SARS-COV-2 antibodies and methods of use thereof

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. These antibodies can be used, for example, in the prevention, post-exposure prevention or treatment of SARS-CoV-2 infection. These antibodies may also be used to detect, for example, SARS-CoV-2 infection in a subject.

Description

[0001] 1. Cross-reference to related applications

[0002] This application claims the benefit of priority to U.S. Provisional Application No. 63 / 336,332, filed on April 29, 2022, and U.S. Provisional Application No. 63 / 371,454, filed on August 15, 2022, each of which is incorporated herein by reference in its entirety.

[0003] 2. Reference to a sequence listing submitted electronically

[0004] The contents of the electronically submitted Sequence Listing in the form of an ASCII text file submitted with this application (file name: 2943_218PC02_SequenceListing_ST26; size: 89,789 bytes; creation date: April 24, 2023) are incorporated herein by reference in their entirety. 3. Technical field

[0005] 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 using the same. 4. Background Technology

[0006] A pandemic of coronavirus 2019 (COVID 19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged. SARS-CoV-2 has rapidly caused infection worldwide. The mortality rate of the virus is currently uncertain, but the number of cases and deaths worldwide is alarming. The virus is able to spread from person to person through small droplets expelled from the nose or mouth when an infected person coughs, sneezes, or talks. The incubation period (the time from exposure to symptom onset) ranges from 0 to 24 days, with an average of 3-5 days, but people can be contagious during this period after recovery. Most people infected with SARS-CoV-2 show 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 infected with the virus have mild symptoms, but in some patients, lung infection is severe, leading to severe respiratory distress and even death.

[0007] Several vaccines for the prevention of COVID-19 have been approved, and an antibody combination (Evusheld) is available for pre-exposure prophylaxis, but these are less effective against variants that emerged after its development than against earlier variants. Therefore, there is an urgent need for drugs that can prevent and treat COVID-19, including those cases caused by new viral variants. 5. Summary of the invention

[0008] 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 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 comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having 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, wherein the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 69, 70, 71, 73, 74, and 75, respectively.

[0009] 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 competitively inhibits the binding of 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 to the spike protein of SARS-CoV-2, 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 competitively inhibits the binding of 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 to the spike protein of SARS-CoV-2.

[0010] 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.

[0011] In some aspects, the first antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively. In some aspects, 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.

[0012] In some aspects, the second antibody or antigen-binding fragment thereof comprises a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 69, 70, 71, 73, 74, and 74, respectively. In some aspects, 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 aspects, 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.

[0013] In some aspects, 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 aspects, 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 aspects, 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.

[0014] In some aspects, the composition or combination neutralizes the SARS-CoV-2 virus, optionally wherein 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 aspects, the composition or combination neutralizes SARS-CoV-2 D614G, SARS-CoV-2α, SARS-CoV-2δ+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 viruses with an IC50 of 75 ng / mL or less, or an IC50 of less than 60 ng / mL or less.

[0015] In some aspects, the first antibody or antigen binding fragment is fully human and / or the second antibody or antigen binding fragment is fully human. In some aspects, 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 aspects, 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. In some aspects, 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 aspects, 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 wherein 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 wherein the heavy chain constant region is human IgG1 heavy chain constant region. In some aspects, the first antibody or antigen binding fragment comprises (i) human IgG1 heavy chain constant region and (ii) human IgGκ light chain constant region, and / or the second antibody or antigen binding fragment comprises (i) human IgG1 heavy chain constant region and (ii) human IgGκ light chain constant region.

[0016] In some aspects, 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. In some aspects, 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 IgGκ 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.

[0017] In some aspects, 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.

[0018] In some aspects, 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.

[0019] In some aspects, the first antibody or antigen binding fragment is an antigen binding fragment and / or wherein the second antibody or antigen binding fragment is an antigen binding fragment. In some aspects, the first antigen binding fragment comprises Fab, Fab′, F(ab′)2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, miniantibody, F(ab′)3, tetraantibody, triantibody, double antibody, single domain antibody, (scFv)2 or scFv-Fc and / or the second antigen binding fragment comprises Fab, Fab′, F(ab′)2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, miniantibody, F(ab′)3, tetraantibody, triantibody, double antibody, single domain antibody, (scFv)2 or scFv-Fc.

[0020] In some aspects, the first antibody or Fab is isolated and / or the second antibody or Fab is isolated. In some aspects, the first antibody or Fab is monoclonal and / or the second antibody or Fab is monoclonal. In some aspects, the first antibody or Fab is recombinant and / or the second antibody or Fab is recombinant.

[0021] 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, wherein the first antibody or antigen-binding fragment comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 63, a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 61, a heavy chain constant region and a light chain constant region comprising 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, wherein the second antibody or antigen-binding fragment comprises a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 68, a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 72, a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation. In some aspects, 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 aspects, 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.

[0022] In some aspects, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0023] In some aspects, the first antibody or its antigen binding fragment and the second antibody or its antigen binding fragment are in the same composition, optionally wherein the composition is a pharmaceutical composition. In some aspects, the first antibody or its antigen binding fragment and the second antibody or its antigen binding fragment are in separate compositions, optionally wherein the first antibody or its antigen binding fragment and / or the second antibody or its antigen binding fragment are in a pharmaceutical composition.

[0024] In some aspects, an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2 comprises a VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having the following sequences, respectively: (i) SEQ ID NOs: 31, 32, 37, 34, 35, and 36; (ii) SEQ ID NOs: 25, 26, 30, 28, 67, and 29; or (iii) SEQ ID NOs: 13, 14, 19, 16, 17, and 18.

[0025] In some aspects, 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 an amino acid sequence of SEQ ID NO: 40 and / or a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 41; (b) a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 44 and / or a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 45; (c) a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 48 and / or a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 47; (d) a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 49 and / or a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 47; (e) a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 52 and / or a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 53; (f) a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 54 and / or a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 55. 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: 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; or (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.

[0026] In some aspects, the antibody or antigen-binding fragment neutralizes SARS-CoV-2 pseudovirus. In some aspects, 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-2δ and / or SARS-CoV-2 D614G pseudovirus with an EC50 of 100 ng / mL or less.

[0027] In some aspects, the antibody or antigen-binding fragment is fully human.

[0028] In some aspects, the antibody or antigen-binding fragment comprises a light chain constant region. In some aspects, 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.

[0029] In some aspects, the antibody or antigen binding fragment comprises a heavy chain constant region. In some aspects, 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 aspects, the heavy chain constant region is a human IgG1 heavy chain constant region.

[0030] In some aspects, the antibody or antigen-binding fragment comprises (i) a human IgG1 heavy chain constant region and (ii) a human IgGκ light chain constant region.

[0031] In some aspects, the 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 aspects, the 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. In some aspects, the antibody or antigen binding fragment comprises a heavy chain constant region comprising a YTE mutation and a TM mutation, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region. In some aspects, the antibody or antigen binding fragment comprises a heavy chain constant region comprising an amino acid sequence of SEQ ID NO: 66.

[0032] In some aspects, the antibody or its antigen binding fragment is a full-length antibody. In some aspects, the antibody or its antigen binding fragment is an antigen binding fragment. In some aspects, the antigen binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, mini antibody, F(ab')3, tetrabodies, tribodies, double antibodies, single domain antibodies, (scFv)2 or scFv-Fc.

[0033] In some aspects, the antibody or Fab is isolated. In some aspects, the antibody or Fab is monoclonal. In some aspects, the antibody or Fab is recombinant.

