, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region comprising a TM mutation, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region and a light chain constant region, optionally wherein the light chain constant region is a human IgGK light chain constant region, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region comprising a TM mutation, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region.

[0015] In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:66, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:66.

[0016] In some embodiments, the first antibody or antigen-binding fragment is a full-length antibody and / or the second antibody or antigen-binding fragment is a full-length antibody.

[0017] In some embodiments, the first antibody or antigen-binding fragment is an antigen-binding fragment and / or the second antibody or antigen-binding fragment is an antigen-binding fragment. In some embodiments, the first antigen-binding fragment is a Fab, Fab', F(ab') 2 , single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, minibody, F(ab') 3 , tetrabodies, triabodies, bispecific antibodies, single domain antibodies, (scFv) 2 or scFv-Fc, and / or the second antigen-binding fragment is a Fab, Fab', F(ab') 2, single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, minibody, F(ab') 3 , tetrabodies, triabodies, bispecific antibodies, single domain antibodies, (scFv) 2 , or scFv-Fc.

[0018] In some embodiments, the first antibody or antigen-binding fragment is isolated and / or the second antibody or antigen-binding fragment is isolated. In some embodiments, the first antibody or antigen-binding fragment is monoclonal and / or the second antibody or antigen-binding fragment is monoclonal. In some embodiments, the first antibody or antigen-binding fragment is recombinant and / or the second antibody or antigen-binding fragment is recombinant.

[0019] In some aspects, the composition or combination comprises: (i) a first human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the first antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61, and heavy and light chain constant regions that comprise a YTE mutation and a TM mutation; and (ii) a second human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the second antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 68, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 72, and heavy and light chain constant regions that comprise a YTE mutation and a TM mutation. In some embodiments, the heavy chain constant region of the first human IgG1 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 66 and / or the heavy chain constant region of the first human IgG1 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 66. In some embodiments, the second human IgG1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76 and a light chain comprising the amino acid sequence of SEQ ID NO: 77.

[0020] In some embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0021] In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in the same composition, optionally, the composition is a pharmaceutical composition. In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in separate compositions, optionally, the first antibody or antigen-binding fragment thereof and / or the second antibody or antigen-binding fragment thereof are in a pharmaceutical composition.

[0022] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to spike protein of SARS-CoV-2 comprises (i) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively; (ii) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of SEQ ID NOs: 25, 26, 30, 28, 67, and 29, respectively; or (iii) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of SEQ ID NOs: 13, 14, 19, 16, 17, and 18, respectively.

[0023] In some embodiments, an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2 comprises: (a) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 40 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 41; (b) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 44 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 45; (c) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 48 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47; (d) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 49 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47; (e) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 52 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53; (f) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 56 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 57; (g) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 58 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 57; (h) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 59 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 55; (i) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 62 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61; (j) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61; (k) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 60 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 64; (l) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 62 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 64; (m) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and / or a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 64.

[0024] In some embodiments, the antibody or antigen-binding fragment thereof neutralizes SARS-CoV-2 pseudoviruses. In some embodiments, the antibody or antigen-binding fragment thereof neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2 delta, and / or SARS-CoV-2 D614G pseudoviruses with an EC50 of 100 ng / mL or less.

[0025] In some embodiments, the antibody or antigen-binding fragment is fully human.

[0026] In some embodiments, the antibody or antigen-binding fragment comprises a light chain constant region, hi some embodiments, the light chain constant region is selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions, optionally wherein the light chain constant region is a human IgGκ light chain constant region.

[0027] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region. In some embodiments, the heavy chain constant region is selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions. In some embodiments, the heavy chain constant region is a human IgG1 heavy chain constant region.

[0028] In some embodiments, the antibody or antigen-binding fragment comprises (i) a human IgG1 heavy chain constant region, and (ii) a human IgGκ light chain constant region.

[0029] In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising a YTE mutation, and optionally the heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising a TM mutation, and optionally the heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain constant region comprising a YTE mutation and a TM mutation, and optionally the heavy chain constant region is a human IgG1 heavy chain constant region. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:66.

[0030] In some embodiments, the antibody or antigen-binding fragment thereof is a full-length antibody. In some embodiments, the antibody or antigen-binding fragment thereof is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is a Fab, Fab', F(ab') 2 , single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, minibody, F(ab') 3 , tetraspecific antibodies, trispecific antibodies, bispecific antibodies, single domain antibodies, (scFv) 2 , or scFv-Fc.

[0031] In some embodiments, the antibody or antigen-binding fragment is isolated. In some embodiments, the antibody or antigen-binding fragment is monoclonal. In some embodiments, the antibody or antigen-binding fragment is recombinant.

[0032] In some embodiments, the antibody or antigen-binding fragment thereof further comprises a detectable label.

[0033] Also provided herein are polynucleotides.

[0034] In some embodiments, the isolated polynucleotide comprises a nucleic acid molecule encoding a heavy chain variable region and / or a nucleic acid molecule encoding a light chain variable region of an antibody or antigen-binding fragment thereof provided herein.

[0035] Vectors are also provided herein. In some embodiments, an isolated vector comprises a polynucleotide provided herein.

[0036] Host cells are also provided herein. In some embodiments, the host cells comprise a polynucleotide provided herein, a vector provided herein, or a first vector comprising a nucleic acid molecule encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof provided herein, and a second vector comprising a nucleic acid molecule encoding a light chain variable region.

[0037] Also provided herein are methods of producing antibodies and antigen-binding fragments thereof. In some embodiments, a method of generating an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 comprises culturing a host cell provided herein such that a nucleic acid molecule is expressed and an antibody or antigen-binding fragment thereof is produced. In some embodiments, the method further comprises isolating the antibody or antigen-binding fragment. Also provided herein are antibodies and antigen-binding fragments thereof produced by the methods provided herein.

[0038] Also provided herein are compositions. In some embodiments, the compositions comprise an antibody or antigen-binding fragment provided herein. In some embodiments, the compositions are pharmaceutical compositions further comprising a pharma- ceutically acceptable excipient.

[0039] Also provided herein are compositions and combinations. In some embodiments, the compositions or combinations include a first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, where the first antibody or antigen-binding fragment thereof competitively inhibits binding to the spike protein of SARS-CoV-2 of an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47; and a second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the second antibody or antigen-binding fragment thereof being (i) an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61, or (ii) a second antibody or antigen-binding fragment thereof that competitively inhibits binding to the spike protein of SARS-CoV-2 of an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53.

[0040] In some embodiments, the composition or combination comprises a first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the first antibody or antigen-binding fragment thereof binding to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47; and a second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the second antibody or antigen-binding fragment thereof binding to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising (i) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61, or (ii) a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53.

[0041] In some embodiments, the first antibody or antigen-binding fragment thereof comprises the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively. In some embodiments, the first antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 47.

[0042] In some embodiments, the second antibody or antigen-binding fragment thereof comprises the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, respectively, of SEQ ID NOs: 31, 32, 37, 34, 35, and 36. In some embodiments, the second antibody or antigen-binding fragment thereof comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 61, (ii) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 61, (iii) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 61, (iv) a VH comprising the amino acid sequence of SEQ ID NO: 60 and a VL comprising the amino acid sequence of SEQ ID NO: 64, (v) a VH comprising the amino acid sequence of SEQ ID NO: 62 and a VL comprising the amino acid sequence of SEQ ID NO: 64, or (vi) a VH comprising the amino acid sequence of SEQ ID NO: 63 and a VL comprising the amino acid sequence of SEQ ID NO: 64.

[0043] In some embodiments, the second antibody or antigen-binding fragment thereof comprises the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3, respectively, of SEQ ID NOs: 20, 21, 22, 23, 67, and 24. In some embodiments, the second antibody or antigen-binding fragment thereof comprises (i) a VH comprising the amino acid sequence of SEQ ID NO: 52 and a VL comprising the amino acid sequence of SEQ ID NO: 53, or (ii) a VH comprising the amino acid sequence of SEQ ID NO: 50 and a VL comprising the amino acid sequence of SEQ ID NO: 51.

[0044] In some embodiments, the composition or combination comprises a first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, the first antibody or antigen-binding fragment thereof comprising VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively, and a second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2. and a second antibody or antigen-binding fragment comprising: (i) the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively; or (ii) the VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 20, 21, 22, 23, 67, and 24, respectively.

[0045] In some embodiments, the first antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:46 and / or a VL comprising the amino acid sequence of SEQ ID NO:47.

[0046] In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:63 and / or a VL comprising the amino acid sequence of SEQ ID NO:61.

[0047] In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:52 and / or a VL comprising the amino acid sequence of SEQ ID NO:53.

[0048] In some embodiments, the composition or combination neutralizes SARS-CoV-2 pseudoviruses, and optionally the composition or combination neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2 delta, and / or SARS-CoV-2 D614G pseudoviruses with an EC50 of 75 ng / mL or less. In some embodiments, the composition or combination (i) neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, and / or SARS-CoV-2 BA.2 pseudoviruses with an EC50 of 25 ng / mL or less, and / or (ii) neutralizes SARS-CoV-2 delta and / or SARS-CoV-2 D614G pseudoviruses with an EC50 of 75 ng / mL or less.

[0049] In some embodiments, the first antibody or antigen-binding fragment is fully human and / or the second antibody or antigen-binding fragment is fully human.

[0050] In some embodiments, the first antibody or antigen-binding fragment comprises a light chain constant region, and / or the second antibody or antigen-binding fragment comprises a light chain constant region. In some embodiments, the first antibody or antigen-binding fragment comprises a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions, optionally wherein the light chain constant region is a human IgGκ light chain constant region, and / or the second antibody or antigen-binding fragment comprises a light chain constant region selected from the group consisting of human immunoglobulin IgGκ and IgGλ light chain constant regions, optionally wherein the light chain constant region is a human IgGκ light chain constant region.

[0051] In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region. In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions, optionally where the heavy chain constant region is human IgG1, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region selected from the group consisting of human immunoglobulin IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2 heavy chain constant regions, optionally where the heavy chain constant region is a human IgG1 heavy chain constant region.

[0052] In some embodiments, the first antibody or antigen-binding fragment comprises (i) a human IgG1 heavy chain constant region and (ii) a human IgGκ light chain constant region, and / or the second antibody or antigen-binding fragment comprises (i) a human IgG1 heavy chain constant region and (ii) a human IgGκ light chain constant region.

[0053] In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region comprising a YTE mutation, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region, and a light chain constant region, optionally wherein the light chain constant region is a human IgGκ light chain constant region, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region comprising a YTE mutation, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region.

[0054] In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region comprising TM mutations, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region, and a light chain constant region, optionally wherein the light chain constant region is a human IgGκ light chain constant region, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region comprising TM mutations, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region.

[0055] In some embodiments, the first antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:66, and / or the second antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:66.

[0056] In some embodiments, the first antibody or antigen-binding fragment is a full-length antibody and / or the second antibody or antigen-binding fragment is a full-length antibody. In some embodiments, the first antibody or antigen-binding fragment is an antigen-binding fragment and / or the second antibody or antigen-binding fragment is an antigen-binding fragment. In some embodiments, the first antigen-binding fragment is a Fab, Fab', F(ab') 2 , single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, minibody, F(ab') 3 , tetrabodies, triabodies, bispecific antibodies, single domain antibodies, (scFv) 2 or scFv-Fc, and / or the second antigen-binding fragment is a Fab, Fab', F(ab') 2 , single chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, minibody, F(ab') 3 , tetrabodies, triabodies, bispecific antibodies, single domain antibodies, (scFv) 2 , or scFv-Fc.

[0057] In some embodiments, the first antibody or antigen-binding fragment is isolated and / or the second antibody or antigen-binding fragment is isolated. In some embodiments, the first antibody or antigen-binding fragment is monoclonal and / or the second antibody or antigen-binding fragment is monoclonal. In some embodiments, the first antibody or antigen-binding fragment is recombinant and / or the second antibody or antigen-binding fragment is recombinant.

[0058] In some aspects, the composition or combination comprises a first human IgG1 antibody or antigen-binding fragment thereof that specifically binds to spike protein of SARS-CoV-2, the first antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, a heavy chain constant region comprising a YTE mutation and a TM mutation, and a light chain constant region, and a second human IgG1 antibody or antigen-binding fragment thereof that specifically binds to spike protein of SARS-CoV-2, the second antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61, a heavy chain constant region comprising a YTE mutation ...

[0059] In some aspects, the composition or combination comprises a first human IgG1 antibody or antigen-binding fragment thereof that specifically binds to spike protein of SARS-CoV-2, the first antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, a heavy chain constant region comprising a YTE mutation and a TM mutation, and a light chain constant region, and a second human IgG1 antibody or antigen-binding fragment thereof that specifically binds to spike protein of SARS-CoV-2, the second antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 52, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53, a heavy chain constant region comprising a YTE mutation ...

[0060] In some embodiments, the composition or combination comprises a first human IgG1 antibody or antigen-binding fragment thereof that specifically binds to spike protein of SARS-CoV-2, the first antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, a heavy chain constant region comprising a YTE mutation and a TM mutation, and a light chain constant region, and a second human IgG1 antibody or antigen-binding fragment thereof that specifically binds to spike protein of SARS-CoV-2, the second antibody or antigen-binding fragment comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 50, a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 51, a heavy chain constant region comprising a YTE mutation ...

[0061] In some embodiments, the heavy chain constant region of the first human IgG1 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:66 and / or the heavy chain constant region of the first human IgG1 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO:66.

[0062] In some embodiments, the composition is a pharmaceutical composition further comprising a pharma- ceutically acceptable carrier.

[0063] In some embodiments of the combinations provided herein, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in the same composition, and optionally, the composition is a pharmaceutical composition. In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in separate compositions, and optionally, the first antibody or antigen-binding fragment thereof and / or the second antibody or antigen-binding fragment thereof are in a pharmaceutical composition.

[0064] Also provided herein are methods for neutralizing SARS-CoV-2. In some embodiments, the methods for neutralizing SARS-CoV-2 include contacting SARS-CoV-2 with an antibody or antigen-binding fragment thereof provided herein, or a composition or combination provided herein. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in a subject.

[0065] Also provided herein are methods of pre-exposure prophylaxis of SARS-CoV-2. In some embodiments, a method for providing pre-exposure prophylaxis of SARS-CoV-2 in a subject comprises administering to the subject an effective amount of a composition or combination provided herein. In some embodiments, the subject is not currently infected with SARS-CoV-2. In some embodiments, the subject is not known to have been recently exposed to an individual infected with SARS-CoV-2. In some embodiments, the subject has moderate to severe immunocompromise. In some embodiments, the moderate to severe immunocompromise is due to a medical condition or receiving an immunosuppressant drug or treatment. In some embodiments, the subject may not mount an adequate immune response to COVID-19 vaccination. In some embodiments, COVID-19 vaccination is not recommended for the subject. In some embodiments, COVID-19 vaccination is not recommended due to a history of severe adverse reactions to COVID-19 vaccines and / or COVID-19 vaccine components. In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are administered sequentially.

[0066] Also provided herein are methods of treating or preventing SARS-CoV-2 infection. In some embodiments, a method of treating or preventing SARS-CoV-2 infection in a subject comprises administering to a subject an effective amount of a combination provided herein, wherein a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof are administered sequentially. In some embodiments, a first antibody or antigen-binding fragment thereof is administered prior to administration of a second antibody or antigen-binding fragment thereof. In some embodiments, a first antibody or antigen-binding fragment thereof is administered after administration of a second antibody or antigen-binding fragment thereof. In some embodiments, a first antibody or antigen-binding fragment thereof is administered intramuscularly and / or a second antibody or antigen-binding fragment thereof is administered intramuscularly.

[0067] In some embodiments, a method for providing pre-exposure prophylaxis of SARS-CoV-2 in a subject comprises administering to the subject an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, a method for treating or preventing SARS-CoV-2 infection in a subject comprises administering to the subject an effective amount of an antibody or antigen-binding fragment thereof provided herein. In some embodiments, the administration is intramuscular.

[0068] In some embodiments of the methods provided herein, the subject has been exposed to or is at risk of being exposed to SARS-CoV-2.

[0069] In some embodiments of the methods provided herein, the subject is a human.

[0070] In some embodiments of the methods provided herein, the SARS-CoV-2 is SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2 beta, SARS-CoV-2 delta, and / or SARS-CoV-2 D614G. In some embodiments of the methods provided herein, the SARS-CoV-2 is SARS-CoV-2 BA.2.12.1.

[0071] Also provided herein are methods for detecting SARS-CoV-2. In some embodiments, the method for detecting SARS-CoV-2 in a sample comprises contacting the sample with an antibody or antigen-binding fragment provided herein, or a composition or combination provided herein. [Brief description of the drawings]

[0072] [Figure 1] Neutralization of SARS-CoV-2 pseudovirus by RQ33, RQ40, RQ41, and RQ43 antibodies (see Example 17). [Diagram 2] Neutralization of SARS-CoV-2 pseudovirus by antibody combinations (see Example 17). [Diagram 3] Figure 1 shows the in vitro neutralizing activity of RQ33, RQ43-GL-H-LO1 and the combination of RQ33+RQ43-GL-H-LO1 against SARS-CoV-2 BA.2.12.1 VOC by focus reduction neutralization test (FRNT). The assay was repeated in two independent replicates, each in duplicate. Nonlinear regression dose-response curves show the mean and standard deviation of both replicates at each dilution. mAb: monoclonal antibody, VOC: variants of concern (see Example 18). [Figure 4]Neutralization curves from an in vitro neutralization assay of SARS-CoV-2 Omicron BA.2.12.1 VOC spiked pseudovirus. The assay was repeated in three independent replicates, each in triplicate. Nonlinear regression dose-response curves show representative replicates with mean and standard deviation at each dilution. mAb: monoclonal antibody, VOC: variants of concern (see Example 18). [Diagram 5] Figure 1 shows sequential binding of silgavimab and RQ43-GL-H-LO1 to the receptor-binding domain of the SARS-CoV-2 spike protein. Biolayer interferometry was performed to assess simultaneous binding of silgavimab, RQ43-GL-H-LO1 and CR3022 (non-competitive control mAb) to the SARS-CoV-2 spike RBD. Binding of mAb1 (black) is followed by an increase in signal upon binding of buffer (no visible signal), CR3022 (grey), silgavimab (shaded in the left graph), or RQ43-GL-H-LO1 (shaded in the right graph). Binding signals were averaged from data points acquired for 10 s at equilibrium for each binding step. mAbs were considered bound if the increase in binding signal was >0.3 nM. Data are representative of two independent experiments. mAb: monoclonal antibody, SARS-CoV-2: severe acute respiratory syndrome coronavirus 2. (See Example 18.) [Figure 6] The silgavimab binding site (top) and the RQ43 binding site (bottom) mapped onto the RBD of SARS-CoV-2 are depicted in a linear fashion. The silgavimab and RQ43 binding sites are discontinuous and are indicated by boxes and underlined letters, whereas residues outside the respective binding sites are not underlined. (See Example 18.) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Provided herein are antibodies (e.g., monoclonal antibodies) and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2, and methods of use thereof.

[0074] 7.1 Terminology The term "antibody" refers to an immunoglobulin molecule that recognizes and specifically binds to a target (e.g., a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid, or a combination of the above) through at least one antigen recognition site in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact polyclonal antibodies, intact monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins containing antibodies, and any other modified immunoglobulin molecule, so long as the antibody exhibits the desired biological activity. Antibodies can be any of the five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, or subclasses (isotypes) thereof (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) (based on the identity of the heavy chain constant domains, designated alpha, delta, epsilon, gamma, and mu, respectively). Different classes of immunoglobulins have different well-known subunit structures and three-dimensional structures. The antibodies may be naked or may be conjugated to other molecules, such as toxins or radioisotopes.

[0075] The term "antibody fragment" refers to a portion of an intact antibody. An "antigen-binding fragment", "antigen-binding domain" or "antigen-binding region" refers to a portion of an intact antibody that binds to an antigen. An antigen-binding fragment may include antigen-determining regions of an intact antibody (e.g., complementarity-determining regions (CDRs)). Examples of antigen-binding fragments of antibodies include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, and single-chain antibodies. Antigen-binding fragments of antibodies may be derived from any animal species, such as rodents (e.g., mice, rats, or hamsters) and humans, or may be artificially generated.

[0076] The terms "anti-spike protein of SARS-CoV-2 antibody," "SARS-CoV-2 spike protein antibody," and "antibody that binds to spike protein of SARS-CoV-2" are used interchangeably herein to refer to an antibody capable of binding to the spike protein of SARS-CoV-2 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent in targeting SARS-CoV-2. The extent of binding of a SARS-CoV-2 spike protein antibody to an unrelated, non-SARS-CoV-2 spike protein may be less than about 10% of the binding of the antibody to the SARS-CoV-2 spike protein, as measured, for example, using ForteBio or Biacore.

[0077] A "monoclonal" antibody or antigen-binding fragment thereof refers to a population of homogeneous antibodies or antigen-binding fragments involved in highly specific recognition and binding to a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies, which typically contain different antibodies directed against different antigenic determinants. The term "monoclonal" antibody or antigen-binding fragment thereof encompasses both intact and full-length monoclonal antibodies, as well as antibody fragments (Fab, Fab', F(ab')2, Fv, etc.), single-chain (scFv) variants, fusion proteins containing antibody portions, and any other modified immunoglobulin molecule that contains an antigen recognition site. Furthermore, a "monoclonal" antibody or antigen-binding fragment thereof refers to antibodies and antigen-binding fragments thereof that are produced in any number of ways, including, but not limited to, by hybridoma, phage selection, recombinant expression, and transgenic animals.

[0078] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. A variable region typically refers to a portion of an antibody, generally a light or heavy chain, typically about the amino-terminal 110-120 or 110-125 amino acids in a mature heavy chain and about 90-115 amino acids in a mature light chain, which differ extensively in sequence between antibodies and are used in the binding and specificity of a particular antibody to its particular antigen. The sequence variability is concentrated in regions called complementarity determining regions (CDRs), while the more highly conserved regions in the variable domain are called framework regions (FRs). Without wishing to be bound by any particular mechanism or theory, it is believed that the CDRs of the light and heavy chains are primarily responsible for the interaction and specificity of the antibody with the antigen. In some embodiments, the variable region is a human variable region. In some embodiments, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some embodiments, the variable region is a primate (e.g., non-human primate) variable region. In some embodiments, the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).

[0079] The term "complementarity determining region" or "CDR" as used herein refers to each of the regions of an antibody variable domain that are hypervariable in sequence and / or form structurally defined loops (hypervariable loops) and / or contain antigen contact residues. An antibody may contain six CDRs, e.g., three in VH and three in VL.

[0080] The terms "VL" and "VL domain" are used interchangeably and refer to the light chain variable region of an antibody.

[0081] The terms "VH" and "VH domain" are used interchangeably and refer to the variable region of an antibody heavy chain.

[0082] The term "Kabat numbering" and similar terms are recognized in the art and refer to a system for numbering amino acid residues in the heavy and light chain variable regions of an antibody or an antigen-binding fragment thereof. In some embodiments, CDRs can be determined according to the Kabat numbering system (see, for example, Kabat EA & Wu TT (1971) Ann NY Acad Sci 190:382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242). Using the Kabat numbering system, the CDRs in an antibody heavy chain molecule are typically located at amino acids 31-35 (which may optionally include one or two additional amino acids following 35, designated 35A and 35B in the Kabat numbering scheme) (CDR1), 50-65 (CDR2), and 95-102 (CDR3). Using the Kabat numbering system, the CDRs in an antibody light chain molecule are typically located at amino acids 24-34 (CDR1), 50-56 (CDR2), and 89-97 (CDR3).

[0083] Chothia, on the other hand, refers to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop when numbered using the Kabat numbering convention vary from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34). The AbM hypervariable regions represent a compromise between the Kabat CDRs and the Chothia structural loops and are used by Oxford Molecular's AbM antibody modeling software.

[0084] [Table 1]

[0085] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meaning in the art. The constant region is the carboxyl-terminal portion of an antibody portion, e.g., the light chain and / or the heavy chain, that is not directly involved in binding the antibody to an antigen, but may exhibit various effector functions, such as interacting with Fc receptors. The constant region of an immunoglobulin molecule generally has a conserved amino acid sequence compared to the immunoglobulin variable domain. In some embodiments, the antibody or antigen-binding fragment comprises a constant region or a portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0086] As used herein, the term "heavy chain", when used in reference to an antibody, can refer to any distinct type, e.g., alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), based on the amino acid sequence of the constant domain, which gives rise to the IgA, IgD, IgE, IgG and IgM classes of antibodies, respectively, including subclasses of IgG, e.g., IgG1, IgG2, IgG3 and IgG4. Heavy chain amino acid sequences are well known in the art. In some embodiments, the heavy chain is a human heavy chain.

[0087] As used herein, the term "light chain" when used in reference to an antibody can refer to any distinct type, e.g., κ (kappa) or λ (lambda), based on the amino acid sequence of the constant domain. Light chain amino acid sequences are well known in the art. In some embodiments, the light chain is a human light chain.

[0088] The term "humanized" antibody or antigen-binding fragment thereof refers to a form of a non-human (e.g., murine) antibody or antigen-binding fragment thereof that is a specific immunoglobulin chain or fragment thereof that contains minimal non-human (e.g., murine) sequence. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues of the complementarity determining region (CDR) are replaced with residues from the CDR of a non-human species (e.g., mouse, rat, rabbit, hamster) having the desired specificity, affinity, and capacity ("CDR-grafted") (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)). In some cases, Fv framework region (FR) residues of a human immunoglobulin are replaced with corresponding residues in an antibody or fragment from a non-human species having the desired specificity, affinity, and capacity. The humanized antibody or antigen-binding fragment thereof can be further modified by substitution of additional residues in the Fv framework regions and / or within the substituted non-human residues to refine and optimize the specificity, affinity, and / or capacity of the antibody or antigen-binding fragment thereof. Generally, a humanized antibody or antigen-binding fragment thereof will contain substantially all of at least one, and typically two or three, variable domains that contain all or substantially all of the CDR regions corresponding to a non-human immunoglobulin, while all or substantially all of the FR regions are of human immunoglobulin consensus sequences. The humanized antibody or antigen-binding fragment thereof may also contain at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. Examples of methods used to generate humanized antibodies are described in U.S. Pat. No. 5,225,539, Roguska et al., Proc. Natl. Acad. Sci., USA, 91(3):969-973 (1994), and Roguska et al., Protein Eng. 9(10):895-904 (1996).In some embodiments, a "humanized antibody" is a resurfaced antibody.

[0089] The terms "human" or "fully human" antibody, or antigen-binding fragment thereof, mean an antibody or antigen-binding fragment thereof having an amino acid sequence derived from the human immunoglobulin locus, and such an antibody or antigen-binding fragment thereof is made using any technique known in the art. This definition of human antibody or antigen-binding fragment thereof includes intact or full-length antibodies and fragments thereof.

[0090] "Binding affinity" generally refers to the overall strength of non-covalent interactions between a single binding site of a molecule (e.g., an antibody or antigen-binding fragment thereof) and its binding partner (e.g., an antigen). Unless otherwise specified, "binding affinity" as used herein refers to the inherent binding affinity exhibiting a 1:1 interaction between members of a binding pair (e.g., an antibody or antigen-binding fragment thereof and an antigen). The affinity of a molecule X for its partner Y is generally expressed as a function of the dissociation constant (K D Affinity can be expressed as the equilibrium dissociation constant (K D ) and the equilibrium binding constant (K A ) can be measured and / or expressed in a number of ways known in the art, including, but not limited to, K D k off / k on While it is calculated from the quotient of A k on / k off It is calculated from the quotient of k on k refers to the binding rate constant of, for example, an antibody or an antigen-binding fragment thereof to an antigen, off refers, for example, to the dissociation of an antibody or antigen-binding fragment thereof from an antigen. on and k off can be determined by techniques known to those skilled in the art, such as BIAcore® or KinExA.

[0091] As used herein, "epitope" is a term of the art and refers to a localized region of an antigen to which an antibody or antigen-binding fragment thereof can specifically bind. An epitope can be, for example, consecutive amino acids of a polypeptide (linear or continuous epitope), or can be, for example, joined together from two or more non-contiguous regions of a polypeptide (conformational, non-linear, discontinuous or non-contiguous epitope). In some embodiments, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined, for example, by NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen / deuterium exchange mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligopeptide scanning assays, and / or mutagenesis mapping (e.g., site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be achieved using any of the methods known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr 50(Pt 4):339-350; McPherson A(1990) Eur J Biochem 189:1-23; Chayen NE(1997) Structure 5:1269-1274; McPherson A(1976) J Biol Chem 251:6300-6303).Crystals of the antibody / antigen-binding fragment thereof:antigen can be examined using well-known X-ray diffraction techniques and computer software such as X-PLOR (Yale University, 1992, distributed by Molecular Simulations, Inc; see, e.g., Meth Enzymol (1985) volumes 114 & 115, eds Wyckoff HW et al.,; see U.S. Patent Application Publication No. 2004 / 0014194), and BUSTER (Bricogne G (1993) Acta Crystallogr D Biol Crystallogr 49(Pt 1):37-60; Bricogne G (1997) Meth Enzymol 276A:361-423, ed Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 56(Pt 10):1316-1323). Mutagenesis mapping studies can be accomplished using any method known to those of skill in the art. For example, see Champe M et al., (1995) J Biol Chem 270:1388-1394 and Cunningham BC & Wells JA (1989) Science 244:1081-1085 for a description of mutagenesis techniques, including alanine scanning mutagenesis techniques.

[0092] An antibody that "binds to the same epitope" as a reference antibody refers to an antibody that binds to the same amino acid residue as the reference antibody. The ability of an antibody to bind to the same epitope as a reference antibody can be determined by hydrogen / deuterium exchange assay (see, for example, Coales et al. Rapid Commun. Mass Spectrom. (2009) 23:639-647).

[0093] As used herein, the terms "immunospecifically bind", "immunospecifically recognize", "specifically bind" and "specifically recognize" are similar terms in the context of an antibody or antigen-binding fragment thereof. These terms indicate that the antibody or antigen-binding fragment thereof binds to an epitope via its antigen-binding domain, and that binding requires some complementarity between the antigen-binding domain and the epitope. Thus, in some embodiments, an antibody that "specifically binds" to the spike protein of SARS-CoV-2 may also bind to the spike protein of one or more related viruses (e.g., SARS-1) and / or may bind to variants of the spike protein of SARS-CoV-2, but the extent of binding to unrelated, non-SARS-CoV-2 spike proteins is less than about 10% of the binding of the antibody to the spike protein of SARS-CoV-, e.g., as measured using ForteBio or Biacore.

[0094] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if it preferentially binds to that epitope or an overlapping epitope to the extent that it blocks, to some extent, binding of the reference antibody to that epitope. Competitive inhibition can be determined by any method known in the art, such as a competitive ELISA assay. An antibody can be said to competitively inhibit binding of the reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0095] An "isolated" polypeptide, antibody, polynucleotide, vector, cell, or composition is a polypeptide, antibody, polynucleotide, vector, cell, or composition in a form not found in nature. Isolated polypeptides, antibodies, polynucleotides, vectors, cells, or compositions include those that have been purified to the extent that they are no longer in a form found in nature. In some embodiments, an isolated antibody, polynucleotide, vector, cell, or composition is substantially pure. As used herein, "substantially pure" refers to a material that is at least 50% pure (i.e., free from contaminants), at least 90% pure, at least 95% pure, at least 98% pure, or at least 99% pure.

[0096] The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. The term also includes amino acid polymers that are modified, naturally or by intervention, for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Also included within the definition are polypeptides that contain, for example, one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Because the polypeptides of the present invention are based on antibodies, it is understood that in some embodiments the polypeptides can occur as single chains or associated chains.

[0097] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity can be determined by aligning two sequences and introducing gaps to maximize the identity between the sequences. Alignment can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed with the blastn program set to default parameters, and alignment of amino acid sequences can be performed with the blastp program set to default parameters (see National Center for Biotechnology Information (NCBI) on the World Wide Web at ncbi.nlm.nih.gov).