[0034] In some aspects, the antibody or antigen-binding fragment thereof further comprises a detectable label.

[0035] Also provided herein are polynucleotides.

[0036] In some aspects, the isolated polynucleotide comprises a nucleic acid molecule encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof provided herein and / or a nucleic acid molecule encoding a light chain variable region of an antibody or antigen-binding fragment thereof provided herein.

[0037] Also provided herein are vectors, and in some aspects, isolated vectors comprise a polynucleotide provided herein.

[0038] Host cells are also provided herein. In some aspects, the host cell comprises 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 its antigen-binding fragment provided herein and a second vector comprising a nucleic acid molecule encoding a light chain variable region of an antibody or its antigen-binding fragment provided herein.

[0039] Also provided herein are methods for producing antibodies and antigen-binding fragments thereof. In some aspects, the method for producing an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 includes culturing a host cell provided herein so that the nucleic acid molecule is expressed and produces the antibody or antigen-binding fragment thereof. In some aspects, the method further includes separating the antibody or antigen-binding fragment. Also provided herein are antibodies and antigen-binding fragments produced by the methods provided herein.

[0040] Compositions are also provided herein. In some aspects, the composition comprises an antibody or antigen-binding fragment thereof provided herein. In some aspects, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

[0041] Compositions and combinations are also provided herein. In some aspects, the composition or combination comprises: 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 competitively inhibits the binding of an antibody comprising a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 46 and a variable light chain (VL) comprising an 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 thereof competitively inhibits the binding of an antibody comprising (i) a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 63 and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 61 or (ii) a variable heavy chain (VH) comprising an amino acid sequence of SEQ ID NO: 52 and a variable light chain (VL) comprising an amino acid sequence of SEQ ID NO: 53 to the spike protein of SARS-CoV-2.

[0042] In some aspects, the composition or combination comprises: 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: 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, 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 (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.

[0043] In some aspects, the first antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 13, 14, 15, 16, 17, and 18, respectively. In some aspects, 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.

[0044] In some aspects, the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35 and 36, respectively. In some aspects, 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.

[0045] In some aspects, the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 20, 21, 22, 23, 67, and 24, respectively. In some aspects, 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.

[0046] In some aspects, the composition or combination comprises: 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 comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having 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, wherein the second antibody or antigen-binding fragment thereof comprises (i) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively or (ii) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively. VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 of NO: 20, 21, 22, 23, 67 and 24.

[0047] In some aspects, 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.

[0048] In some aspects, 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.

[0049] In some aspects, 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.

[0050] In some aspects, the composition or combination neutralizes SARS-CoV-2 pseudovirus, optionally wherein the composition or combination neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2δ and / or SARS-CoV-2 D614G pseudovirus with an EC50 of 75 ng / mL or less. In some aspects, the composition or combination (i) neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1 and / or SARS-CoV-2 BA.2 pseudovirus with an EC50 of 25 ng / mL or less, and / or (ii) neutralizes SARS-CoV-2δ and / or SARS-CoV-2 D614G pseudovirus with an EC50 of 75 ng / mL or less.

[0051] In some aspects, the first antibody or antigen-binding fragment is fully human and / or the second antibody or antigen-binding fragment is fully human.

[0052] In some aspects, 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 aspects, 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 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 human IgGκ light chain constant region.

[0053] In some aspects, the first antibody or Fab comprises a heavy chain constant region and / or the second antibody or Fab comprises a heavy chain constant region. In some aspects, the first antibody or Fab 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 wherein the heavy chain constant region is human IgG1 and / or the second antibody or Fab 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 wherein the heavy chain constant region is human IgG1 heavy chain constant region.

[0054] In some aspects, 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.

[0055] In some aspects, 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.

[0056] In some aspects, 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 IgGκ 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.

[0057] In some aspects, 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.

[0058] In some aspects, 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 aspects, 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 aspects, the first antigen binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, mini antibody, F(ab')3, tetra antibody, tri antibody, double antibody, single domain antibody, (scFv)2 or scFv-Fc and / or the second antigen binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, mini antibody, F(ab')3, tetra antibody, tri antibody, double antibody, single domain antibody, (scFv)2 or scFv-Fc.

[0059] In some aspects, the first antibody or Fab is isolated and / or the second antibody or Fab is isolated. In some aspects, the first antibody or Fab is monoclonal and / or the second antibody or Fab is monoclonal. In some aspects, the first antibody or Fab is recombinant and / or the second antibody or Fab is recombinant.

[0060] In some aspects, the composition or combination comprises: a first human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation; and a second human IgG1 antibody or an 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) 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 a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation.

[0061] In some aspects, the composition or combination comprises: a first human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation; and a second human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation.

[0062] In some aspects, the composition or combination comprises: a first human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation; and a second human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation.

[0063] In some aspects, 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.

[0064] In some aspects, the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

[0065] In some aspects of the combinations provided herein, the first antibody or its antigen binding fragment and the second antibody or its antigen binding fragment are in the same composition, optionally wherein the composition is a pharmaceutical composition. In some aspects, the first antibody or its antigen binding fragment and the second antibody or its antigen binding fragment are in separate compositions, optionally wherein the first antibody or its antigen binding fragment and / or the second antibody or its antigen binding fragment are in a pharmaceutical composition.

[0066] Also provided herein are methods for neutralizing SARS-CoV-2. In some aspects, the method for neutralizing SARS-CoV-2 comprises contacting SARS-CoV-2 with an antibody or antigen-binding fragment thereof provided herein or a composition or combination provided herein. In some aspects, the contacting is performed in vitro. In some aspects, the contacting is in vivo in a subject.

[0067] Also provided herein is a method for pre-exposure prophylaxis of SARS-CoV-2. In some aspects, a method for providing pre-exposure prophylaxis of SARS-CoV-2 in a subject includes administering to the subject an effective amount of a composition or combination provided herein. In some aspects, the subject is not currently infected with SARS-CoV-2. In some aspects, the subject is known to have not been recently exposed to an individual infected with SARS-CoV-2. In some aspects, the subject has moderate to severe immunocompromise. In some aspects, moderate to severe immunocompromise is due to a medical condition or receiving immunosuppressive drugs or treatment. In some aspects, the subject may not produce sufficient immune response to COVID-19 vaccination. In some aspects, COVID-19 vaccination is not recommended for the subject. In some aspects, 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 aspects, the first antibody or its antigen-binding fragment and the second antibody or its antigen-binding fragment are administered sequentially.

[0068] Also provided herein is a method for treating or preventing SARS-CoV-2 infection. In some aspects, the method for treating or preventing SARS-CoV-2 infection in a subject includes administering to the subject an effective amount of a combination provided herein, wherein the first antibody or its antigen binding fragment and the second antibody or its antigen binding fragment are administered sequentially. In some aspects, the first antibody or its antigen binding fragment is administered before the second antibody or its antigen binding fragment is administered. In some aspects, the first antibody or its antigen binding fragment is administered after the second antibody or its antigen binding fragment is administered. In some aspects, the first antibody or its antigen binding fragment is administered intramuscularly and / or the second antibody or its antigen binding fragment is injected intramuscularly.

[0069] In some aspects, the method of 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 aspects, the method of 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 aspects, the administration is intramuscular.

[0070] In some aspects of the methods provided herein, the subject has been exposed to or is at risk of being exposed to SARS-CoV-2.

[0071] In some aspects of the methods provided herein, the subject is a human.

[0072] In some aspects of the methods provided herein, SARS-CoV-2 is SARS-CoV-2 BA.1, SARS-CoV-2BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2β, SARS-CoV-2δ and / or SARS-CoV-2 D614G. In some aspects of the methods provided herein, SARS-CoV-2 is SARS-CoV-2 BA.2.12.1.

[0073] Also provided herein are methods for detecting SARS-CoV-2. In some aspects, 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. 6. Description of the drawings

[0074] Figure 1 Neutralization of SARS-CoV-2 pseudovirus by RQ33, RQ40, RQ41 and RQ43 antibodies was shown. (See Example 17.)

[0075] Figure 2 Neutralization of SARS-CoV-2 pseudovirus by antibody combinations was shown. (See Example 17.)

[0076] Figure 3 In vitro neutralization 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 as determined by plaque reduction neutralization assay (FRNT) is shown. The assay was repeated in two independent replicates, each with two replicates. The nonlinear regression dose-response curves depict the mean and standard deviation of two replicates at each dilution. mAb, monoclonal antibody; VOC, variant of interest (see Example 18.)

[0077] Figure 4 In vitro neutralization curves of pseudoviruses bearing SARS-CoV-2 omicron BA.2.12.1 VOC spikes by pseudovirus neutralization assay are shown. The assay was repeated in three independent replicates, each with three replicates. Nonlinear regression dose-response curves depict representative replicates with mean and standard deviation at each dilution. mAb, monoclonal antibody; VOC, variant of interest (see Example 18.)

[0078] Figure 5 Sequential binding of segavir and RQ43-GL-H-LO1 to the SARS-CoV-2 spike protein receptor binding domain is shown. Biolayer interferometry was performed to assess simultaneous binding of segavir, RQ43-GL-H-LO1, and CR3022 (non-competitive control mAb) to the SARS-CoV-2 spike RBD. Binding signals (nm) are shown for mAb1 binding (black), followed by additions of buffer (no visible signal), CR3022 (grey), segavir (slashed stripes in the left panel), or RQ43-GL-H-LO1 (slashed stripes in the right panel). Binding signals are averaged over 10 seconds of data points at equilibrium for each binding step. mAbs were considered bound if the increase in binding signal was greater than 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.)

[0079] Figure 6 Linear depictions of the sigmavir binding site (top) and RQ43 binding site (bottom) mapped to the SARS-CoV-2 RBD are provided. The sigmavir and RQ43 binding sites are discontinuous, indicated by boxes and underlined letters, while residues outside of the respective binding sites are not underlined. (See Example 18.) 7. Specific implementation methods

[0080] Provided herein are antibodies (e.g., monoclonal antibodies) that specifically bind to the spike protein of SARS-CoV-2 and antigen-binding fragments thereof, and methods of using the same.

[0081] 7.1 Terminology

[0082] The term "antibody" means an immunoglobulin molecule that recognizes and specifically binds to a target (such as a protein, polypeptide, peptide, carbohydrate, polynucleotide, lipid or a combination of the foregoing) by at least one antigen recognition site in the variable region of an immunoglobulin molecule. As used herein, the term "antibody" encompasses complete polyclonal antibodies, complete monoclonal antibodies, chimeric antibodies, humanized antibodies, human antibodies, fusion proteins comprising antibodies, and any other modified immunoglobulin molecules, as long as these antibodies exhibit the desired biological activity. The antibody can be any of the following five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, or its subclass (isotype) (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2), which are referred to as α, δ, ε, γ and μ based on the characteristics of their heavy chain constant domains. Different classes of immunoglobulins have different and well-known subunit structures and three-dimensional configurations. Antibodies can be naked or conjugated to other molecules such as toxins, radioisotopes, etc.

[0083] The term "antibody fragment" refers to a portion of a complete antibody. "Antigen binding fragment", "antigen binding domain" or "antigen binding region" refers to a portion of a complete antibody that binds to an antigen. The antigen binding fragment may contain the antigenic determining region (e.g., complementary determining region (CDR)) of the complete antibody. 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 produced.

[0084] The terms "anti-SARS-CoV-2 spike protein antibody", "SARS-CoV-2 spike protein antibody", and "antibody that binds to the SARS-CoV-2 spike protein" are used interchangeably herein to refer to an antibody that is capable of binding to the spike protein of SARS-CoV-2 with sufficient affinity such that the antibody can be used as a diagnostic and / or therapeutic agent in targeting SARS-CoV-2. The extent of binding of the SARS-CoV-2 spike protein antibody to an unrelated, non-SARS-CoV-2 spike protein can be less than about 10% of the extent of binding of the antibody to the SARS-CoV-2 spike protein, as measured, for example, using ForteBio or Biacore.

[0085] "Monoclonal" antibody or its antigen-binding fragment refers to a homologous antibody or antigen-binding fragment group that is involved in the highly specific recognition and binding of a single antigenic determinant or epitope. This is in contrast to polyclonal antibodies that typically include different antibodies against different antigenic determinants. The term "monoclonal" antibody or its antigen-binding fragment encompasses complete and full-length monoclonal antibodies as well as antibody fragments (such as Fab, Fab', F(ab')2, Fv), single-chain (scFv) mutants, fusion proteins comprising antibody portions, and any other modified immunoglobulin molecules comprising antigen recognition sites. In addition, "monoclonal" antibody or its antigen-binding fragment refers to such antibodies and their antigen-binding fragments prepared in any number of ways (including but not limited to by hybridoma, phage selection, recombinant expression, and transgenic animals).

[0086] As used herein, the terms "variable region" or "variable domain" are used interchangeably and are common in the art. Variable region generally refers to a part of an antibody, typically a part of a light chain or a heavy chain, typically about 110 to 120 amino acids or 110 to 125 amino acids at the amino terminus in a mature heavy chain and about 90 to 115 amino acids in a mature light chain, the sequence of which is widely different between antibodies and is used for the binding and specificity of a particular antibody to its specific antigen. The variability of the sequence is concentrated in those regions referred to as complementary determining regions (CDRs), while the regions with higher conservatism in the variable domains are referred to as framework regions (FRs). It is not desirable to be bound by any particular mechanism or theory, and it is believed that the CDRs of light and heavy chains are primarily responsible for the interaction and specificity of antibodies with antigens. In some respects, the variable region is a human variable region. In some respects, the variable region comprises rodent or murine CDRs and human framework regions (FRs). In some respects, the variable region is a primate (e.g., non-human primate) variable region. In some aspects, the variable regions comprise rodent or murine CDRs and primate (eg, non-human primate) framework regions (FRs).

[0087] As used herein, the term "complementarity determining region" or "CDR" refers to each region of an antibody variable domain that is hypervariable in sequence and / or forms structurally defined loops (hypervariable loops) and / or contains residues that contact the antigen. An antibody may contain six CDRs, e.g., three in VH and three in VL.

[0088] The terms "VL" and "VL domain" are used interchangeably to refer to the light chain variable region of an antibody.

[0089] The terms "VH" and "VH domain" are used interchangeably to refer to the heavy chain variable region of an antibody.