[0098] As used herein, amino acids having hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tryptophan (W), and tyrosine (Y). Amino acids having aliphatic hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), and valine (V). Amino acids having aromatic hydrophobic side chains include phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0099] As used herein, amino acids having a polar neutral side chain include asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), and threonine (T).

[0100] As used herein, amino acids having a charged side chain include aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Amino acids having an acidic charged side chain include aspartic acid (D) and glutamic acid (E). Amino acids having a basic charged side chain include arginine (R), histidine (H), and lysine (K).

[0101] As used herein, the term "host cell" may be any type of cell, such as a primary cell, a cultured cell, or a cell from a cell line. In some embodiments, the term "host cell" refers to a cell that has been transfected with a nucleic acid molecule, and the progeny or potential progeny of such a cell. The progeny of such a cell may not be identical to the parent cell transfected with the nucleic acid molecule, for example, due to mutations or environmental influences that may occur in subsequent generations or upon integration of the nucleic acid molecule into the host cell genome.

[0102] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and that does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation will be administered. The formulation may be sterile.

[0103] As provided herein, a "combination" of an antibody or antigen-binding fragment thereof refers to two or more antibodies or antigen-binding fragments thereof used together. The combination of antibodies or antigen-binding fragments thereof can be contained together in a single pharmaceutical composition or in separate pharmaceutical compositions. The combination of antibodies or antigen-binding fragments thereof contained in separate pharmaceutical compositions can be administered simultaneously or sequentially.

[0104] As used herein, the terms "administer," "administering," "administration," and the like refer to methods (e.g., intravenous administration) that can be used to enable delivery of an agent, such as an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein are described, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0105] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be an animal. In some embodiments, a subject is a mammal, e.g., a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some embodiments, a subject is a human.

[0106] The term "therapeutically effective amount" refers to an amount of a drug, such as one or more antibodies or antigen-binding fragments thereof, effective to treat a disease or disorder in a subject.

[0107] Terms such as "treating" or "treatment" or "to treat" or "alleviating" or "to alleviate" refer to therapeutic measures that cure, slow, relieve symptoms, and / or halt the progression of a diagnosed condition or disorder. Thus, those in need of treatment include those already diagnosed with a disorder or those suspected of having a disorder. Patients or subjects in need of treatment may include those diagnosed with coronavirus 2019 (COVID-19) and those infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0108] Alternatively, the pharmacological and / or physiological effect may be prophylactic, i.e., the effect completely or partially prevents a disease or its symptoms. In this regard, the methods of the disclosure include administering a "prophylactically effective amount" of an agent (e.g., one or more antibodies or antigen-binding fragments thereof). A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve a desired prophylactic outcome (e.g., prevention of SARS-CoV-2 infection or disease development).

[0109] As used in this disclosure and claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0110] Whenever an embodiment is described herein with the word "comprising," it is understood that similar embodiments are also provided that are separately described with the terms "consisting of" and / or "consisting essentially of." In this disclosure, "comprises," "comprising," "containing," and "having" can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" is open-ended, allowing for the presence of other things than what is recited, but excluding prior art aspects, so long as the basic or novel characteristics of what is recited are not altered by the presence of other things than what is recited.

[0111] As used herein, unless otherwise specified or clear from the context, the term "or" is understood to be inclusive. When used herein in phrases such as "A and / or B," the term is intended to include both "A and B," "A or B," "A" and "B." Similarly, when used in phrases such as "A, B and / or C," the term is intended to include each of the following aspects: A, B and C; A, B or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0112] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate that a deviation of up to 10% above and below that value or range remains within the intended meaning of the recited value or range. Whenever embodiments are described herein with the phrase "about" or "approximately" in reference to a numerical value or range, it is understood that otherwise similar embodiments that refer to that particular numerical value or range (without "about") are also provided.

[0113] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0114] 7.2 Antibodies and Antigen-Binding Fragments In certain embodiments, provided herein are antibodies (e.g., monoclonal antibodies, such as human antibodies) and antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2. The amino acid sequence of the spike protein of SARS-CoV-2 is provided in SEQ ID NO:1. [ka]

[0115] Amino acids 1-12 of SEQ ID NO:1 are the signal peptide of the spike protein. Thus, the mature version of the spike protein of SARS-CoV-2 includes amino acids 13-1273 of SEQ ID NO:1. Amino acids 13-1213 of SEQ ID NO:1 correspond to the extracellular domain, amino acids 1214-1234 correspond to the transmembrane domain, and amino acids 1235-1273 correspond to the cytoplasmic domain.

[0116] In some aspects, the antibodies or antigen-binding fragments thereof described herein bind to the spike protein of SARS-CoV-2 and specifically bind to the receptor binding domain (RBD) of the spike protein of SARS-CoV-2.

[0117] In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the spike protein of SARS-CoV-2 and contain the six CDRs of the antibodies listed in Table 1 (i.e., the three VH CDRs of the antibodies and the three VL CDRs of the antibodies).

[0118] [Table 2]

[0119] [Table 3]

[0120] [Table 4]

[0121] [Table 5]

[0122] [Table 6]

[0123] [Table 7]

[0124] [Table 8]

[0125] [Table 9]

[0126] [Table 10]

[0127] In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the spike protein of SARS-CoV-2 and comprise the VH of an antibody described in Table 1. In some embodiments, the antibodies or antigen-binding fragments thereof described herein bind to the spike protein of SARS-CoV-2 and comprise the VL of an antibody described in Table 1.

[0128] In some aspects, the antibodies or antigen-binding fragments thereof described herein bind to the spike protein of SARS-CoV-2 and comprise the VH and VL of an antibody listed in 1 (i.e., the VH of an antibody and the VL of the same antibody).

[0129] In some embodiments, the antibody or antigen-binding fragment thereof described herein may be described by its VL domain only, or by its VH domain only, or by its three VL CDRs only, or by its three VH CDRs only. See, for example, Rader C et al., (1998) PNAS 95:8910-8915, incorporated herein by reference in its entirety, which describes the humanization of a murine anti-αvβ3 antibody by identifying a complementary light or heavy chain from a human light or heavy chain library, respectively, resulting in a humanized antibody variant with high or higher affinity than that of the original antibody. See also Clackson T et al., (1991) Nature 352:624-628, incorporated herein by reference in its entirety, which describes a method of generating an antibody that binds to a specific antigen by using a specific VL domain (or VH domain) to screen a library for the presence or absence of a complementary variable domain. Screening resulted in 14 new partners for a particular VH domain and 13 new partners for a particular VL domain that were strong binders as determined by ELISA. See also Kim SJ & Hong HJ, (2007) J Microbiol 45:572-577, incorporated herein by reference in its entirety, which describes a method for generating antibodies that bind to a specific antigen by using a specific VH domain to screen a library (e.g., a human VL library) for the presence or absence of complementary VL domains, where the selected VL domains can then be used to guide the selection of additional complementary (e.g., human) VH domains.

[0130] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the Chothia numbering scheme, which refers to the location of the immunoglobulin structural loops (see, e.g., Chothia C & Lesk AM, (1987), J Mol Biol 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol 273:927-948; Chothia C et al., (1992) J Mol Biol 227:799-817; Tramontano A et al., (1990) J Mol Biol 215(1):175-82; and U.S. Pat. No. 7,709,226). Typically, using Kabat numbering, a Chothia CDR-H1 loop is located at heavy chain amino acids 26-32, 33, or 34, a Chothia CDR-H2 loop is located at heavy chain amino acids 52-56, and a Chothia CDR-H3 loop is located at heavy chain amino acids 95-102, while a Chothia CDR-L1 loop is located at light chain amino acids 24-34, a Chothia CDR-L2 loop is located at light chain amino acids 50-56, and a Chothia CDR-L3 loop is located at light chain amino acids 89-97. The ends of the Chothia CDR-H1 loop, when numbered using the Kabat numbering convention, vary from H32 to H34 depending on the length of the loop (this is because the Kabat numbering scheme places insertions at H35A and H35B; if neither 35A nor 35B are present, the loop ends at 32, if only 35A is present, the loop ends at 33, and if both 35A and 35B are present, the loop ends at 34).

[0131] In some embodiments, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and comprise the Chothia VH and VL CDRs of the antibodies listed in Table 1. In some embodiments, the antibodies or antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 comprise one or more CDRs, wherein the Chothia and Kabat CDRs have the same amino acid sequence. In some embodiments, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and comprise a combination of the Kabat and Chothia CDRs.

[0132] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the IMGT numbering system as described in Lefranc MP, (1999) The Immunologist 7:132-136 and Lefranc MP et al., (1999) Nucleic Acids Res 27:209-212. According to the IMGT numbering scheme, VH-CDR1 is at positions 26-35, VH-CDR2 is at positions 51-57, VH-CDR3 is at positions 93-102, VL-CDR1 is at positions 27-32, VL-CDR2 is at positions 50-52, and VL-CDR3 is at positions 89-97. In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and contain the IMGT VH and VL CDRs of the antibodies listed in Table 1, e.g., as described in Lefranc MP (1999) supra and Lefranc MP et al., (1999) supra.

[0133] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol 262:732-745. See also, e.g., Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering, Kontermann and Duebel, eds., Chapter 31, pp.422-439, Springer-Verlag, Berlin (2001). In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and comprise the VH and VL CDRs of an antibody listed in Table 1, as determined by the method in MacCallum RM et al.

[0134] In some embodiments, the CDRs of an antibody or antigen-binding fragment thereof refer to the AbM hypervariable regions, which are taken between the Kabat CDRs and the Chothia structural loops, and can be determined according to the AbM numbering scheme used in Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In some embodiments, provided herein are antibodies or antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and comprise the VH and VL CDRs of an antibody listed in Table 1, as determined by the AbM numbering scheme.

[0135] In some aspects, provided herein is an antibody comprising a heavy chain and / or a light chain. Non-limiting examples of human constant region sequences are described in the art, see, e.g., U.S. Patent No. 5,693,780 and Kabat EA et al. (1991), supra.

[0136] With respect to the heavy chain, in some embodiments, the heavy chain of the antibodies described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the heavy chain of the antibodies described herein can comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments, the antibodies described herein that immunospecifically bind to the spike protein of SARS-CoV-2 comprise a heavy chain, wherein the amino acid sequence of the VH domain comprises an amino acid sequence set forth in Table 1, and the constant region of the heavy chain comprises an amino acid sequence of a human gamma (γ) heavy chain constant region (e.g., a human IgG1 heavy chain constant region). In some embodiments, the antibodies described herein that specifically bind to the spike protein of SARS-CoV-2 comprise a heavy chain, wherein the amino acid sequence of the VH domain comprises a sequence set forth in Table 1, and the constant region of the heavy chain comprises an amino acid sequence of a human heavy chain described herein or known in the art.

[0137] As is well known in the art, the C-terminal lysine of the heavy chain can be removed, for example, in cell culture. Meanwhile, the C-terminal lysine is conserved in the heavy chain genes of all subclasses of human IgG (i.e., IgG1, IgG2, IgG3, IgG4), but this residue is generally absent in serum IgG. It is also known that processing and cleavage of C-terminal lysine from antibodies is one of the most common causes of product charge heterogeneity in cell culture. The lysine residue at the C-terminus of the heavy chain of recombinant IgG can be removed during cell culture by carboxypeptidases endogenous to mammalian host cells. Thus, in some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain having a C-terminal lysine. In some embodiments, the antibody or antigen-binding fragment provided herein comprises a heavy chain without a C-terminal lysine.

[0138] In some embodiments, the light chain of the antibodies or antigen-binding fragments thereof described herein is a human kappa light chain or a human lambda light chain. In some embodiments, the antibodies described herein that immunospecifically bind to the spike protein of SARS-CoV-2 comprise a light chain, wherein the amino acid sequence of the VL domain comprises a sequence set forth in Table 1, and the constant region of the light chain comprises the amino acid sequence of a human kappa or lambda light chain constant region.

[0139] In some embodiments, an antibody or antigen-binding fragment thereof described herein that immunospecifically binds to the spike protein of SARS-CoV-2 comprises a light chain, wherein the amino acid sequence of the VL domain comprises a sequence set forth in Table 1, and the constant region of the light chain comprises the amino acid sequence of the human kappa light chain constant region. The amino acid sequence of the human kappa light chain constant region is: It may be composed of the amino acid sequence RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 64).

[0140] In some embodiments, the light chain of the antibody described herein is a lambda light chain. In some embodiments, the antibody described herein that immunospecifically binds to the spike protein of SARS-CoV-2 comprises a light chain, the amino acid sequence of the VL domain comprises a sequence set forth in Table 1, and the constant region of the light chain comprises the amino acid sequence of the human lambda light chain constant region. The amino acid sequence of the human kappa light chain constant region is It may be composed of the amino acid sequence of GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 65).

[0141] In some embodiments, the antibodies described herein that immunospecifically bind to the spike protein of SARS-CoV-2 comprise a VH domain and a VL domain comprising any amino acid sequence described herein, and the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, or the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some embodiments, the antibodies described herein that immunospecifically bind to the spike protein of SARS-CoV-2 comprise a VH domain and a VL domain comprising any amino acid sequence described herein, and the constant region comprises the amino acid sequence of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b) constant region. In some embodiments, the constant region comprises the amino acid sequence of the constant region of a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecule (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b).

[0142] Genetic engineering of the Fc region has been used in the art, for example, to extend the half-life of therapeutic antibodies and antigen-binding fragments thereof and to prevent in vivo degradation. In some embodiments, the Fc region of an IgG antibody or antigen-binding fragment can be modified to increase the affinity of the IgG molecule to the fetal Fc receptor (FcRn), which mediates catabolism of IgG and prevents degradation of the IgG molecule. Suitable Fc region amino acid substitutions or modifications are known in the art, including, for example, the triple substitution M252Y / S254T / T256E numbered according to the EU index of Kabat (referred to as "YTE") (see, for example, U.S. Pat. No. 7,658,921; U.S. Patent Application Publication No. 2014 / 0302058; and Yu et al., Antimicrob. Agents Chemother., 61(1):e01020-16 (2017)). In some embodiments, an antibody or antigen-binding fragment (e.g., a monoclonal antibody or fragment) that binds to the spike protein of SARS-CoV-2 comprises an Fc region that includes a YTE mutation.

[0143] The triple mutation (TM) L234F / L235E / P331S (following the European Union numbering convention; Sazinsky et al. Proc Natl Acad Sci USA, 105:20167-20172 (2008)) in the heavy chain constant region can significantly reduce IgG effector function.

[0144] In some aspects, the IgG1 CH1-CH3 sequence comprising the YTE and TM mutations consists of the following amino acid sequence: [ka]

[0145] In some embodiments, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1), and / or the CH3 domain (residues 341-447 of human IgG1), and / or the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) of an antibody or antigen-binding fragment thereof described herein to alter one or more functional properties of the antibody or antigen-binding fragment thereof, such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity.

[0146] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) may be introduced into the hinge region of the Fc region (CH1 domain) to alter (e.g., increase or decrease) the number of cysteine ​​residues in the hinge region, e.g., as described in U.S. Patent No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, for example, to facilitate association of the light and heavy chains or to alter (e.g., increase or decrease) the stability of the antibody or antigen-binding fragment thereof.

[0147] In some embodiments, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., the CH2 domain (residues 231-340 of human IgG1), and / or the CH3 domain (residues 341-447 of human IgG1), and / or the hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) of an antibody or antigen-binding fragment thereof described herein to increase or decrease the affinity of the antibody or antigen-binding fragment thereof for an Fc receptor (e.g., an activating Fc receptor) on the surface of an effector cell. Mutations in Fc regions that reduce or increase affinity for Fc receptors, and techniques for introducing such mutations into Fc receptors or fragments thereof, are known to those of skill in the art. Examples of Fc receptor mutations that can alter the affinity of an antibody or antigen-binding fragment thereof for an Fc receptor are described, for example, in Smith P et al., (2012) PNAS 109:6181-6186, U.S. Pat. No. 6,737,056, and WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated by reference in their entireties.

[0148] In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to change (e.g., shorten or extend) the half-life of the antibody or antigen-binding fragment thereof in vivo. For examples of mutations that change (e.g., shorten or extend) the half-life of the antibody or antigen-binding fragment thereof in vivo, see, for example, WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631; and U.S. Patent Nos. 5,869,046, 6,121,022, 6,277,375 and 6,165,745. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to shorten the half-life of the antibody or antigen-binding fragment thereof in vivo. In some embodiments, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into the IgG constant domain or FcRn-binding fragment thereof (preferably, Fc or hinge-Fc domain fragment) to increase the half-life of the antibody or antigen-binding fragment thereof in vivo. In some embodiments, the antibody or antigen-binding fragment thereof may have one or more amino acid mutations (e.g., substitutions) in the second constant (CH2) domain (residues 231-340 of human IgG1) and / or the third constant (CH3) domain (residues 341-447 of human IgG1), numbered according to the EU index in Kabat (Kabat EA et al., (1991) supra). In some embodiments, the IgG1 constant region comprises a methionine (M) to tyrosine (Y) substitution at position 252, a serine (S) to threonine (T) substitution at position 254, and a threonine (T) to glutamic acid (E) substitution at position 256, numbered according to the EU index as in Kabat. See U.S. Patent No. 7,658,921, incorporated herein by reference.This type of mutant IgG, termed "YTE mutants," has been shown to exhibit a four-fold increase in half-life compared to the wild-type version of the antibody (see Dall'Acqua WF et al., (2006) J. Biol. Chem. 281:23514-24). In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgG constant domain comprising one, two, three or more amino acid substitutions of amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436, numbered according to the EU index in Kabat.

[0149] In some embodiments, one, two or more amino acid substitutions are introduced into the Fc region of the IgG constant domain to alter the effector function of the antibody or antigen-binding fragment thereof. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322, numbered according to the EU index as in Kabat, can be replaced with different amino acid residues such that the affinity of the antibody or antigen-binding fragment thereof to an effector ligand is altered but the antigen-binding ability of the parent antibody is retained. The effector ligand to which the affinity is altered can be, for example, an Fc receptor or the C1 component of complement. This approach is described in more detail in U.S. Pat. Nos. 5,624,821 and 5,648,260. In some embodiments, the constant region domain may be deleted or inactivated (by point mutation or other means) to reduce Fc receptor binding of circulating antibodies or antigen-binding fragments thereof, thereby increasing tumor localization. For example, see U.S. Patent Nos. 5,585,097 and 8,591,886 for a description of mutations that increase tumor localization by deleting or inactivating constant domains. In some embodiments, one or more amino acid substitutions can be introduced in the Fc region to remove potential glycosylation sites on the Fc region, which may reduce Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276:6591-604).

[0150] In some embodiments, one or more amino acids selected from amino acid residues 322, 329, and 331 of the constant region, numbered according to the EU index as in Kabat, may be replaced with a different amino acid residue such that the antibody or antigen-binding fragment thereof has altered C1q binding and / or reduced or abolished complement dependent cytotoxicity (CDC). This approach is described in further detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some embodiments, one or more amino acid residues within amino acids 231-238 of the N-terminal region of the CH2 domain are altered, thereby altering the ability of the antibody to bind complement. This approach is further described in WO 94 / 29351. In some embodiments, the ability of an antibody or antigen-binding fragment thereof to mediate antibody-dependent cellular cytotoxicity (ADCC) is improved and / or a nucleotide sequence is provided that is selected from the following positions numbered according to the EU index as in Kabat: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 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, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 358, 365, 367, 368, 369, 370, 372, 376 The Fc region has been engineered to increase the affinity of the antibody or antigen-binding fragment thereof for an Fcγ receptor by mutating (e.g., introducing amino acid substitutions) one or more amino acids at positions 9, 312, 315, 320, 322, 324, 326, 327, 328, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, ​​388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438, or 439. This approach is further described in WO 00 / 42072.

[0151] In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an IgG1 constant domain with a mutation (e.g., substitution) at position 267, position 328, or a combination thereof, numbered according to the EU index as in Kabat. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an IgG1 constant domain with a mutation (e.g., substitution) selected from the group consisting of S267E, L328F, and combinations thereof. In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an IgG1 constant domain with S267E / L328F mutations (e.g., substitutions). In some embodiments, the antibodies or antigen-binding fragments thereof described herein comprising an IgG1 constant domain with S267E / L328F mutations (e.g., substitutions) have increased binding affinity to FcγRIIA, FcγRIIB, or FcγRIIA and FcγRIIB.

[0152] Engineered glycoforms may be useful for a variety of purposes, including but not limited to, enhancing or decreasing effector function. Methods for generating engineered glycoforms of the antibodies or antigen-binding fragments thereof described herein include, for example, those described in Umana P et al., (1999) Nat Biotechnol 17:176-180; Davies J et al., (2001) Biotechnol Bioeng 74:288-294; Shields RL et al., (2002) J Biol Chem 277:26733-26740; Shinkawa T et al., (2003) J Biol Chem 278:3466-3473; Niwa R et al., (2004) Clin Cancer Res 1:6248-6255; Presta LG et al., (2002) Biochem Soc Trans 30:487-490; Kanda Y et al., (2007) Glycobiology 17:104-118; U.S. Patent Nos. 6,602,684; 6,946,292; and 7,214,775; U.S. Patent Application Publication Nos. 2007 / 0248600; 2007 / 0178551; 2008 / 0060092; and 2006 / 0253928; WO 00 / 61739; WO 01 / 292246; WO 02 / 311140; and WO 02 / 30954; Potillegent™ technology (Biowa, Inc. Princeton, NJ); and GlycoMAb® glycosylation engineering technology (Glycart biotechnology AG, Zurich, Switzerland.See, e.g., Ferrara C et al., (2006) Biotechnol Bioeng 93:851-861; WO 07 / 039818; WO 12 / 130831; WO 99 / 054342; WO 03 / 011878; and WO 04 / 065540.

[0153] In some embodiments, any of the constant region mutations or modifications described herein may be introduced into one or both heavy chain constant regions of an antibody or antigen-binding fragment thereof described herein that has two heavy chain constant regions.

[0154] In some embodiments, the antibodies or antigen-binding fragments thereof described herein that specifically bind to the spike protein of SARS-CoV-2 neutralize SARS-CoV-2. In some embodiments, the antibodies or antigen-binding fragments thereof described herein that specifically bind to the spike protein of SARS-CoV-2 neutralize SARS-CoV-2 pseudoviruses. In some embodiments, the antibodies or antigen-binding fragments thereof neutralize SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2 delta, and / or SARS-CoV-2 D614G pseudoviruses with an EC50 of 100 ng / mL or less (e.g., an EC50 of about 1 ng / mL to about 100 ng / mL, or about 2.5 ng / mL to about 100 ng / mL).

[0155] Competitive binding assays can be used to determine whether two antibodies bind to overlapping epitopes, where an immunoglobulin inhibits specific binding of a reference antibody to a common antigen, such as SARS-CoV-2 or the spike protein of SARS-CoV-2, under test. There are many types of competitive binding assays, such as the Octet competitive binding assay, solid-phase direct or indirect radioimmunoassays (RIA), solid-phase direct or indirect enzyme immunoassays (EIA), sandwich competitive assays (see Stahli C et al., (1983) Methods Enzymol 9:242-253); solid-phase direct biotin-avidin EIA (see Kirkland TN et al., (1986) J Immunol 137:3614-9); solid-phase direct label assays, solid-phase direct label sandwich assays (see Harlow E & Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid-phase direct label RIA using I-125 label (see Morel GA et al., (1988) Mol Immunol 25(1):7-15); solid-phase direct biotin-avidin EIA (Cheung RC et al., (1988) Mol Immunol 25(1):7-15); al., (1990) Virology 176:546-52); and direct labeling RIA (Moldenhauer G et al., (1990) Scand J Immunol 32:77-82). Typically such assays involve the use of purified antigen bound to a solid surface or cells bearing either an unlabeled test immunoglobulin and a labeled reference immunoglobulin. Competitive inhibition can be measured by determining the amount of label bound to the solid surface or cells in the presence of the test immunoglobulin. Usually the test immunoglobulin is present in excess. Usually the competing antibody, when present in excess, will inhibit specific binding of the reference antibody to the common antigen by at least 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, or more. Competitive binding assays can be configured in a number of different formats, using either labeled antigen or labeled antibody. In a common version of this assay, the antigen is immobilized on a 96-well plate.The ability of unlabeled antibodies to block the binding of labeled antibodies to antigens is then measured using radioactive or enzymatic labels. For further details, see, e.g., Wagener C et al., (1983) J Immunol 130:2308-2315; Wagener C et al., (1984) J Immunol Methods 68:269-274; Kuroki M et al., (1990) Cancer Res 50:4872-4879; Kuroki M et al., (1992) Immunol Invest 21:523-538; Kuroki M et al., (1992) Hybridoma 11:391-407, and Antibodies: A Laboratory Manual, Ed Harlow E & Lane D editors supra, pp.386-389. An example of a competitive binding assay is shown in Example 15 herein.

[0156] In some embodiments, an antibody or antigen-binding fragment thereof that competitively inhibits binding of another antibody or antigen-binding fragment thereof competitively inhibits binding in an Octet competitive binding assay, such as the Octet competitive binding assay provided in Example 15 herein.

[0157] In some embodiments, the competitive assay is performed using surface plasmon resonance (BIAcore®) by an "in-tandem approach" such as that described by Abdiche YN et al., (2009) Analytical Biochem 386:172-180, whereby the antigen is immobilized on a chip surface, e.g., a CM5 sensor chip, and then the antibody or antigen-binding fragment is flowed over the chip. To determine whether an antibody or antigen-binding fragment thereof competes with an antibody binding to the spike protein of SARS-CoV-2 described herein, the antibody or antigen-binding fragment is first flowed over the chip surface to achieve saturation, and then a potential competing antibody is added. Binding of the competing antibody or antigen-binding fragment thereof can then be determined and quantified relative to a non-competitive control.

[0158] In another aspect, provided herein are antibodies that competitively (e.g., dose-dependently) inhibit the binding of a described antibody or antigen-binding fragment thereof to the spike protein of SARS-CoV-2 or to SARS-CoV-2 as determined using an assay known to one of skill in the art or described herein (e.g., an ELISA competition assay, or a suspension array or surface plasmon resonance assay).

[0159] In some embodiments, an antigen-binding fragment as described herein that specifically binds to the spike protein of SARS-CoV-2 is a Fab, Fab', F(ab') 2 , and scFv; Fab, Fab', F(ab') 2 Fab, Fab', F(ab'), or scFv comprises the heavy chain variable region sequence and the light chain variable region sequence of an antibody or antigen-binding fragment thereof described herein that specifically binds to the spike protein of SARS-CoV-2 or SARS-CoV-2. 2 , or scFv can be produced by any technique known to one of skill in the art, including, but not limited to, those discussed herein. In some embodiments, Fab, Fab', F(ab') 2 The Fab, Fab', F(ab') or scFv further comprises a moiety that extends the half-life of the antibody in vivo. This moiety is also referred to as a "half-life extending moiety." 2or any moiety known to one of skill in the art to extend the half-life of scFvs may be used. For example, half-life extending moieties may include Fc regions, polymers, albumin, or albumin binding proteins or compounds. Polymers may include natural or synthetic, optionally substituted linear or branched polyalkylenes, polyalkenylenes, polyoxyalkylenes, polysaccharides, polyethylene glycols, polypropylene glycols, polyvinyl alcohols, methoxypolyethylene glycols, lactose, amylose, dextran, glycogen, or derivatives thereof. Substituents may include one or more hydroxy, methyl, or methoxy groups. In some embodiments, Fab, Fab', F(ab') 2 In some embodiments, the Fab, Fab', F(ab') or scFv can be modified by adding one or more C-terminal amino acids to attach a half-life extending moiety. In some embodiments, the half-life extending moiety is polyethylene glycol or human serum albumin. In some embodiments, the Fab, Fab', F(ab') 2 or the scFv is fused to an Fc region.

[0160] An antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 can be fused or conjugated (e.g., covalently or non-covalently linked) to a detectable label or substance. Examples of detectable labels or substances include enzyme labels, such as glucose oxidase; iodine ( 125 I, 121 I), Carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 Labels that can be used to detect SARS-CoV-2 spike protein or SARS-CoV-2 include radioisotopes such as 3Tc; luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine, and biotin. Such labeled antibodies or antigen-binding fragments thereof can be used to detect the spike protein of SARS-CoV-2 or SARS-CoV-2. See, e.g., Section 7.6.2 below.

[0161] 7.3 Combinations of antibodies and antigen-binding fragments thereof The methods provided herein use a combination of antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, such as a combination of a first antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 and a second antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2. In some embodiments, the first and second antibodies or antigen-binding fragments in the combination are a single composition. In some embodiments, the first and second antibodies or antigen-binding fragments in the combination are each separate compositions. For combinations in which the first and second antibodies, or antigen-binding fragments thereof, are separate compositions, the first and second antibodies, or antigen-binding fragments thereof, can be administered simultaneously or sequentially.

[0162] Also provided herein are compositions, e.g., pharmaceutical compositions, comprising antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, e.g., a first antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 and a second antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2. Such compositions include both the first antibody or antigen-binding fragment and the second antibody or antigen-binding fragment.

[0163] In some embodiments, the combinations or compositions provided herein comprise a first antibody or antigen-binding fragment that binds to the spike protein of SARS-CoV-2 and a second antibody or antigen-binding fragment that binds to the spike protein of SARS-CoV-2. In some embodiments, the methods provided herein use a combination or composition of antibodies and antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, such as a combination or composition comprising a first antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 and a second antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2.

[0164] In some embodiments of the combinations, compositions, or methods provided herein, the first and second antibodies, or antigen-binding fragments thereof, bind to non-overlapping epitopes of the spike protein of SARS-CoV-2. In some embodiments of the combinations, compositions, or methods provided herein, the first and second antibodies, or antigen-binding fragments thereof, can simultaneously bind to the RBD of the spike protein of SARS-CoV-2.

[0165] In some embodiments of the combinations, compositions or methods provided herein, the first antibody or antigen-binding fragment thereof comprises the six CDR sequences of an antibody provided in Table 1, and / or the second antibody or antigen-binding fragment thereof comprises the six CDR sequences of an antibody provided in Table 1.

[0166] The first antibody or antigen-binding fragment thereof can include the six CDRs of the RQ43-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof can include the six CDRs of the silgavimab antibody.

[0167] The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ29 or RQ29-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof can include the six CDRs of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the six CDRs of the RQ43 antibody, the RQ43-GL-H antibody, the RQ43-GL-H-LO1 antibody, the RQ43-GL-L antibody, the RQ43-GL-LH antibody, or the RQ43-GL-LH-LO1 antibody.

[0168] The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ33 antibody, the RQ33-GL-H antibody, the RQ33-GL-H-LO1 antibody, or the RQ33-GL-H-LO1 antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ43 antibody, the RQ43-GL-H antibody, the RQ43-GL-H-LO1 antibody, the RQ43-GL-L antibody, the RQ43-GL-LH antibody, or the RQ43-GL-LH-LO1 antibody.

[0169] The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ40 antibody or the RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof can include the six CDRs of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof can include the six CDRs of the RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH, or RQ43-GL-LH-LO1 antibody.

[0170] The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ43 antibody, the RQ43-GL-H antibody, the RQ43-GL-H-LO1 antibody, the RQ43-GL-L antibody, the RQ43-GL-LH antibody, or the RQ43-GL-LH-LO1 antibody.

[0171] The first antibody or antigen-binding fragment thereof can include the six CDRs of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody, and the second antibody or antigen-binding fragment thereof can include the six CDRs of the RQ43 antibody, the RQ43-GL-H antibody, the RQ43-GL-H-LO1 antibody, the RQ43-GL-L antibody, the RQ43-GL-LH antibody, or the RQ43-GL-LH-LO1 antibody.

[0172] In some embodiments of the combinations, compositions or methods provided herein, the first antibody or antigen-binding fragment thereof comprises the VH and / or VL sequence of an antibody provided in Table 1, and / or the second antibody or antigen-binding fragment thereof comprises the VH and / or VL sequence of an antibody provided in Table 1.

[0173] The first antibody or antigen-binding fragment thereof can comprise the VH and / or VL of the RQ43-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof can comprise the VH and / or VL of the silgavimab antibody. In some embodiments, the second antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:76 and a light chain comprising the amino acid sequence of SEQ ID NO:77.