[0090] The term "Kabat numbering" and similar terms are well-known 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 its antigen-binding fragment. In some aspects, CDRs can be determined according to the Kabat numbering system (see, e.g., Kabat EA and Wu TT (1971) Ann NY Acad Sci [Annuals of the New York Academy of Sciences] 190: 382-391 and Kabat EA et al., (1991) Sequences of Proteins of Immunological Interest [immunology-related protein sequences], Fifth Edition, U.S. Department of Health and Human Services (USDepartment of Health and Human Services), NIH Publication No. 91-3242). Using the Kabat numbering system, CDRs within an antibody heavy chain molecule are typically present at amino acid positions 31 to 35 (optionally one or two additional amino acids after position 35 may be included (referred to as 35A and 35B in the Kabat numbering scheme)) (CDR1), amino acid positions 50 to 65 (CDR2), and amino acid positions 95 to 102 (CDR3). Using the Kabat numbering system, CDRs within an antibody light chain molecule generally occur at amino acid positions 24 to 34 (CDR1), amino acid positions 50 to 56 (CDR2), and amino acid positions 89 to 97 (CDR3).

[0091] In contrast, Chothia refers to the location of structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The ends of the Chothia CDR-H1 loop vary between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion 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; 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.

[0092]

[0093] As used herein, the terms "constant region" or "constant domain" are interchangeable and have their common meanings in the art. The constant region is a portion of an antibody, such as the carboxyl terminal portion of a light chain and / or a heavy chain, which is not directly involved in the binding of the antibody to an antigen, but can exhibit various effector functions, such as interactions with Fc receptors. Relative to immunoglobulin variable domains, the constant region of an immunoglobulin molecule typically has a more conservative amino acid sequence. In some aspects, an antibody or antigen-binding fragment comprises a constant region or a portion thereof sufficient for antibody-dependent cell-mediated cytotoxicity (ADCC).

[0094] As used herein, the term "heavy chain" when used to refer to an antibody can refer to any different types based on the amino acid sequence of the constant domain, such as alpha (α), delta (δ), epsilon (ε), gamma (γ) and mu (μ), which produce IgA, IgD, IgE, IgG and IgM class antibodies, respectively, including IgG subclasses, such as IgG1, IgG2, IgG3 and IgG4. Heavy chain amino acid sequences are well known in the art. In some aspects, the heavy chain is a human heavy chain.

[0095] As used herein, the term "light chain" when used to refer to an antibody can refer to any of the different types based on the amino acid sequence of the constant domain, for example, kappa (κ) or lambda (λ). Light chain amino acid sequences are well known in the art. In some aspects, the light chain is a human light chain.

[0096] The term "humanized" antibody or antigen-binding fragment thereof refers to forms of non-human (e.g., murine) antibodies or antigen-binding fragments that are specific immunoglobulin chains or fragments thereof that contain minimal non-human (e.g., murine) sequences. Typically, a humanized antibody or antigen-binding fragment thereof is a human immunoglobulin in which residues from a complementarity determining region (CDR) are replaced with residues from a CDR of a non-human species (e.g., mouse, rat, rabbit or hamster) having the desired specificity, affinity and capacity ("CDR grafting") (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, the Fv framework region (FR) residues of human immunoglobulin are replaced by the corresponding residues in the antibody or fragment with desired specificity, affinity and ability from non-human species. Humanized antibody or its Fab can be further modified by replacing other residues in the Fv framework region and / or the non-human residues replaced, to improve and optimize antibody or its Fab specificity, affinity and / or ability. Usually, humanized antibody or its Fab will comprise substantially all at least one (and typically two or three) variable domains, which comprise all or substantially all CDR regions corresponding to non-human immunoglobulins, and all or substantially all FR regions are those of human immunoglobulin consensus sequences. Humanized antibody or its Fab can also comprise immunoglobulin constant region or domain (Fc), typically at least a portion of a constant region or domain of a human immunoglobulin. Examples of methods for producing 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 aspects, a "humanized antibody" is a resurfaced antibody.

[0097] The term "human" or "fully human" antibody or antigen-binding fragment thereof means an antibody or antigen-binding fragment thereof having an amino acid sequence derived from a human immunoglobulin locus, wherein such antibody or antigen-binding fragment is prepared 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.

[0098] "Binding affinity" generally refers to the strength of the sum 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 indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity that reflects 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 can generally be expressed in terms of the dissociation constant (K D Affinity can be measured and / or expressed in a variety of ways known in the art, including but not limited to the equilibrium dissociation constant (K D ) and equilibrium association constant (K A ). D According to k off / k on The quotient of is calculated, and K A According to k on / k off k on refers to the association rate constant of, for example, an antibody or its antigen-binding fragment with an antigen, while k off Refers to, for example, the dissociation of an antibody or its antigen-binding fragment from an antigen. on and k off It can be determined by techniques known to those of ordinary skill in the art, such as or KinExA.

[0099] As used herein, "epitope" is a term in the art and refers to a local region in an antigen to which an antibody or its Fab can specifically bind. An epitope can be, for example, a continuous amino acid (linear or continuous epitope) of a polypeptide, or an epitope can be, for example, from two or more discontinuous regions (conformational, nonlinear, discontinuous or discontinuous epitopes) of one or more polypeptides together. In some aspects, the epitope that an antibody or its Fab is combined can be, for example, by NMR spectroscopy, X-ray diffraction crystallography, ELISA assay, hydrogen / deuterium exchange combined mass spectrometry (for example, liquid chromatography-electrospray mass spectrometry), array-based oligopeptide scanning assay and / or mutagenesis mapping (for example, site-directed mutagenesis mapping). For X-ray crystallography, crystallization can be accomplished using any method known in the art (e.g., Giege R et al., (1994) Acta Crystallogr D Biol Crystallogr [Acta Crystallographica Sinica, Series D: Biological Crystallography] 50(Pt 4): 339-350; McPherson A (1990) Eur J Biochem [European Journal of Biochemistry] 189: 1-23; Chayen NE (1997) Structure [Structure] 5: 1269-1274; McPherson A (1976) J Biol Chem [Journal of Biological Chemistry] 251: 6300-6303). Antibodies / antigen-binding fragments thereof: Antigen crystals can be studied using well-known X-ray diffraction techniques and can be improved using computer software, such as X-PLOR (Yale University, 1992, published by Molecular Simulations, Inc.; see, e.g., Meth Enzymol (1985) Vols. 114 and 115, ed. Wyckoff HW et al.; US2004 / 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 50(Pt 2): 37-60; Bricogne G (1997) Meth Enzymol 51(Pt 3): 361-423, ed. Carter CW; Roversi P et al., (2000) Acta Crystallogr D Biol Crystallogr 60(Pt 1): 37-60; Bricogne G (1997) Meth Enzymol 6 ... Crystallogr [Acta Crystallogrica Sinica, Series D: Biological Crystallography] 56(Pt 10): 1316-1323). Mutagenesis mapping studies can be performed using any method known to those skilled in the art.For a description of mutagenesis techniques, including alanine scanning mutagenesis, see, for example, Champe M et al. (1995) J Biol Chem 270: 1388-1394 and Cunningham BC and Wells JA (1989) Science 244: 1081-1085.

[0100] An antibody that "binds to the same epitope" as a reference antibody is 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 assays (see, e.g., Coales et al. Rapid Commun. Mass Spectrom. 2009; 23: 639-647).

[0101] As used herein, the terms "immunospecific binding," "immunospecific recognition," "specific binding," and "specific recognition" 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 the binding requires some complementarity between the antigen-binding domain and the epitope. Thus, in some aspects, an antibody that "specifically binds" to the spike protein of SARS-CoV-2 may also bind to spike proteins of one or more related viruses (e.g., SARS-1) and / or may also bind to variants of the SARS-CoV-2 spike protein, 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 SARS-CoV spike protein, as measured, for example, using ForteBio or Biacore.