[0174] The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ29 or RQ29-GL-LH antibody. The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody. The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ41 antibody, RQ41-GL-LH antibody, RQ41-GL-LH-LO1 antibody, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof can comprise the VH and VL of the RQ20, RQ20-GL-LH, or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can comprise the VH and VL of the RQ43 antibody, the RQ43-GL-H antibody, the RQ43-GL-H-LO1 antibody, the RQ43-GL-L antibody, the RQ43-GL-LH antibody, or the RQ43-GL-LH-LO1 antibody.

[0175] The first antibody or antigen-binding fragment thereof may comprise the VH and VL of the RQ29 antibody or the RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the VH and VL of the RQ33 antibody, the RQ33-GL-H antibody, the RQ33-GL-H-LO1 antibody, or the RQ33-GL-H-LO1 antibody. The first antibody or antigen-binding fragment thereof may comprise the VH and VL of the RQ29 antibody or the RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the VH and VL of the RQ40 or the RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ29 antibody or the RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ43 antibody, the RQ43-GL-H antibody, the RQ43-GL-H-LO1 antibody, the RQ43-GL-L antibody, the RQ43-GL-LH antibody, or the RQ43-GL-LH-LO1 antibody.

[0176] The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof can comprise the VH and VL of the RQ33, RQ33-GL-H, RQ33-GL-H-LO1, or RQ33-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof can comprise the VH and VL of the RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH, or RQ43-GL-LH-LO1 antibody.

[0177] The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof can include the VH and VL of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen-binding fragment thereof can include the VH and VL of the RQ43 antibody, the RQ43-GL-H antibody, the RQ43-GL-H-LO1 antibody, the RQ43-GL-L antibody, the RQ43-GL-LH antibody, or the RQ43-GL-LH-LO1 antibody.

[0178] The first antibody or antigen-binding fragment thereof can comprise the VH and VL of the RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1, or RQ41-LO1 antibody, and the second antibody or antigen-binding fragment thereof can comprise the VH and VL of the RQ43 antibody, RQ43-GL-H antibody, RQ43-GL-H-LO1 antibody, RQ43-GL-L antibody, RQ43-GL-LH antibody, or RQ43-GL-LH-LO1 antibody.

[0179] In some embodiments of the combinations, compositions, or methods provided herein, a first antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:61, which competitively inhibits binding to the spike protein of SARS-CoV-2, and a second antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:68 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:72, which competitively inhibits binding to the spike protein of SARS-CoV-2. In some embodiments, the first antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:63 and a VL comprising the amino acid sequence of SEQ ID NO:61. In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:68 and a VL comprising the amino acid sequence of SEQ ID NO:72. In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:76 and a light chain comprising the amino acid sequence of SEQ ID NO:77.

[0180] In some embodiments of the combinations, compositions, or methods provided herein, a first antibody or antigen-binding fragment thereof binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:61, and a second antibody or antigen-binding fragment thereof, which binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO:68 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO:72.

[0181] In some embodiments of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively, and the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 69, 70, 71, 73, 74, and 75, respectively.

[0182] In some embodiments of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61, and the second antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 68 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 72. In some embodiments, the second antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76 and a light chain comprising the amino acid sequence of SEQ ID NO: 77.

[0183] In some embodiments of the combinations, compositions, or methods provided herein, a first antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, which competitively inhibits binding to the spike protein of SARS-CoV-2, and a second antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61, which competitively inhibits binding to the spike protein of SARS-CoV-2. In some embodiments, the first antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 47, a VH comprising the amino acid sequence of SEQ ID NO: 48 and a VL comprising the amino acid sequence of SEQ ID NO: 47, or a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:63 and a VL comprising the amino acid sequence of SEQ ID NO:61, a VH comprising the amino acid sequence of SEQ ID NO:60 and a VL comprising the amino acid sequence of SEQ ID NO:61, a VH comprising the amino acid sequence of SEQ ID NO:62 and a VL comprising the amino acid sequence of SEQ ID NO:61, a VH comprising the amino acid sequence of SEQ ID NO:60 and a VL comprising the amino acid sequence of SEQ ID NO:64, a VH comprising the amino acid sequence of SEQ ID NO:62 and a VL comprising the amino acid sequence of SEQ ID NO:64, or a VH comprising the amino acid sequence of SEQ ID NO:63 and a VL comprising the amino acid sequence of SEQ ID NO:64.

[0184] In some embodiments of the combinations, compositions, or methods provided herein, a first antibody or antigen-binding fragment thereof binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, and a second antibody or antigen-binding fragment thereof, which binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61.

[0185] In some embodiments of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively, and the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of SEQ ID NOs: 31, 32, 37, 34, 35 and 36, respectively.

[0186] In some embodiments of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, and the second antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61.

[0187] In some embodiments of the combinations, compositions, or methods provided herein, a first antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, which competitively inhibits binding to the spike protein of SARS-CoV-2, and a second antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53, which competitively inhibits binding to the spike protein of SARS-CoV-2. In some embodiments, the first antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO: 46 and a VL comprising the amino acid sequence of SEQ ID NO: 47, a VH comprising the amino acid sequence of SEQ ID NO: 48 and a VL comprising the amino acid sequence of SEQ ID NO: 47, or a VH comprising the amino acid sequence of SEQ ID NO: 49 and a VL comprising the amino acid sequence of SEQ ID NO: 47. In some embodiments, the second antibody or antigen-binding fragment thereof comprises a VH comprising the amino acid sequence of SEQ ID NO:52 and a VL comprising the amino acid sequence of SEQ ID NO:53, or a VH comprising the amino acid sequence of SEQ ID NO:50 and a VL comprising the amino acid sequence of SEQ ID NO:51.

[0188] In some embodiments of the combinations, compositions, or methods provided herein, a first antibody or antigen-binding fragment thereof binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, and a second antibody or antigen-binding fragment thereof, which binds to the same epitope of the spike protein of SARS-CoV-2 as an antibody comprising a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53.

[0189] In some embodiments of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively, and the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs: 20, 21, 22, 23, 67, and 24, respectively.

[0190] In some embodiments of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 47, and the second antibody or antigen-binding fragment thereof comprises a variable heavy chain (VH) comprising the amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53.

[0191] In some embodiments of the combinations, compositions or methods provided herein, the combination or composition neutralizes SARS-CoV-2 pseudoviruses. In some embodiments, the combination or composition neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2 delta, and / or SARS-CoV-2 D614G pseudoviruses with an EC50 of 75 ng / mL or less (e.g., an EC50 of about 1 ng / mL to about 75 ng / mL, or about 2.5 ng / mL to about 75 ng / mL). In some embodiments, the combination or composition neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, and / or SARS-CoV-2 BA.2 pseudoviruses with an EC50 of 25 ng / mL or less (e.g., an EC50 of about 1 ng / mL to about 25 ng / mL, or about 2.5 ng / mL to about 25 ng / mL). In some embodiments, the combination or composition neutralizes SARS-CoV-2 delta and / or SARS-CoV-2 D614G pseudoviruses with an EC50 of 75 ng / mL or less (e.g., an EC50 of about 1 ng / mL to about 75 ng / mL, about 2.5 ng / mL to about 75 ng / mL, about 10 ng / mL to about 75 ng / mL, or about 20 ng / mL to about 75 ng / mL). In some embodiments, the combination or composition (i) neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, and / or SARS-CoV-2 BA.2 pseudoviruses with an EC50 of 25 ng / mL or less (e.g., an EC50 of about 1 ng / mL to about 25 ng / mL, or about 2.5 ng / mL to about 25 ng / mL); and (ii) neutralizes SARS-CoV-2 delta and / or SARS-CoV-2 D614G pseudoviruses with an EC50 of 75 ng / mL or less (e.g., an EC50 of about 1 ng / mL to about 75 ng / mL, about 2.5 ng / mL to about 75 ng / mL, about 10 ng / mL to about 75 ng / mL, or about 20 ng / mL to about 75 ng / mL).

[0192] In some embodiments of the combinations, compositions or methods provided herein, the combination or composition neutralizes the SARS-CoV-2 virus. In some embodiments, the combination or composition neutralizes SARS-CoV-2 D614G, SARS-CoV-2 alpha, SARS-CoV-2 delta+T51I+T95I, SARS-CoV-2 BA.1, SARS-CoV-2 BA.1.1, SARS-CoV-2 BA.2, SARS-CoV-2 BA.2.12.1, and / or SARS-CoV-2 BA.5 with an IC50 of 75 ng / mL or less (e.g., an IC50 of about 1 ng / mL to about 75 ng / mL, about 2.5 ng / mL to about 75 ng / mL, or about 5 ng / mL to about 75 ng / mL). In some embodiments, the combination or composition neutralizes SARS-CoV-2 D614G, SARS-CoV-2 alpha, SARS-CoV-2 delta+T51I+T95I, SARS-CoV-2 BA.1, SARS-CoV-2 BA.1.1, SARS-CoV-2 BA.2, SARS-CoV-2 BA.2.12.1, and / or SARS-CoV-2 BA.5 with an IC50 of 60 ng / mL or less (e.g., an IC50 of about 1 ng / mL to about 60 ng / mL, about 2.5 ng / mL to about 60 ng / mL, or about 5 ng / mL to about 60 ng / mL); or

[0193] In some embodiments of the combinations, compositions, or methods provided herein, the combination or composition neutralizes the SARS-CoV-2 BA.2.12.1 virus. In some embodiments of the combinations, compositions, or methods provided herein, the combination or composition neutralizes the SARS-CoV-2 BA.2.12.1 virus with an IC50 of 25 ng / mL or less (e.g., an IC50 of about 1 ng / mL to about 25 ng / mL, about 5 ng / mL to about 25 ng / mL, about 10 ng / mL to about 25 ng / mL, or about 15 ng / mL to about 25 ng / mL). In some embodiments of the combinations, compositions, or methods provided herein, the combination or composition neutralizes SARS-CoV-2 BA.2.12.1 virus with an IC50 of 20 ng / mL or less (e.g., an IC50 of about 1 ng / mL to about 20 ng / mL, about 5 ng / mL to about 20 ng / mL, about 10 ng / mL to about 20 ng / mL, or about 15 ng / mL to about 20 ng / mL).

[0194] In some embodiments of the combinations and methods provided herein, the first and second antibodies or antigen-binding fragments thereof are in the same composition. In some embodiments of the combinations and methods provided herein, the first and second antibodies or antigen-binding fragments thereof are in separate compositions.

[0195] 7.4 Antibody production Antibodies and antigen-binding fragments thereof that immunospecifically bind to the spike protein of SARS-CoV-2 can be produced by any method known in the art for synthesizing antibodies and antigen-binding fragments thereof, for example, by chemical synthesis or by recombinant expression techniques. The methods described herein employ, unless otherwise indicated, conventional techniques in molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields that are within the skill of the art. These techniques are described, for example, in the references cited herein and are described in detail in the literature. See, e.g., Sambrook J et al., (2001) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY; Ausubel FM et al., Current Protocols in Molecular Biology, John Wiley & Sons (1987 and annually revised editions); Current Protocols in Immunology, John Wiley & Sons (1987 and annually revised editions) Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren B et al., (eds.) (1999) Genome Analysis: A Laboratory Manual, Cold Spring Harbor Laboratory Press.

[0196] In some embodiments, provided herein are methods of producing an antibody or antigen-binding fragment thereof that immunospecifically binds to a spike protein of SARS-CoV-2, comprising culturing a cell or host cell described herein. In some embodiments, provided herein are methods of producing an antibody or antigen-binding fragment thereof that immunospecifically binds to a spike protein of SARS-CoV-2, comprising expressing (e.g., recombinantly expressing) the antibody or antigen-binding fragment thereof using a cell or host cell described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody or antigen-binding fragment thereof described herein). In some embodiments, the cell is an isolated cell. In some embodiments, an exogenous polynucleotide has been introduced into the cell. In some embodiments, the method further comprises isolating or purifying the antibody or antigen-binding fragment obtained from the cell, host cell, or culture.

[0197] Methods for generating polyclonal antibodies are known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., eds., John Wiley and Sons, New York).

[0198] Monoclonal antibodies or antigen-binding fragments thereof can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technology, yeast-based display technology, or a combination thereof. For example, monoclonal antibodies or antigen-binding fragments thereof can be produced using hybridoma techniques known in the art, including those taught in, for example, Harlow E & Lane D, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563 681 (Elsevier, NY, 1981), or those described in Kohler G & Milstein C (1975) Nature 256:495. Examples of yeast-based display methods that can be used to select and generate the antibodies described herein include, for example, those disclosed in WO 2009 / 036379 A2, WO 2010 / 105256, and WO 2012 / 009568, each of which is incorporated by reference in its entirety.

[0199] In some embodiments, the monoclonal antibody or antigen-binding fragment is an antibody or antigen-binding fragment produced by a clonal cell (e.g., a hybridoma or host cell that produces a recombinant antibody or antigen-binding fragment), and the antibody or antigen-binding fragment immunospecifically binds to the spike protein of SARS-CoV-2, e.g., as determined by ELISA or other antigen-binding assays known in the art. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can be a human antibody or antigen-binding fragment thereof. In some embodiments, the monoclonal antibody or antigen-binding fragment thereof can be a Fab fragment or a F(ab') 2The monoclonal antibodies or antigen-binding fragments thereof described herein can be produced, for example, by the hybridoma method described in Kohler G & Milstein C (1975) Nature 256:495, or can be isolated from phage libraries, for example, using the techniques described herein. Other methods for the preparation of clonal cell lines and the monoclonal antibodies and antigen-binding fragments thereof expressed thereby are well known in the art (see, for example, Chapter 11 of Short Protocols in Molecular Biology, (2002) 5th Ed., Ausubel FM et al., supra).

[0200] Antigen-binding fragments of the antibodies described herein can be produced by any technique known to those of skill in the art, including, for example, the Fab and F(ab') fragments described herein. 2 Fragments can be produced by the action of enzymes such as papain (to produce Fab fragments) or F(ab') 2 An F(ab') fragment can be generated by proteolytic cleavage of an immunoglobulin molecule with pepsin (to generate a Fab fragment). A Fab fragment corresponds to one of the two identical arms of a tetrameric antibody molecule and contains a complete light chain paired with the VH and CH1 domains of a heavy chain. 2 The fragment contains the two antigen-binding arms of a tetrameric antibody molecule linked by disulfide bonds in the hinge region.

[0201] Furthermore, the antibodies or antigen-binding fragments thereof described herein can also be generated using various phage display and / or yeast-based display methods known in the art. In phage display methods, proteins are displayed on the surface of phage particles carrying the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from an animal cDNA library (e.g., a human or mouse cDNA library of diseased tissue). The DNA encoding the VH and VL 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, e.g., fd and M13, and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing antibodies or antigen-binding fragments thereof that bind to a particular antigen can be selected or identified by 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 or fragments described herein include those described in Brinkman U et al., (1995) J Immunol Methods 182:41-50; Ames RS et al., (1995) J Immunol Methods 184:177-186; Kettleborough CA et al., (1994) Eur J Immunol 24:952-958; Persic L et al., (1997) Gene 187:9-18; Burton DR & Barbas CF (1994) Advan Immunol 57:191-280; International Application PCT / GB91 / 001134, 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. Pat. No. 5,698,426 and U.S. Pat. No. 5,223,409. , U.S. Pat. Nos. 5,403,484, 5,580,717, 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.

[0202] 7.4.1 Polynucleotides In some aspects, provided herein are polynucleotides comprising nucleotide sequences encoding the antibodies or antigen-binding fragments thereof described herein, or domains thereof (e.g., variable light chain regions and / or variable heavy chain regions) that immunospecifically bind to the spike protein of SARS-CoV-2, as well as vectors, e.g., vectors comprising such polynucleotides, for recombinant expression in host cells (e.g., E. coli and mammalian cells).

[0203] In some aspects, provided herein are antibodies or antigen-binding fragments thereof that immunospecifically bind to the spike protein of SARS-CoV-2 and comprise an amino acid sequence described herein, as well as polynucleotides comprising a nucleotide sequence encoding an antibody or antigen-binding fragment that competes (e.g., in a dose-dependent manner) with such an antibody or antigen-binding fragment for binding to SARS-CoV-2 or that binds to the same epitope as such an antibody or antigen-binding fragment.