[0102] An antibody is said to "competitively inhibit" binding of a reference antibody to a given epitope if the antibody preferentially binds to that epitope or an overlapping epitope to the extent that it blocks binding of the reference antibody to the epitope to some extent. 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 a reference antibody to a given epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.

[0103] "Isolated" polypeptide, antibody, polynucleotide, vector, cell or composition is a polypeptide, antibody, polynucleotide, vector, cell or composition that is not found in nature. Isolated polypeptide, antibody, polynucleotide, vector, cell or composition include those that have been purified to the extent that they are no longer in the form found in nature. In some aspects, the isolated antibody, polynucleotide, vector, cell or composition is substantially pure. As used herein, "substantially pure" refers to at least 50% pure (i.e., free of contaminants), at least 90% pure, at least 95% pure, at least 98% pure or at least 99% pure material.

[0104] The terms "polypeptide," "peptide," and "protein," used interchangeably herein, refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. The definition also includes, for example, polypeptides containing one or more amino acid analogs (including, for example, non-natural amino acids, etc.), as well as other modifications known in the art. It should be understood that because the polypeptides of the present invention are antibody-based, in some aspects, the polypeptides may appear as single chains or related chains.

[0105] "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 comparing two sequences and introducing spaces to maximize the identity between the sequences. Comparisons can be generated using programs known in the art. For purposes herein, comparisons of nucleotide sequences can be performed using the blastn program set to default parameters, while comparisons of amino acid sequences can be performed using the blastp program set to default parameters (see National Center for Biotechnology Information (NCBI) on the World Wide Web, ncbi.nlm.nih.gov).

[0106] As used herein, amino acids with hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), valine (V), phenylalanine (F), tryptophan (W), and tyrosine (Y). Amino acids with aliphatic hydrophobic side chains include alanine (A), isoleucine (I), leucine (L), methionine (M), and valine (V). Amino acids with aromatic hydrophobic side chains include phenylalanine (F), tryptophan (W), and tyrosine (Y).

[0107] As used herein, amino acids having polar neutral side chains include asparagine (N), cysteine ​​(C), glutamine (Q), serine (S), and threonine (T).

[0108] As used herein, amino acids with charged side chains include aspartic acid (D), glutamic acid (E), arginine (R), histidine (H), and lysine (K). Amino acids with charged acidic side chains include aspartic acid (D) and glutamic acid (E). Amino acids with charged basic side chains include arginine (R), histidine (H), and lysine (K).

[0109] As used herein, the term "host cell" can be any type of cell, such as a primary cell, a cell in culture, or a cell from a cell line. In some aspects, the term "host cell" refers to a cell 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 during successful passage or integration of the nucleic acid molecule into the host cell genome.

[0110] The term "pharmaceutical formulation" or "pharmaceutical composition" refers to a preparation that is in such form as to permit the biological activity of the active ingredient to be effective and contains no additional components that are unacceptably toxic to a subject to which the formulation would be administered. The formulation may be sterile.

[0111] As provided herein, a "combination" of antibodies or antigen-binding fragments thereof refers to two or more antibodies or antigen-binding fragments thereof that are used together. The antibodies or antigen-binding fragments thereof in the combination can be included together in a single pharmaceutical composition or can be included in separate pharmaceutical compositions. The antibodies or antigen-binding fragments of the combination in separate pharmaceutical compositions can be administered simultaneously or sequentially.

[0112] As used herein, the terms "administering" and the like refer to methods (e.g., intravenous administration) that can be used to enable delivery of a drug (e.g., an antibody or antigen-binding fragment thereof that specifically binds to the SARS-CoV-2 spike protein) to a desired site of biological action. Administration techniques that can be used with the agents and methods described herein can be found, 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.

[0113] As used herein, the terms "subject" and "patient" are used interchangeably. The subject can be an animal. In some aspects, the subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey or other primate, etc.). In some aspects, the subject is a human.

[0114] The term "therapeutically effective amount" refers to an amount of a drug (eg, one or more antibodies or antigen-binding fragments thereof) that is effective to treat a disease or condition in a subject.

[0115] Terms such as "treating or treatment or to treat" or "alleviating or toalleviate" refer to therapeutic measures that cure, slow down, relieve the symptoms of, and / or stop the progression of a diagnosed pathological condition or disorder. Therefore, those patients in need of treatment include those who have been diagnosed with or suspected of having the disorder. Patients or subjects in need of treatment can include patients diagnosed with coronavirus 2019 (COVID 19) and patients infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0116] Alternatively, the pharmacological and / or physiological effects may be preventive, i.e., the effects prevent the disease or its symptoms in whole or in part. In this regard, the disclosed methods include administering a "preventive effective amount" of a drug (e.g., one or more antibodies or their antigen binding fragments). "Preventive effective amount" refers to an amount that is effective to achieve the desired preventive outcome (e.g., preventing SARS-CoV-2 infection or disease onset) at the necessary dosage for the necessary time period.

[0117] As used in the disclosure and claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0118] It should be understood that wherever the language "comprising" is used herein to describe aspects, other similar aspects described with respect to "consisting of" and / or "consisting essentially of" are also provided. In the present disclosure, "comprises or comprising", "containing", and "having" and the like may mean "includes or including" and the like; "consisting essentially of or consists essentially of" is open-ended, allowing for existence beyond what is recited, as long as the basic or novel features recited are not altered by existence beyond what is recited, but excluding prior art aspects.

[0119] Unless expressly stated or obvious from the context, as used herein, the term "or" is understood to be inclusive. The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B." Similarly, the term "and / or" as used in phrases such as "A, B, and / or C" is intended to cover each of the following: 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).

[0120] As used herein, the terms "about" and "approximately" when used to modify a numerical value or numerical range indicate that deviations of 10% above and 10% below the numerical value or range are still within the intended meaning of the recited value or range. It should be understood that wherever the language "about" or "approximately" numerical values ​​or ranges are used herein to describe various aspects, similar aspects referring to specific numerical values ​​or ranges (without "about") are also provided.

[0121] Any composition or method provided herein can be combined with one or more of any other compositions and methods provided herein.

[0122] 7.2 Antibodies and Antigen-binding Fragments

[0123] In certain aspects, antibodies (e.g., monoclonal antibodies, such as human antibodies) and antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 are provided herein. The amino acid sequence of the SARS-CoV-2 spike protein is provided in SEQ ID NO: 1:

[0124]

[0125]

[0126] 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 contains 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.

[0127] 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.

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

[0129] Table 1. Antibody sequences with Kabat-defined CDRs

[0130]

[0131]

[0132]

[0133]

[0134]

[0135]

[0136]

[0137] 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 of the antibodies listed in Table 1. In some aspects, the antibodies or antigen-binding fragments thereof described herein bind to the spike protein of SARS-CoV-2 and comprise the VL of the antibodies listed in Table 1.

[0138] 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 the antibodies listed in 1 (i.e., the VH of an antibody and the VL of the same antibody).

[0139] In some aspects, the antibodies or antigen-binding fragments thereof described herein can be described by their VL domain alone or their VH domain alone, or by their three VL CDRs alone or their three VH CDRs alone. See, e.g., Rader C et al., (1998) PNAS [Proceedings of the National Academy of Sciences of the United States of America] 95:8910-8915, which is incorporated herein by reference in its entirety, which describes the humanization of mouse anti-αvβ3 antibodies by identifying complementary light or heavy chains from human light or heavy chain libraries, respectively, to produce humanized antibody variants with affinities as high as or higher than the affinity of the original antibody. See also Clackson T et al., (1991) Nature [Nature] 352:624-628, which is incorporated herein by reference in its entirety, which describes methods for producing antibodies that bind to a specific antigen by using a specific VL domain (or VH domain) and screening the library for complementary variable domains. The screening yielded 14 new partners for specific VH domains and 13 new partners for specific VL domains that were strong binders as determined by ELISA. See also Kim SJ and Hong HJ, (2007) J Microbiol [Microbiology Journal] 45: 572-577, which is incorporated herein by reference in its entirety, describing a method for generating antibodies that bind to a specific antigen by using a specific VH domain and screening a library (e.g., a human VL library) for a complementary VL domain; the selected VL domain can in turn be used to guide the selection of additional complementary (e.g., human) VH domains.

[0140] In some aspects, the CDRs of an antibody or antigen-binding fragment thereof can be identified according to the Chothia numbering scheme, which refers to the positions of immunoglobulin structural loops (see, e.g., Chothia C and Lesk AM, (1987), J Mol Biol [Journal of Molecular Biology] 196:901-917; Al-Lazikani B et al., (1997) J Mol Biol [Journal of Molecular Biology] 273:927-948; Chothia C et al., (1992) J Mol Biol [Journal of Molecular Biology] 227:799-817; Tramontano A et al., (1990) J Mol Biol [Journal of Molecular Biology] 215(1):175-82; and U.S. Pat. No. 7,709,226). Typically, when using the Kabat numbering convention, the Chothia CDR-H1 loop occurs at heavy chain amino acids 26 to 32, 33, or 34, the Chothia CDR-H2 loop occurs at heavy chain amino acids 52 to 56, and the Chothia CDR-H3 loop occurs at heavy chain amino acids 95 to 102, while the Chothia CDR-L1 loop occurs at light chain amino acids 24 to 34, the Chothia CDR-L2 loop occurs at light chain amino acids 50 to 56, and the Chothia CDR-L3 loop occurs at light chain amino acids 89 to 97. The ends of the Chothia CDR-H1 loop vary between H32 and H34 when numbered using the Kabat numbering convention, depending on the length of the loop (this is because the Kabat numbering scheme places the insertion at H35A and H35B; if both 35A and 35B are absent, the loop ends at 32; if only 35A is present, the loop ends at 33; if both 35A and 35B are present, the loop ends at 34).

[0141] In some aspects, 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 are provided herein. In some aspects, 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 aspects, antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and comprise a combination of Kabat CDRs and Chothia CDRs are provided herein.

[0142] In some aspects, 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 to 35, VH-CDR2 is at positions 51 to 57, VH-CDR3 is at positions 93 to 102, VL-CDR1 is at positions 27 to 32, VL-CDR2 is at positions 50 to 52, and VL-CDR3 is at positions 89 to 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 comprise 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).

[0143] In some aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to MacCallum RM et al., (1996) J Mol Biol [Journal of Molecular Biology] 262: 732-745. See also, for example, Martin A. "Protein Sequence and Structure Analysis of Antibody Variable Domains," in Antibody Engineering [Antibody Engineering], Kontermann and Dübel, eds., Chapter 31, pp. 422-439, Springer-Verlag, Berlin (2001). In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and comprise the VH and VL CDRs of the antibodies listed in Table 1 as determined by the method of MacCallum RM et al.

[0144] In some aspects, the CDRs of an antibody or antigen-binding fragment thereof can be determined according to the AbM numbering scheme, which refers to the AbM hypervariable regions that represent a compromise between Kabat CDRs and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software (Oxford Molecular Group, Inc.). In some aspects, provided herein are antibodies and antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 and comprise the VH and VL CDRs of the antibodies listed in Table 1 as determined by the AbM numbering scheme.

[0145] In some aspects, provided herein are antibodies comprising heavy chains and / or light chains. Non-limiting examples of human constant region sequences have been described in the art, for example, see U.S. Pat. No. 5,693,780 and Kabat EA et al., (1991) supra.

[0146] Regarding the heavy chain, in some aspects, the heavy chain of the antibodies described herein may be an alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some aspects, the heavy chain of the antibody may comprise a human alpha (α), delta (δ), epsilon (ε), gamma (γ) or mu (μ) heavy chain. In some aspects, 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 the amino acid sequence listed in Table 1 and wherein the constant region of the heavy chain comprises the amino acid sequence of a human gamma (γ) heavy chain constant region (e.g., a human IgG1 heavy chain constant region). In some aspects, 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 the sequence listed in Table 1, and wherein the constant region of the heavy chain comprises the amino acids of a human heavy chain described herein or known in the art.

[0147] As known in the art, the heavy chain C-terminal lysine can be removed, for example, in cell culture. Although the C-terminal lysine is conservative in the heavy chain genes of all human IgG subclasses (i.e., IgG1, IgG2, IgG3, and IgG4), this residue is not usually present in the IgG found in serum. It is also known that the C-terminal lysine is one of the most commonly detected product charge heterogeneity sources in cell culture processes from antibody processing and cutting. The lysine residue at the C-terminal end of the heavy chain of recombinant IgG can be removed by endogenous carboxypeptidases of mammalian host cells during cell culture. Therefore, in some aspects, the antibody or antigen-binding fragment provided herein comprises a heavy chain with a C-terminal lysine. In some aspects, the antibody or antigen-binding fragment provided herein comprises a heavy chain without a C-terminal lysine.

[0148] In some aspects, 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 aspects, 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 listed in Table 1, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa or lambda light chain constant region.

[0149] In some aspects, the antibodies or antigen-binding fragments thereof 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 listed in Table 1, and wherein the constant region of the light chain comprises the amino acid sequence of a human kappa light chain constant region. The amino acid sequence of the human kappa light chain constant region may comprise the following amino acid sequence

[0150]

[0151] In some aspects, the light chain of the antibodies described herein is a lambda light chain. In some aspects, 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 listed in Table 1, and wherein the constant region of the light chain comprises the amino acid sequence of a human lambda light chain constant region. The amino acid sequence of a human kappa light chain constant region may comprise the following amino acid sequence

[0152]

[0153] In some aspects, 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 of the amino acid sequences described herein, and wherein the constant region comprises an amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule or a human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule. In some aspects, 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 of the amino acid sequences described herein, and wherein the constant region comprises an amino acid sequence of a constant region of an IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecule, any class of immunoglobulin molecules (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or any subclass (e.g., IgG2a and IgG2b). In some aspects, the constant region comprises the amino acid sequence of a constant region of any class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2) or any subclass (e.g., IgG2a and IgG2b) of human IgG, IgE, IgM, IgD, IgA, or IgY immunoglobulin molecules.

[0154] Fc region engineering is used in the art, for example, to extend the half-life of therapeutic antibodies and their antigen-binding fragments and to avoid degradation in vivo. In some aspects, the Fc region of an IgG antibody or antigen-binding fragment can be modified to increase the affinity of IgG molecules to neonatal Fc receptors (FcRn), thereby mediating IgG catabolism and avoiding IgG molecule degradation. Suitable Fc region amino acid substitutions or modifications are known in the art and include, for example, tri-substituted M252Y / S254T / T256E (referred to as "YTE"), numbered according to the EU index as in Kabat (see, for example, U.S. Patent No. 7,658,921; U.S. Patent Application Publication No. 2014 / 0302058; and Yu et al., Antimicrob. Agents Chemother. [Antimicrobial Preparations and Chemotherapy], 61 (1): e01020-16 (2017)). In some aspects, 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 containing a YTE mutation.