[0204] Also provided herein are polynucleotides encoding the antibodies or antigen-binding fragments thereof described herein that specifically bind to the spike protein of SARS-CoV-2, optimized, for example, by codon / RNA optimization, replacement with heterologous signal sequences, and elimination of mRNA instability elements. Methods for generating optimized nucleic acids encoding antibodies or antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 or a domain thereof (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes that code for the same amino acid due to the degeneracy of the genetic code) and / or removing inhibitory regions of the mRNA can be performed by appropriately adapting the optimization methods described, for example, in U.S. Patent Nos. 5,965,726, 6,174,666, 6,291,664, 6,414,132, and 6,794,498.

[0205] Polynucleotides encoding the antibodies or antigen-binding fragments thereof, or domains thereof described herein, can be generated from nucleic acid from a suitable source (e.g., a hybridoma) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of a known sequence can be performed using genomic DNA obtained from a hybridoma cell producing the antibody of interest. Using such PCR amplification methods, nucleic acid comprising sequences encoding the light and / or heavy chains of an antibody or antigen-binding fragment thereof can be obtained. Using such PCR amplification methods, nucleic acid comprising sequences encoding the variable light and / or variable heavy chain regions of an antibody or antigen-binding fragment thereof can be obtained. The amplified nucleic acid can be cloned into a vector for expression in a host cell and for further cloning to generate, for example, chimeric and humanized antibodies or antigen-binding fragments thereof.

[0206] The polynucleotides provided herein may be, for example, in the form of RNA or DNA. DNA includes cDNA, genomic DNA, and synthetic DNA, and the DNA may be double-stranded or single-stranded. If single-stranded, the DNA may be the coding strand or the non-coding (antisense) strand. In some embodiments, the polynucleotide is a cDNA or DNA lacking another endogenous intron. In some embodiments, the polynucleotide is a non-naturally occurring polynucleotide. In some embodiments, the polynucleotide is recombinantly produced. In some embodiments, the polynucleotide is isolated. In some embodiments, the polynucleotide is substantially pure. In some embodiments, the polynucleotide is purified from natural components.

[0207] 7.4.2 Cells and Vectors In some embodiments, provided herein are vectors (e.g., expression vectors) comprising a polynucleotide comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof or a domain thereof that binds to the spike protein of SARS-CoV-2, for recombinant expression in a host cell, e.g., a mammalian cell. Also provided herein are cells, e.g., host cells, comprising such vectors for recombinant expression of an antibody or antigen-binding fragment thereof (e.g., a human antibody or antigen-binding fragment thereof) described herein that binds to the spike protein of SARS-CoV-2. In certain embodiments, provided herein are methods for producing an antibody or antigen-binding fragment thereof described herein, comprising expressing such an antibody or antigen-binding fragment thereof in a host cell.

[0208] In some embodiments, recombinant expression of an antibody or antigen-binding fragment thereof or domain thereof (e.g., a heavy or light chain described herein) that specifically binds to the spike protein of SARS-CoV-2 involves the construction of an expression vector containing a polynucleotide encoding the antibody or antigen-binding fragment thereof or domain thereof. Once a polynucleotide encoding an antibody or antigen-binding fragment thereof or domain thereof (e.g., a heavy or light chain variable domain) described herein is obtained, a vector for the production of the antibody or antigen-binding fragment thereof can be generated by recombinant DNA technology using techniques well known in the art. Thus, methods for preparing a protein by expressing a polynucleotide containing an antibody or antigen-binding fragment thereof or domain thereof (e.g., a light or heavy chain) encoding nucleotide sequence are described herein. Methods well known to those skilled in the art can be used to construct expression vectors containing the antibody or antigen-binding fragment thereof or domain thereof (e.g., a light or heavy chain) coding 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 techniques. Also provided herein is a replicable vector comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof, heavy or light chain, heavy or light chain variable domain, or heavy, light or light chain CDR operably linked to a promoter, as described herein. Such a vector can, for example, comprise a nucleotide sequence encoding the constant region of an antibody or antigen-binding fragment thereof (see, for example, WO 86 / 05807 and WO 89 / 01036; and U.S. Pat. No. 5,122,464), and the variable domain of an antibody or antigen-binding fragment thereof 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.

[0209] The expression vector can be transferred into a cell (e.g., a host cell) by conventional techniques, and the resulting cells can then be cultured by conventional techniques to produce an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of the antibodies provided in Table 1, i.e., VH, VL, VH and VL, a heavy chain, a light chain, or a heavy chain and a light chain), or a domain thereof (e.g., the VH, VL, VH and VL, a heavy chain, or ...)) described herein, operably linked to a promoter for expression of such sequences in In some embodiments, for expression of a double-chain antibody or antigen-binding fragment thereof, vectors encoding both the heavy and light chains can be individually co-expressed in a host cell for expression of the whole immunoglobulin, as described in detail below. In some embodiments, the host cell contains a vector containing a polynucleotide encoding both the heavy and light chains of an antibody described herein (e.g., the heavy and light chains of an antibody provided in Table 1), or a domain thereof (e.g., the VH and VL of an antibody provided in Table 1). In some embodiments, the host cell contains two different vectors, a first vector containing a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein, and a second vector containing a polynucleotide encoding the light chain or light chain variable region of an antibody or domain thereof described herein (e.g., an antibody comprising the six CDRs of an antibody provided in Table 1).In some embodiments, a first host cell comprises a first vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein, and a second host cell comprises a second vector comprising a polynucleotide encoding a light chain or light chain variable region of an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of an antibody provided in Table 1). In some embodiments, the heavy chain or heavy chain variable region expressed by the first cell associates with the light chain or light chain variable region of the second cell to form an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the six CDRs of an antibody provided in Table 1). In some embodiments, provided herein is a population of host cells comprising such a first host cell and such a second host cell.

[0210] In some aspects, provided herein is a population of vectors comprising a first vector comprising a polynucleotide encoding a light chain or light chain variable region of an antibody or antigen-binding fragment thereof described herein, and a second vector comprising a polynucleotide encoding a heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising the CDRs of an antibody provided in Table 1). Alternatively, a single vector encoding and capable of expressing both heavy and light chain polypeptides may be used.

[0211] A variety of host-expression vector systems can be utilized to express the antibodies and antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising the CDRs of the antibodies provided in Table 1) (see, e.g., U.S. Pat. No. 5,807,715). Such host-expression systems represent vehicles in which a coding sequence of interest can be produced and subsequently purified, but also cells which, when transfected or transfected with the appropriate nucleotide coding sequence, can express the antibodies or antigen-binding fragments thereof described herein in situ.These include microorganisms such as bacteria (e.g., E. coli and B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA, or cosmid DNA expression vectors containing the antibody coding sequences; yeast (e.g., Saccharomyces and Pichia) transformed with recombinant yeast expression vectors containing the antibody coding sequences; insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing the antibody coding sequences; plant cell systems (e.g., Chlamydomonas reinhardtii) infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmids) containing the antibody coding sequences. reinhardtii); or mammalian cell lines (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7O3O, HsS78Bst, HeLa, and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, and BMT10 cells) harboring a recombinant expression construct containing a promoter derived from the genome of a mammalian cell (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter). In some embodiments, cells for expressing the antibodies and antigen-binding fragments thereof described herein (e.g., antibodies or antigen-binding fragments thereof comprising the CDRs of an antibody provided in Table 1) are CHO cells, e.g., CHO cells from CHO GS System™ (Lonza). In some embodiments, cells for expressing the antibodies described herein are human cells, e.g., human cell lines.In some embodiments, the mammalian expression vector is pOptiVEC™ or pcDNA3.3. In some embodiments, bacterial cells such as Escherichia coli, especially for the expression of whole recombinant antibody molecules, or eukaryotic cells (e.g., mammalian cells) are used for the expression of a recombinant antibody molecule. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, in conjunction with vectors such as the major intermediate early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK & Hofstetter H (1986) Gene 45:101-105; and Cockett MI et al., (1990) Biotechnology 8:662-667). In some embodiments, the antibodies or antigen-binding fragments thereof described herein are produced by CHO cells or NS0 cells.

[0212] In addition, a host cell strain can be chosen which modulates the expression of the inserted sequences or modifies and processes the gene product in the defined fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of the protein product may contribute to the function of the protein. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product can be used. Such mammalian host cells include, but are not limited to, CHO, VERO, BHK, Hela, MDCK, HEK 293, NIH 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NS0 (a mouse myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL7O3O, COS (e.g., COS1 or COS), PER.C6, VERO, HsS78Bst, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20, BMT10, and HsS78Bst cells. In some embodiments, the antibodies or antigen-binding fragments thereof described herein that specifically bind to the spike protein of SARS-CoV-2 are produced in mammalian cells, such as CHO cells.

[0213] Once the antibodies or antigen-binding fragments thereof described herein have been produced by recombinant expression, they can be purified by any method of purifying immunoglobulin molecules known in the art, such as, for example, chromatography (e.g., ion exchange, affinity, particularly by affinity for specific antigens following Protein A, and size exclusion chromatography), centrifugation, differential solubility, or any other standard protein purification technique. Additionally, the antibodies or antigen-binding fragments thereof described herein can be fused to heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0214] In some embodiments, the antibodies or antigen-binding fragments thereof described herein are isolated or produced. Generally, an isolated antibody or antigen-binding fragment thereof is substantially free of other antibodies or antigen-binding fragments thereof that have antigen specificity different from that of the isolated antibody or antigen-binding fragment thereof. For example, in some embodiments, preparations of the antibodies or antigen-binding fragments thereof described herein are substantially free of cellular material and / or chemical precursors.

[0215] 7.5 Pharmaceutical Compositions Provided herein are compositions comprising an antibody or antigen-binding fragment thereof described herein, or a combination of antibodies or antigen-binding fragments thereof described herein, with the desired degree of purity in a physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, Pa.). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed.

[0216] In some embodiments, a composition comprising at least one antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 is provided in a formulation with a pharma- ceutically acceptable carrier (see, e.g., Gennaro, Remington: The Science and Practice of Pharmacy with Facts and Comparisons: Drugfacts Plus, 20th ed. (2003); Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 7th ed., Lippencott Williams and Wilkins (2004); Kibbe et al., Handbook of Pharmaceutical Excipients, 3rd ed., Pharmaceutical Press (2000)). In some embodiments, the pharmaceutical compositions described herein comprise two antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, e.g., two antibodies or antigen-binding fragments thereof that bind to different epitopes of the spike protein of SARS-CoV-2. In some embodiments, the pharmaceutical compositions described herein comprise two antibodies or antigen-binding fragments that bind to different epitopes of the receptor binding domain (RBD) of the spike protein of SARS-CoV-2. In some embodiments, the pharmaceutical compositions described herein comprise two antibodies or antigen-binding fragments that bind to non-overlapping epitopes of the RBD of the spike protein of SARS-CoV-2. In some embodiments, the pharmaceutical compositions described herein comprise two antibodies or antigen-binding fragments that can simultaneously bind to SARS-CoV-2.

[0217] The pharmaceutical compositions described herein may be useful for neutralizing SARS-CoV-2.

[0218] The pharmaceutical compositions described herein may be useful for preventing and / or treating SARS-CoV-2 infection in a patient, or one or more conditions or complications associated with SARS-CoV-2 infection in a patient. In some embodiments, the patient may have already been exposed to SARS-CoV-2. Examples of SARS-CoV-2 infection, or one or more conditions or complications associated with SARS-CoV-2 infection that can be prevented and / or treated according to the methods described herein include, but are not limited to, fever, cough, fatigue, shortness of breath, difficulty breathing, muscle pain, chills, muscle pain, chills, sore throat, loss of taste or smell, headache, chest pain, nausea, vomiting, and diarrhea. Further examples of one or more conditions or complications associated with SARS-CoV-2 infection in a patient that can be treated according to the methods described herein include, but are not limited to, cardiac complications, respiratory complications, diabetic complications, organ failure, and blood clotting. In some embodiments, the pharmaceutical compositions provided herein may be useful for treating or preventing a SARS-CoV-2 infection, or one or more conditions or complications associated with a SARS-CoV-2 infection, as described herein, in a patient having one or more risk factors for SARS-CoV-2 infection, including, but not limited to, being over 65 years old, being immunocompromised, and suffering from one or more of the following: chronic lung disease, asthma, or diabetes.

[0219] The pharmaceutical compositions described herein are, in some embodiments, intended for use as medicaments. The pharmaceutical compositions described herein are, in some embodiments, intended for use as diagnostics, for example, for detecting the presence of SARS-CoV-2 in a sample obtained from a patient (e.g., a human patient).

[0220] The compositions provided herein can be formulated for intramuscular (IM) administration, for example, IM injection.

[0221] Compositions provided herein for use in the context of in vivo administration can be sterile, which is readily accomplished, for example, by filtration through sterile filtration membranes.

[0222] 7.6 Use and Materials 7.6.1 Therapeutic Uses and Methods In some embodiments, provided herein are methods of preventing and / or treating SARS-CoV-2 infection in a patient, or one or more conditions or complications associated with SARS-CoV-2 infection in a patient. The method of treating or preventing SARS-CoV-2 infection can include administering to a patient (e.g., a human patient) in need thereof one or more antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2.

[0223] In some embodiments, provided herein are methods for reducing the likelihood of infection in a subject at risk of contracting a SARS-CoV-2 infection. The methods for reducing the likelihood of infection in a subject at risk of contracting a SARS-CoV-2 infection may include administering one or more antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2.

[0224] In some embodiments, provided herein are methods of preventing and / or treating SARS-CoV-2 infection or one or more conditions or complications associated with SARS-CoV-2 infection, including, but not limited to, fever, cough, fatigue, shortness of breath, difficulty breathing, muscle pain, chills, muscle pain, chills, sore throat, loss of taste or smell, headache, chest pain, nausea, vomiting, and diarrhea. In some embodiments, provided herein are methods of preventing and / or treating SARS-CoV-2 infection in patients with one or more risk factors for SARS-CoV-2 infection. In some embodiments, the risk factors include, but are not limited to, being 65 years of age or older, having an immunodeficiency, having one or more of chronic lung disease, asthma, or diabetes, and / or having an immunodeficiency. In some embodiments, such methods include administering to a patient (e.g., a human patient) in need thereof an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 provided herein, or a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 provided herein.

[0225] In some embodiments, such methods include administering to a patient (e.g., a human patient) in need thereof two antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 provided herein, or a pharmaceutical composition comprising two antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 provided herein.

[0226] In some embodiments, such methods include administering a composition comprising one or more antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 herein to a patient (e.g., a human patient) in need thereof. In some embodiments, the patient includes, but is not limited to, being 65 years of age or older, being immunocompromised, suffering from one or more of chronic lung disease, asthma, or diabetes.

[0227] In some embodiments, one or more antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, or pharmaceutical compositions, are administered to a subject (e.g., a human subject) at risk of contracting SARS-CoV-2.

[0228] In some embodiments, one or more antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 are administered intramuscularly, for example via intramuscular injection.

[0229] Usually, the patient is a human, but non-human mammals, including transgenic mammals, can also be treated.

[0230] In some aspects, the present invention relates to an antibody or antigen-binding fragment thereof, a combination of antibodies or antigen-binding fragments thereof, or a pharmaceutical composition provided herein for use as a medicament. In some aspects, the present invention relates to an antibody or antigen-binding fragment thereof, a combination of antibodies or antigen-binding fragments thereof, or a pharmaceutical composition provided herein for use in a method for the prevention or treatment of SARS-CoV-2 infection. In some aspects, the present invention relates to an antibody or antigen-binding fragment thereof, a combination of antibodies or antigen-binding fragments thereof, or a pharmaceutical composition provided herein for use in a method for the treatment of SARS-CoV-2 infection in a subject comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof, a combination of antibodies or antigen-binding fragments thereof, or a pharmaceutical composition provided herein.

[0231] The amount of the antibody or antigen-binding fragment thereof, or composition that will be effective in treating a condition will vary depending on the nature of the disease. The exact dosage employed in the composition will also vary depending on the route of administration, and the severity of the disease.

[0232] 7.6.2 Detection and diagnostic applications Antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 described herein (see, e.g., Section 7.2) can be used to assay SARS-CoV-2 protein levels, or levels of SARS-CoV-2, in biological samples using classical methods known to those of skill in the art, including immunoassays such as enzyme-linked immunosorbent assay (ELISA), immunoprecipitation, or Western blotting. Suitable antibody assay labels are known in the art and include enzyme labels, such as glucose oxidase; iodine ( 125 I, 121 I), Carbon ( 14 C), Sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In), and technetium ( 99 These labels include radioisotopes such as Tc; luminescent labels such as luminol; and fluorescent labels such as fluorescein and rhodamine. Such labels can be used to label the antibodies or antigen-binding fragments thereof described herein. Alternatively, a second antibody or antigen-binding fragment thereof that recognizes an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 described herein can be labeled with and used in conjunction with an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 to detect SARS-CoV-2 protein levels.

[0233] Assaying the expression level of a SARS-CoV-2 protein is intended to include qualitatively or quantitatively measuring or estimating the level of a SARS-CoV-2 protein in a first biological sample, either directly (e.g., by determining or estimating absolute protein levels) or relatively (e.g., by comparing the level of a protein associated with a disease in a second biological sample). The SARS-CoV-2 protein expression level in a first biological sample can be measured or estimated and compared to a standard SARS-CoV-2 protein level, which can be taken from a second biological sample obtained from an individual without the disorder or determined by the average level of a population of individuals without the disorder.

[0234] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cellular source that potentially expresses SARS-CoV-2. Methods for obtaining tissue biopsies and body fluids from animals (e.g., humans) are well known in the art.

[0235] The antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 described herein may have a detectable or functional label. When a fluorescent label is used, specific binding members can be identified and quantified using currently available microscopy and fluorescence activated cell sorter analysis (FACS), or a combination of procedures of both methods known in the art. The antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 described herein may have a fluorescent label. Exemplary fluorescent labels include, for example, reactive binding probes such as aminocoumarins, fluorescein, and Texas Red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. Antibodies or antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 may be isotopically labeled. 3 H, 14 C. 32 P, 35 S, 36 Cl, 51 Cr,57 Co, 58 Co, 59 Fe, 67 Cu, 90 Y, 99 Tc, 111 In, 117 Lu, 121 I, 124 I, 125 I, 131 I, 198 Au, 211 At, 213 Bi, 225 Ac, and 186 The antibody or antigen-binding fragment thereof may have a radioactive label such as Re. When a radioactive label is used, currently available counting procedures known in the art can be used to identify and quantify specific binding of the antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2. If the label is an enzyme, detection can be achieved by any of the currently available colorimetric, spectrophotometric, fluorospectrophotometric, amperometric, or gasometric techniques known in the art. This can be achieved by contacting the sample or control sample with an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 under conditions that allow the formation of a complex between the antibody or antigen-binding fragment thereof and the spike protein of SARS-CoV-2. Any complexes formed between the antibody or antigen-binding fragment and the spike protein of SARS-CoV-2 can be detected and compared in the sample (and optionally the control). Given the specific binding of the antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 described herein to SARS-CoV-2, the antibodies or antigen-binding fragments thereof can be used to specifically detect SARS-CoV-2 (e.g., in a subject).

[0236] Also included herein are assay systems that can be prepared in the form of test kits for quantitatively analyzing the presence of, for example, the SARS-CoV-2 spike protein. Such systems or test kits can include a labeled component, such as a labeled antibody or antigen-binding fragment, and one or more additional immunochemical reagents. For details of kits, see, for example, Section 7.7 below.

[0237] In some embodiments, provided herein is a method for detecting SARS-CoV-2 spike protein in a sample in vitro, comprising contacting the sample with an antibody or antigen-binding fragment thereof. In some embodiments, provided herein is a use of an antibody or antigen-binding fragment thereof provided herein for detecting SARS-CoV-2 spike protein in a sample in vitro. In one embodiment, provided herein is an antibody or antigen-binding fragment thereof, or a pharmaceutical composition provided herein for use in detecting SARS-CoV-2 spike protein in a subject or a sample obtained from a subject. In one embodiment, provided herein is an antibody or antigen-binding fragment thereof, or a pharmaceutical composition provided herein for use as a diagnostic agent. In some embodiments, the antibody comprises a detectable label. In some embodiments, the subject is a human.

[0238] 7.7 Kits Provided herein is a kit comprising one or more antibodies or antigen-binding fragments thereof as described herein. In some embodiments, provided herein is a pharmaceutical pack or kit comprising one or more containers filled with one or more of the components of the pharmaceutical compositions described herein, such as one or more antibodies or antigen-binding fragments thereof as provided herein. Optionally, such containers may be accompanied by a notice in a form prescribed by a governmental agency regulating the manufacture, use, or sale of pharmaceutical or biological products, which notice indicates approval by the agency of the manufacture, use, or sale for administration to humans.

[0239] Also provided herein are kits that can be used in diagnostic methods. In some embodiments, the kits include, in one or more containers, an antibody or antigen-binding fragment thereof described herein, e.g., a purified antibody or antigen-binding fragment thereof. In some embodiments, the kits described herein contain a substantially isolated SARS-CoV-2 spike protein antigen that can be used as a control. In some embodiments, the kits described herein further include a control antibody or antigen-binding fragment thereof that does not react with the SARS-CoV-2 spike protein antigen. In some embodiments, the kits described herein contain one or more elements for detecting binding of the antibody or antigen-binding fragment thereof to the SARS-CoV-2 spike protein antigen (e.g., the antibody or antigen-binding fragment thereof may be conjugated to a detectable substrate, such as a fluorescent compound, an enzymatic substrate, a radioactive compound, or a luminescent compound, or a second antibody or antigen-binding fragment thereof that recognizes the first antibody or antigen-binding fragment thereof may be conjugated to a detectable substrate). In some embodiments, the kits provided herein may include a recombinantly produced or chemically synthesized SARS-CoV-2 spike protein antigen. The SARS-CoV-2 spike protein antigen provided in the kit may also be bound to a solid support. In some embodiments, the detection means of the above kit comprises a solid support to which the SARS-CoV-2 spike protein antigen is bound. Such kits may also comprise a non-binding reporter-labeled anti-human antibody or antigen-binding fragment thereof, or an anti-mouse / rat antibody or antigen-binding fragment thereof. In this embodiment, the binding of an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 to the SARS-CoV-2 spike protein antigen is detectable by the binding of the reporter-labeled antibody or antigen-binding fragment thereof.

[0240] The following examples are offered by way of illustration and not by way of limitation. EXAMPLES

[0241] 8. Working Example The examples in this embodiment section (ie, Section 8) are provided by way of illustration and not by way of limitation.

[0242] 8.1 Example 1: Antibody and Protein Generation Antibody VH and VL DNA fragments were cloned into a CMV-driven mammalian expression plasmid containing human IgG1 Fc modified for serum half-life extension with YTE and loss of TM effector function. Plasmids were sequenced by Sanger sequencing. Antibodies were transiently expressed in HEK293F cells using 293Fectin (Gibco #12347019) according to the manufacturer's instructions and cultured in Freestyle medium (Gibco #12338018) for 6 days. On day 6, the medium was clarified by centrifugation, filtered, and purified using MabSelect SuRe resin (Cytiva #11-0034-94). Antibody purity was determined using HP-SEC as described below, and the accurate mass of the antibodies was verified by mass spectrometry. Exemplary antibody sequences are listed in Table 1.

[0243] SARS-CoV-2 RBD (residues 334-526) was cloned with an N-terminal CD33 leader sequence and a C-terminal GSSG linker, an AviTag, a GSSG linker, and an 8xHisTag, expressed in FreeStyle 293 cells (Thermo Fisher), and isolated by affinity chromatography using a HisTrap column (GE Healthcare) followed by size-exclusion chromatography using a Superdex200 column (GE Healthcare). Purified proteins were analyzed by SDS-PAGE to confirm purity and appropriate molecular weight. Endotoxin concentrations were <1EU / mg as measured using Charles River Endosafe® cartridges (Charles River).

[0244] 8.2 Example 2: HP-SEC Antibody samples were analyzed using HP-SEC to determine aggregate, monomer, and fragment concentrations. Samples (100 μg in PBS buffer) were injected into an Agilent 1200 series high performance liquid chromatography (HPLC) instrument and separated using a TSKgel G3000SWxl size exclusion column (Tosoh Bioscience #08541). The mobile phase was 100 mM sodium phosphate (pH 6.8) and the sample flow rate was 1 mL / min. Ultraviolet (UV) detection was performed at 280 nm. The results are shown in Table 2.

[0245] 8.3 Example 3: Baculovirus ELISA Baculovirus particle (BVP) ELISA was performed essentially as previously reported (Hotzel I, Theil FP, Bernstein LJ, Prabhu S, Deng R, Quintana L, et al. A strategy for risk mitigation of antibodies with fast clearance. MAbs 2012;4:753-60) with some modifications. Briefly, a 1% bacolvirus (BV) suspension in 50 mM sodium carbonate buffer (pH 9.6) was used to coat half of a 96-well ELISA plate (Nunc Maxisorp) overnight at 4°C, and the second half of the ELISA plate was left uncoated to test plate-bound antibodies. All the following steps were performed at room temperature. The next day, the wells were washed with Dulbecco's PBS (DPBS) and then incubated in blocking buffer (Dulbecco's PBS with 0.5% BSA) for 1 h, followed by washing three times with DPBS. Next, 100 nM and 10 nM of antibody in blocking buffer were added to both BVP-coated and uncoated wells and incubated for 1 hour, followed by washing 3 times with DPBS. Next, goat anti-human IgG-HRP secondary antibody (1:5000 dilution, Sigma-Aldrich #A0170) in blocking buffer was added to the wells and incubated for 1 hour, followed by washing 3 times with DPBS. Finally, TMB substrate (SeraCare #5120-0075) was added to each well and incubated for 2 minutes. The reaction was stopped by adding an equal volume of 0.2 M sulfuric acid to each well. Absorbance was measured at 450 nm. BVP scores and plate binding were determined by normalizing absorbance to control wells without test antibody. Results are shown in Table 2.

[0246] 8.4 Example 4: HEK Binding Assay Nonspecific HEK cell binding was measured using a Mirrorball fluorescence cytometer (SPT Labtech). First, 10 uL of Alexa Fluor 647 goat anti-human IgG (H+L) antibody (Invitrogen #A-21445) diluted to 16 nM in Mirrorball buffer (Hank's balanced salt solution with 0.5% BSA) was added to the wells of a 384-well clear bottom plate. Next, 10 uL of test antibody serially diluted in Mirrorball buffer was added to the wells. Finally, 20 uL of HEK293f cells diluted to 250,000 cells / mL in Mirrorball buffer were added to the wells. The plates were incubated at room temperature for 2 hours and the fluorescence of each well was measured using the Mirrorball fluorescence cytometer. The results are shown in Table 2.

[0247] 8.5 Example 5: AC-SINS AC-SINS was performed essentially as previously described (Geng SB, Wu J, Alam ME, Schultz JS, Dickinson CD, Seminer CR, et al. Facilitated Preparation of Stable Antibody-Gold Conjugates and Application to Affinity-Capture Self-Interaction Nanoparticle Spectroscopy. Bioconjug Chem 2016;27:2287-300) with minor modifications. Briefly, both total goat IgG (Jackson ImmunoResearch #005-000-003) (non-capture) and polyclonal goat anti-human IgG Fc (Jackson ImmunoResearch #109-005-098) (capture) antibodies were dialyzed into 20 mM potassium acetate (pH 4.3) buffer and conjugated to 20 nm gold nanoparticles (Innova Biosciences #3201-0100) in a 3:2 ratio of capture:non-capture antibody. The antibodies were incubated with the gold nanoparticles in a 9:1 ratio for 1 hour at room temperature, followed by blocking with the addition of 0.1 μM poly(ethylene glycol) methyl ether thiol (2000 MW, Sigma-Aldrich #729140) for 1 hour. The coated and blocked nanoparticles were concentrated 12.5-fold by centrifugation and stored at 4°C. To assess self-association, 5 μL of nanoparticles were mixed with 45 μL of 50 ug / mL purified antibody in PBS, pH 7.2 or buffer in a 384-well plate. As a control, nanoparticles were mixed with buffer only (no antibody). Absorbance was measured in the range of 490-700 nm on a SPECTROstar Nano UV / vis plate reader. The wavelength of peak absorbance was calculated with MARS data analysis software and used to determine the wavelength shift compared to the nanoparticle-only control. The results are shown in Table 2.

[0248] 8.6 Example 6: Accelerated Stability Heat Load Test For accelerated stability studies, samples were diluted to 1 mg / mL in PBS (pH 7.2) and incubated at 4°C or 45°C for 2 weeks. Samples at days 0, 7, and 14 were then analyzed by HP-SEC peptide mapping and DELFIA binding assays as described herein. The percentage of monomer, aggregate, and fragment for each sample was calculated based on curve integration using HPLC ChemStation software (Agilent). The change in monomer, aggregate, and fragment content was calculated from the difference in values ​​between each sample incubated at 45°C and 4°C. The results are shown in Table 3.

[0249] 8.7 Example 7: Photostability Assay For photostability testing, antibodies were prepared at a concentration of 2.5 mg / mL in PBS (pH 7.2), filled into 1 cc Schott glass vials, stoppered and sealed, and placed in an ICH compliant photostability chamber (Caron model 6545-2). Samples were exposed to 3000 lux of white light for one week, with a total exposure of approximately 500,000 lux hours. Samples were analyzed by HP-SEC and peptide mapping as described in these sections. Results are shown in Table 3.

[0250] 8.8 Example 8: Serum Stability Assay Antibodies were diluted to 0.5 mg / ml in human serum (Sigma) and incubated at 37° C. for 14 days. Day 0 control samples were taken and stored frozen at −80° C. until testing. Day 14 culture samples were also stored frozen at −80° C. until testing of all samples was completed. Binding of stressed and non-stressed samples was assessed by the DELFIA binding assay against the RBD as described herein. Results are shown in Table 3.

[0251] 8.9 Example 9: DSC Thermal melting transitions were measured using a Microcal VP differential scanning calorimetry system (Malvern, PA). Monoclonal antibody solutions were diluted to 1 mg / ml in the final buffer, and the change in heat capacity (Cp) was measured as the samples were heated from 20°C to 95°C at a temperature gradient of 90°C / h. Normalized heat capacity data were obtained by subtracting the buffer blank and then normalizing to the concentration of monoclonal antibody. Data were analyzed using Microcal LLC origin software to calculate the thermal melting transitions associated with the conformational changes of distinct domains. The results are shown in Table 2.

[0252] 8.10 Example 10: Viscosity The concentrated antibody solution was diluted to a 100 mg / mL solution. Each sample was subjected to a rotational shear stress of 1000 s-1 at 23 °C in an MCR301 rheometer using a CP20-1 cone-plate system (Anton Parr, part number 3274). During the course of data collection, five measurements were taken per minute and averaged to calculate the viscosity value. The results are shown in Table 2.

[0253] 8.11 Example 11: DLS The antibody panel was reconstituted in a different buffer. Measurements of the Z-average apparent diffusion coefficient were performed using a Dynapro plate reader (Wyatt technology, Santabarbara, CA) equipped with a laser source with a wavelength of 833 nm. Scattered light was collected in backscatter mode at an angle of 153°. For each antibody, 40 μl of each concentration of 2, 4, 6, 8, and 10 mg / ml was dispensed in triplicate into a 384-well low volume plate (Corning, Tewskbury, MA), covered with paraffin film, and centrifuged at 3000 RPM for 1 minute to remove air bubbles. The sample chamber was equilibrated for 1 hour before measurements, and 10 data acquisitions were performed at 10 second intervals and averaged for each well. The Z-average translational diffusion coefficient was determined from cumulant analysis of the autocorrelation function, It is modeled as D = D0(1 + kDC), giving the "kD" diffusion virial coefficient (interaction parameter). The results are shown in Table 2.