[0155] The triple mutation (TM) L234F / L235E / P331S in the heavy chain constant region (according to the EU numbering convention; Sazinsky et al. Proc Natl Acad Sci USA, 105:20167-20172 (2008)) can significantly reduce IgG effector function.

[0156] In some aspects, an IgG1 CH1-CH3 sequence comprising a YTE mutation and a TM mutation comprises the following amino acid sequence:

[0157]

[0158] In some aspects, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of an antibody or antigen-binding fragment thereof described herein (e.g., into 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 (EU index in Kabat)) 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.

[0159] In some aspects, one, two or more mutations (e.g., amino acid substitutions) are introduced into the hinge region of the Fc region (CH1 domain) such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased), as described, e.g., in U.S. Pat. No. 5,677,425. The number of cysteine ​​residues in the hinge region of the CH1 domain may be altered, e.g., to facilitate assembly of light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody or its antigen-binding fragment.

[0160] In some aspects, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (e.g., CH2 domain (residues 231-340 of human IgG1) and / or CH3 domain (residues 341-447 of human IgG1) and / or hinge region, numbered according to the Kabat numbering system (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 on the surface of an effector cell (e.g., an activating Fc receptor). Mutations in the Fc region that decrease or increase affinity for an Fc receptor and techniques for introducing such mutations into an Fc receptor or fragment thereof are known to those of skill in the art. Examples of mutations that can be made in Fc receptors to 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 [Proceedings of the National Academy of Sciences of the United States of America] 109: 6181-6186, U.S. Pat. No. 6,737,056, and International Publication Nos. WO 02 / 060919, WO 98 / 23289, and WO 97 / 34631, which are incorporated herein by reference.

[0161] In some aspects, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into IgG constant domains or their FcRn binding fragments (preferably Fc or hinge-Fc domain fragments) to change (e.g., reduce or increase) the half-life of the antibody or its antigen-binding fragment in vivo. See, e.g., International Publication No. 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, which will change (e.g., reduce or increase) the half-life of the antibody or its antigen-binding fragment in vivo. In some aspects, one, two or more amino acid mutations (i.e., substitutions, insertions or deletions) are introduced into IgG constant domains or their FcRn binding fragments (preferably Fc or hinge-Fc domain fragments) to reduce the half-life of the antibody or its antigen-binding fragment in vivo. In some aspects, one, two or more amino acid mutations (i.e., substitution, insertion or deletion) are introduced into IgG constant domains or its FcRn binding fragments (preferably Fc or hinge-Fc domain fragments) to increase the half-life of the antibody or its Fab in vivo. In some aspects, the antibody or its Fab can have one or more amino acid mutations (e.g., substitution) 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), according to the EU index numbering in Kabat (Kabat EA et al., (1991) the same). In some aspects, according to the EU index numbering as in Kabat, the constant region of IgG1 is included in the substitution of methionine (M) at position 252 to tyrosine (Y), serine (S) at position 254 to threonine (T), and threonine (T) at position 256 to glutamic acid (E). See U.S. Pat. No. 7,658,921, which is incorporated herein by reference. This type of mutant IgG (referred to as a "YTE mutant") has been shown to exhibit a four-fold increased half-life compared to the wild-type form of the same antibody (see Dall'Acqua WF et al., (2006) J Biol Chem [Journal of Biological Chemistry] 281: 23514-24). In some aspects, the IgG constant domains comprised by the antibody or antigen-binding fragment thereof comprise one, two, three or more amino acid substitutions at amino acid residues at positions 251-257, 285-290, 308-314, 385-389 and 428-436 numbered according to the EU index as in Kabat.

[0162] In some aspects, one, two or more amino acid substitutions are introduced into the IgG constant domain Fc region to change one or more effector functions of the antibody or its antigen-binding fragment. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322, which are numbered according to the EU index as in Kabat, can be replaced by different amino acid residues so that the affinity of the antibody or its antigen-binding fragment to the effector ligand is changed, but the antigen-binding ability of the parent antibody is retained. The effector ligand to which its affinity is changed can be, for example, the C1 component of an Fc receptor or complement. This method is described in more detail in U.S. Patent Nos. 5,624,821 and 5,648,260. In some aspects, the deletion or inactivation of the constant region domain (by point mutation or other means) can reduce the Fc receptor binding of circulating antibodies or their antigen-binding fragments, thereby increasing tumor localization. For a description of mutations that delete or inactivate constant domains to increase tumor localization, see, e.g., U.S. Pat. Nos. 5,585,097 and 8,591,886. In some aspects, one or more amino acid substitutions can be introduced into the Fc region to remove potential glycosylation sites on the Fc region, thereby reducing Fc receptor binding (see, e.g., Shields RL et al., (2001) J Biol Chem 276: 6591-604).

[0163] In some aspects, one or more amino acids selected from amino acid residues 322, 329 and 331 in the constant region (numbered according to the EU index as in Kabat) can be replaced by 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 more detail in U.S. Pat. No. 6,194,551 (Idusogie et al.). In some aspects, one or more amino acid residues within amino acid positions 231 to 238 in the N-terminal region of the CH2 domain are altered to alter the ability of the antibody to fix complement. This approach is further described in International Publication No. WO 94 / 29351. In some aspects, the Fc region is modified to increase the ability of the antibody or its antigen-binding fragment to mediate antibody-dependent cellular cytotoxicity (ADCC) and / or to increase the affinity of the antibody or its antigen-binding fragment for Fcγ receptors by mutating one or more amino acids (e.g., introducing amino acid substitutions) at the following positions: 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, 309, 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, numbered according to the EU index as in Kabat. This method is further described in International Publication No. WO 00 / 42072.

[0164] In some aspects, antibodies or antigen-binding fragments thereof as described herein are included in positions 267, 328 or combinations thereof numbered according to the EU index as in Kabat with mutations (e.g., substitutions) of IgG1 constant domains. In some aspects, antibodies or antigen-binding fragments thereof as described herein include IgG1 constant domains with mutations (e.g., substitutions) selected from the group consisting of S267E, L328F and combinations thereof. In some aspects, antibodies or antigen-binding fragments thereof as described herein include IgG1 constant domains with S267E / L328F mutations (e.g., substitutions). In some aspects, antibodies or antigen-binding fragments thereof as described herein comprising IgG1 constant domains with S267E / L328F mutations (e.g., substitutions) have increased binding affinity to FcγRIIA, FcγRIIB or FcγRIIA and FcγRIIB.

[0165] Engineered glycoforms can be used for a variety of purposes, including but not limited to enhancing or reducing effector function. Methods for generating engineered glycoforms in the antibodies or antigen-binding fragments thereof described herein include but are not limited to those disclosed in the following: 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 Publication Nos. US 2007 / 0248600; 2007 / 0178551; 2008 / 0060092; and 2006 / 0253928; International Publication Nos. WO 00 / 61739; WO 01 / 292246; WO 02 / 311140; and WO 02 / 30954; Potillegent TM Technology (Biowa, Inc. Princeton, NJ); and Glycosylation engineering technology (Glycartbiotechnology AG, Zurich, Switzerland). See also, for example, Ferrara C et al., (2006) Biotechnol Bioeng 93:851-861; International Publication Nos. WO 07 / 039818; WO 12 / 130831; WO 99 / 054342; WO 03 / 011878; and WO 04 / 065540.