[0254] 8.12 Example 12: Peptide Mapping Tryptic digest peptide mapping was performed according to the manufacturer's recommendations. Briefly, 100 μg of antibody sample was denatured and reduced in guanidine hydrochloride buffer supplemented with dithiothreitol (DTT, 20291, Thermo Scientific) for 30 min at 37 °C. The sample was then alkylated with iodoacetamide (IAM, 786-078, G-Biosciences) for 30 min at room temperature in the dark. The sample mixture was then dialyzed against 6 M urea and diluted with Tris pH 7.5 buffer for tryptic digestion. Trypsin (V5280, Promega) was added at a protease:protein ratio of 1:20 and incubated at 37 °C for 4 h. The reaction was stopped by adding trifluoroacetic acid (TFA, T6508, Sigma-Aldrich). The digests of the samples were analyzed by a Fusion Orbitrap mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA) connected to an AQUITY ultra-performance liquid chromatograph (UPC; Waters). An AQUITY UPLC BEH300 C18 column (1.7 μm, 2.1 × 150 mm, Waters) was used for separation. The column temperature was maintained at 55 °C. Mobile phase A was 0.02% TFA in water, and mobile phase B was 0.02% TFA in acetonitrile. The digested peptides were eluted from the column with a linear gradient of 0–35%, and the chromatographic profile was monitored using UV absorption at 220 nm and mass spectrometry (MS). MS data was processed using BiopharmaFinder 3.0 (Thermo Fisher Scientific). The results are shown in Table 3.

[0255] 8.13 Example 13: DELFIA Binding Assay Antibody binding to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein was measured using the DELFIA® (PerkinElmer) method before and after heat stress. Nunc MaxiSorp™ 96-well plates were coated overnight with purified RBD omicron protein. The assay plates were then washed with 1× TBS-Tween buffer (PerkinElmer) and blocked with bovine serum albumin (Sigma). Antibody samples were serially diluted and added to the plates for 1 h. Bound antibodies were detected using DELFIA® Europium-N1 anti-human IgG (PerkinElmer) and the signal was quantified by time-resolved fluorescence on an Envision plate reader (PerkinElmer). IC 50 was calculated using GraphPad Prism v8.0 with a four-parameter logistic (4PL) curve. IC of stressed samples 50 was compared to a reference sample to obtain percent relative potency.

[0256] 8.14 Example 14: FcRn Affinity Chromatography Approximately 40 μg / 40 μL of antibody was loaded onto a 1 ml huFcRN-bound Sepharose affinity column using an Agilent HPLC, followed by a 3 CV linear gradient from buffer A (20 mM MES, 150 mM NaCl, pH 5.5) to 40% buffer B (20 mM Tris + 150 mM NaCl, pH 8.8) and an 18 CV linear gradient from 40% to 100% buffer B. The experiment was performed at room temperature with a flow rate of 0.5 ml / min, and A280 was measured as the elution profile using an Agilent-DAD. Retention time (RT) was calculated using the following formula: relative RT = ([sample RT - NIP228 RT] / [NIP228-YTE RT - NIP228 RT]). Relative retention times were normalized to 0.05 vs. 0.1 using a well-behaved control antibody, NIP228, in WT and YTE formats. Results are shown in Table 3.

[0257] 8.15 Example 15: Octet Binding Competition Binding competition was performed using an Octet model QK 384 biolayer interferometer (ForteBio). Streptavidin chips were first immersed in 1x kinetics buffer for 10 min, followed by a baseline signal measurement in 1x kinetics buffer for 60 s. Biotinylated SARS-CoV-2 spike RBD in kinetics buffer diluted to 4 nM was then added to the chip for 300 s. The chip containing the antigen was then added to a well containing 1 μM of the first mAb (mAb1) as the mAb1 reference antibody, and binding was measured for 300 s. The chip was then immersed in a well containing 1 μM of the second mAb (mAb2), and a second round of binding was measured for 300 s. The binding signal at the end of each step was averaged over the final 10 s data points at equilibrium to determine the change in signal. If the signal increased by 0.5 nM or more after the addition of mAb2, the mAb was considered not competing. The results are shown in Table 4 and indicate that RQ40 and RQ43 do not compete with RQ33.

[0258] 8.16 Example 16: Binding to RBDs with point mutations Kinetic evaluation of mAb binding to SARS-CoV-2 spike RBD (wt and point mutants) (k a ,k d , and K D) was performed using an Octet model QK 384 Biolayer Interferometer (ForteBio). Anti-penta-His (HIS1K) chips were first soaked in 1x kinetics buffer for 10 min, followed by a baseline signal measurement in 1x kinetics buffer for 60 s. His-tagged SARS-CoV-2 spike RBD in kinetics buffer diluted to 5 μg / ml was then added to the chip for 240 s. The antigen-containing chips were then added to wells containing mAb at a starting concentration of 200 nM, followed by a 2-fold dilution, and binding was measured for 300 s. Dissociation was then measured in 1x kinetics buffer for 300 s. Data were reference subtracted and fitted to a 1:1 binding model using Octet Data Analysis Software 12.0. (The collected data was fitted to a single-site binding equation using Biacore software to obtain measurements of binding kinetics.) The results, shown in Table 5, indicate that RQ43 and RQ33 are insensitive to the K444A and F486A RBD mutations.

[0259] 8.17 Example 17: Pseudovirus Neutralization Assay Freestyle 293X cells were seeded and transfected with a third generation human immunodeficiency virus (HIV)-based lentiviral vector expressing luciferase together with packaging plasmids encoding: SARS-CoV-2 spike protein with a C-terminal 19 amino acid deletion, Rev, and Gag-pol. Viral supernatants were harvested 48 hours later. Cell debris was removed by low speed centrifugation and the supernatant was passed through a 0.45 μM filter unit. The supernatant was concentrated 100-fold by ultracentrifugation to generate stock virus for use in this assay.

[0260] Serial dilutions of mAbs were prepared in 384-well microtiter plates and pre-incubated with pseudovirus for 1 h at 37°C before adding Ad293 cells stably expressing human ACE2. Plates were returned to the 37°C incubator for 48 h and luciferase activity was measured using the Bright-Glo™ Luciferase Assay System (Promega) on an EnVision 2105 Multimode Plate Reader (Perkin Elmer) according to the manufacturer's recommendations. Percent inhibition was calculated relative to the pseudovirus alone control and potency (IC 50 The mean IC values ​​for each mAb were determined by nonlinear regression using GraphPad Prism software version 8.1.0. 50 Values ​​were determined from a minimum of two independent experiments. The results of the pseudovirus neutralization assay are shown in Figures 1 and 2. In the neutralization assays testing the mAb combinations, AZD7442 was used as a control. AZD7442 is a combination of two antibodies (silgavimab (AZD8895) and silgavimab (AZD1061)) derived from B cells provided by a patient in the convalescent stage after SARS-CoV-2 virus infection.

[0261] [Table 11]

[0262] [Table 12]

[0263] [Table 13]

[0264] [Table 14]

[0265] [Table 15]

[0266] [Table 16]

[0267] 8.18 Example 18: Diverse Neutralization Profiles of RQ33 RQ33, RQ43-GL-H-LO1, and their combination (RQ33+RQ43-GL-H-LO1) were evaluated in neutralization assays to measure antiviral activity against SARS-CoV-2 variants of concern (VOCs). Antiviral activity was observed in a dose-dependent manner, with a sigmoidal correlation between antibody concentration and neutralization rate for most VOCs. However, when neutralization was measured against the authentic SARS-CoV-2 virus BA.2.12.1 mutant, negative neutralization (i.e., enhanced infection) was observed with low concentrations of RQ33 (Figure 3). This observation contrasts with that observed with RQ43-GL-H-LO1 and their combination (RQ33+RQ43-GL-H-LO1), where low concentrations of antibodies showed low or no neutralization rate, but no negative neutralization (i.e., enhanced infection). This observation of enhanced infection was only observed when assaying authentic BA.2.12.1 virus, and not with pseudovirus (FIG. 4) or with authentic viruses of other variants.

[0268] The observed enhanced infection of authentic BA.2.12.1 virus at low concentrations of RQ33 is not consistent with the targeted product profile of monoclonal antibody preparations to prevent SARS-CoV-2 infection in pre-exposure prophylaxis. Therefore, we evaluated the combination of silgavimab (instead of RQ33) and RQ43-GL-H-LO1. Silgavimab and RQ43-GL-H-LO1 are biochemically compatible, and RQ43-GL-H-LO1 was shown not to compete with silgavimab in an Octet competition assay with the BA.2 RBD (Figure 5). This was also demonstrated by comparing the binding footprints of RQ43 (the parent of RQ43-GL-H-LO1) and silgavimab on the RBD surface (Figure 6). Furthermore, the combination of silgavimab and RQ43-GL-H-LO1 provides broad and potent coverage of all tested variants of concern (Table 6).

[0269] [Table 17]

[0270] The present invention should not be limited in scope by the embodiments described herein. Indeed, various modifications of the present invention in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to be within the scope of the appended claims.

[0271] All references (e.g., publications, or patents, or patent applications) cited in this specification are incorporated by reference in their entirety for all purposes to the same extent as if each individual reference (e.g., publication, or patent, or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

[0272] Some aspects are within the scope of the claims.

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the antibody or antigen-binding fragment comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs. 31, 32, 37, 34, 35, and 36, respectively.

2. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

61.

3. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment neutralizes the SARS-CoV-2 virus.

4. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment neutralizes SARS-CoV-2 D614G, SARS-CoV-2 alpha, SARS-CoV-2 delta + T51I + T95I, SARS-CoV-2 BA. 1, SARS-CoV-2 BA. 1.1, SARS-CoV-2 BA. 2, SARS-CoV-2 BA. 2.12.1, and / or SARS-CoV-2 BA. 5 virus.

5. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment is of the fully human type.

6. The antibody or antigen-binding fragment according to claim 1, wherein the antibody or antigen-binding fragment comprises the human IgGλ light chain constant region.

7. The antibody or antigen-binding fragment according to claim 6, wherein the antibody or antigen-binding fragment comprises the human immunoglobulin IgG1 heavy chain constant region.

8. The antibody or antigen-binding fragment according to claim 7, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region containing a YTE mutation.

9. The antibody or antigen-binding fragment according to claim 8, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region containing a TM mutation.

10. The antibody or antigen-binding fragment according to claim 9, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region having the amino acid sequence of SEQ ID NO:

66.

11. The antibody or antigen-binding fragment according to claim 10, wherein the antibody or antigen-binding fragment thereof is a full-length antibody.

12. An antibody that specifically binds to the spike protein of SARS-CoV-2, and is a human IgG1 antibody comprising a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 63, a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 61, a heavy chain constant region containing YTE mutations and TM mutations, and an IgGλ light chain constant region.

13. The antibody according to claim 12, wherein the heavy chain constant region comprises the amino acid sequence of SEQ ID NO:

66.

14. A composition comprising an antibody or antigen-binding fragment according to any one of claims 1 to 13, further comprising a pharmaceutically acceptable carrier, which is a pharmaceutical composition.

15. The composition according to claim 14, further comprising an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2 and includes VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of Sequence ID Nos. 69, 70, 71, 73, 74, and 75, respectively.

16. The composition according to claim 14, further comprising an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2 and comprises VH containing the amino acid sequence of SEQ ID NO: 68 and VL containing the amino acid sequence of SEQ ID NO:

72.

17. An antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the antibody or antigen-binding fragment comprises (i) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs. 25, 26, 30, 28, 67, and 29, respectively, or (ii) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 of SEQ ID NOs. 13, 14, 19, 16, 17, and 18, respectively.

18. An antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the antibody or antigen-binding fragment is (a) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 40, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 41; (b) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 44, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 45; (c) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 48, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 47; (d) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 49, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 47; (e) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 52, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 53; (f) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 56, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 57; (g) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 58, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 57; (h) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 59, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 55; (i) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 62, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 61; (j) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 60, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 64; (k) A variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 62, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 64; or (l) An antibody or antigen-binding fragment thereof comprising a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 63, and / or a variable light chain (VL) containing the amino acid sequence of SEQ ID NO:

64.

19. Isolated polynucleotides comprising a nucleic acid molecule encoding the heavy chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 13 and / or a nucleic acid molecule encoding the light chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 13.

20. An isolation vector comprising the polynucleotide described in Claim 19.

21. A host cell comprising a first vector comprising a nucleic acid molecule encoding the heavy chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 13, and a second vector comprising a nucleic acid molecule encoding the light chain variable region of an antibody or antigen-binding fragment according to any one of claims 1 to 13.

22. A method for producing an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2, comprising culturing the host cells described in claim 21 such that the nucleic acid molecule is expressed and the antibody or antigen-binding fragment thereof is produced, wherein the method optionally further comprises isolating the antibody or antigen-binding fragment.

23. An antibody or antigen-binding fragment thereof produced by the method described in Claim 22.

24. A composition or combination, (a) A first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or antigen-binding fragment competitively inhibits the binding of an antibody comprising a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 47 to the spike protein of SARS-CoV-2, and A second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or antigen-binding fragment competitively inhibits the binding of an antibody to the SARS-CoV-2 spike protein by (i) an antibody comprising a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 61, or (ii) an antibody comprising a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 53; (b) A first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or antigen-binding fragment binds to the same SARS-CoV-2 spike protein epitope as an antibody comprising a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 47, and A second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or antigen-binding fragment binds to the same SARS-CoV-2 spike protein epitope as an antibody comprising (i) a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 61, or (ii) an antibody comprising a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 53; (c) A first antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or antigen-binding fragment comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, respectively, whose sequence numbers are 13, 14, 15, 16, 17, and 18, and A second antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or antigen-binding fragment comprises (i) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, respectively, whose sequence numbers are 31, 32, 37, 34, 35, and 36, or (ii) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3, respectively, whose sequence numbers are 20, 21, 22, 23, 67, and 24; (d) A first human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or antigen-binding fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 46, a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 47, a heavy chain constant region containing YTE mutations and TM mutations, and a light chain constant region, and A second human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or antigen-binding fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 63, a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 61, a heavy chain constant region containing YTE mutations and TM mutations, and a light chain constant region; (e) A first human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or antigen-binding fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 46, a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 47, a heavy chain constant region containing YTE mutations and TM mutations, and a light chain constant region, and A second human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or antigen-binding fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 52, a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 53, a heavy chain constant region containing YTE mutations and TM mutations, and a light chain constant region; or (f) A first human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or antigen-binding fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 46, a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 47, a heavy chain constant region containing YTE mutations and TM mutations, and a light chain constant region, and A second human IgG1 antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or antigen-binding fragment comprises a variable heavy chain (VH) containing the amino acid sequence of SEQ ID NO: 50, a variable light chain (VL) containing the amino acid sequence of SEQ ID NO: 51, a heavy chain constant region containing YTE mutations and TM mutations, and a light chain constant region. A composition or combination containing the following:

25. An in vitro method for neutralizing SARS-CoV-2, comprising contacting the SARS-CoV-2 with an antibody or antigen-binding fragment according to any one of claims 1 to 13.

26. A pharmaceutical composition for providing pre-exposure prophylaxis against COVID-19 in a subject, comprising the antibody or antigen-binding fragment described in any one of claims 1 to 13.

27. The pharmaceutical composition according to claim 26, wherein the subject is moderate to severe immunosuppression.

28. The pharmaceutical composition according to claim 27, wherein the moderate to severe immunosuppression is due to a medical condition or administration or treatment of an immunosuppressant.

29. The pharmaceutical composition according to claim 26, wherein the subject may not be able to acquire a sufficient immune response to COVID-19 vaccination.

30. The pharmaceutical composition according to claim 26, wherein COVID-19 vaccination is not recommended for the subject.

31. A pharmaceutical composition for the treatment or prevention of SARS-CoV-2 infection in a subject, comprising the antibody or antigen-binding fragment described in any one of claims 1 to 13.

32. The pharmaceutical composition according to claim 26, wherein the pharmaceutical composition is administered by intramuscular administration.

33. The pharmaceutical composition according to claim 26, wherein the subject is a human.

34. The pharmaceutical composition according to claim 31, wherein SARS-CoV-2 is SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2 beta, SARS-CoV-2 delta, and / or SARS-CoV-2 D614G.

35. The pharmaceutical composition according to claim 31, wherein the SARS-CoV-2 is SARS-CoV-2 BA. 2.12.

1.

36. An in vitro method for detecting SARS-CoV-2 in a sample, comprising contacting the sample with an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 13.

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