[0166] In some aspects, any of the constant region mutations or modifications described herein can be introduced into one or both heavy chain constant regions of an antibody or antigen-binding fragment thereof described herein having two heavy chain constant regions.

[0167] In some aspects, 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 aspects, 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 aspects, 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-2δ, and / or SARS-CoV-2D614G 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).

[0168] Competitive binding assays can be used to determine if two antibodies bind to overlapping epitopes. Competitive binding can be determined in an assay in which a test immunoglobulin inhibits specific binding of a reference antibody to a common antigen, such as the spike protein of SARS-CoV-2 or SARS-CoV-2. Many types of competitive binding assays are known, for example: octet competitive binding assays, solid phase direct or indirect radioimmunoassays (RIA), solid phase direct or indirect enzyme immunoassays (EIA), sandwich competition 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 and Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using an I-125 label (see Morel GA et al., (1988) Mol Immunol 137:3614-9); solid phase direct label assays, solid phase direct label sandwich assays (see Harlow E and Lane D, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using an I-125 label (see Morel GA et al., (1988) Mol Immunol 137:3614-9). Immunol [Molecular Immunology] 25(1): 7-15); solid phase direct biotin-avidin EIA (Cheung RC et al., (1990) Virology [Virology] 176: 546-52); and direct label RIA (Moldenhauer G et al., (1990) Scand J Immunol [Scandinavian Journal of Immunology] 32: 77-82). Typically, such assays involve the use of purified antigen bound to a solid surface or cells loaded with 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. Typically the test immunoglobulin is present in excess. Typically, when the competing antibody is present in excess, it 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 variety of different formats using labeled antigens or labeled antibodies. In a common format of this assay, the antigen is immobilized on a 96-well plate. A radioactive label or enzyme label is then used to measure the ability of the unlabeled antibody to block the binding of the labeled antibody to the antigen.For more 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, Harlow E and Lane D, eds., supra, pp. 386-389. A competitive binding assay is provided herein in Example 15.

[0169] In some aspects, 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 (e.g., an octet competitive binding assay as provided in Example 15 herein).

[0170] In some aspects, surface plasmon resonance is used For example, a competition assay is performed by a "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, such as a CM5 sensor chip, and then the antibody or antigen binding fragment is run on the chip. To determine whether the antibody or antigen binding fragment thereof competes with an antibody that binds to the spike protein of SARS-CoV-2 as described herein, the antibody or antigen binding fragment thereof is first run on the chip surface to saturation, and then a potential competing antibody is added. The binding of the competing antibody or antigen binding fragment thereof can then be determined and quantified relative to a non-competitive control.

[0171] In another aspect, provided herein are antibodies that competitively inhibit (e.g., in a dose-dependent manner) the binding of the antibodies or antigen-binding fragments thereof to the spike protein of SARS-CoV-2 or to SARS-CoV-2 as determined using an assay known to those of skill in the art or described herein (e.g., an ELISA competition assay or a suspension array or surface plasmon resonance assay).

[0172] In some aspects, the antigen-binding fragment as described herein that specifically binds to the spike protein of SARS-CoV-2 is selected from the group consisting of Fab, Fab′, F(ab′)2, and scFv, wherein Fab, Fab′, F(ab′)2, or scFv comprises a heavy chain variable region sequence and a 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 to SARS-CoV-2. Fab, Fab′, F(ab′)2, or scFv can be produced by any technique known to those skilled in the art, including but not limited to those discussed herein. In some aspects, Fab, Fab′, F(ab′)2, or scFv further comprises a portion that extends the half-life of the antibody in vivo. This portion is also referred to as a “half-life extension portion”. Any portion known to those skilled in the art for extending the half-life of Fab, Fab′, F(ab′)2, or scFv in vivo can be used. For example, the half-life extension portion may include an Fc region, a polymer, albumin, or an albumin-binding protein or compound. The polymer may include natural or synthetic, optionally substituted linear or branched polyalkylene, polyalkenylene, polyoxyalkylene, polysaccharide, polyethylene glycol, polypropylene glycol, polyvinyl alcohol, methoxypolyethylene glycol, lactose, amylose, dextran, glycogen or its derivatives. The substituent may include one or more hydroxyl, methyl or methoxy groups. In some aspects, Fab, Fab', F(ab')2 or scFv may be modified by adding one or more C-terminal amino acids for attaching a half-life extension portion. In some aspects, the half-life extension portion is polyethylene glycol or human serum albumin. In some aspects, Fab, Fab', F(ab')2 or scFv is fused to the Fc region.

[0173] The 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; radioactive isotopes, such as iodine ( 125 I. 121 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99 Tc); 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, for example, Section 7.6.2 below.

[0174] 7.3 Combinations of Antibodies and Antigen-Binding Fragments thereof

[0175] Provided herein are combinations of antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, for example, 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 aspects, the first and second antibodies or antigen-binding fragments thereof in the combination are in a single composition. In some aspects, the first and second antibodies or antigen-binding fragments thereof in the combination are in separate compositions. When the first and second antibodies or antigen-binding fragments thereof in the combination are in separate compositions, the first and second antibodies or antigen-binding fragments thereof can be administered simultaneously or sequentially.

[0176] Also provided herein are compositions, such as pharmaceutical compositions, comprising antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2, such as 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 comprise a first antibody or antigen-binding fragment thereof and a second antibody or antibody-binding fragment thereof.

[0177] In some aspects, the combinations or compositions provided herein comprise 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 aspects, the methods provided herein use a combination or composition of antibodies or 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.

[0178] In some aspects 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 aspects 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.

[0179] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises 6 CDR sequences of an antibody provided in Table 1 and / or the second antibody or antigen-binding fragment thereof comprises 6 CDR sequences of an antibody provided in Table 1.

[0180] The first antibody or antigen-binding fragment thereof may comprise the six CDRs of the RQ43-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of the celgavir monoclonal antibody.

[0181] The first antibody or antigen binding fragment thereof may comprise the six CDRs of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the six CDRs of RQ29 or RQ29-GL-LH antibody. The first antibody or antigen binding fragment thereof may comprise the six CDRs of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the six CDRs of 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 RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the six CDRs of RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of 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 RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the six CDRs of RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH or RQ43-GL-LH-LO1 antibody.

[0182] The first antibody or antigen binding fragment thereof may comprise the 6 CDRs of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the 6 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 6 CDRs of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the 6 CDRs of the RQ40 or RQ40-GL-LH antibody. The first antibody or antigen binding fragment thereof may comprise the 6 CDRs of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the 6 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, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH or RQ43-GL-LH-LO1 antibody.

[0183] The first antibody or antigen-binding fragment thereof may comprise the six CDRs of 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 RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the six CDRs of 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 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 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 RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH or RQ43-GL-LH-LO1 antibody.

[0184] The first antibody or antigen binding fragment thereof may comprise the 6 CDRs of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the 6 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 6 CDRs of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the 6 CDRs of the RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH, or RQ43-GL-LH-LO1 antibody.

[0185] The first antibody or its antigen-binding fragment may comprise the six CDRs of RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1 or RQ41-LO1 antibody, and the second antibody or its antigen-binding fragment may comprise the six CDRs of RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH or RQ43-GL-LH-LO1 antibody.

[0186] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof comprises a VH and / or VL sequence of an antibody provided in Table 1 and / or the second antibody or antigen-binding fragment thereof comprises a VH and / or VL sequence of an antibody provided in Table 1.

[0187] The first antibody or antigen-binding fragment thereof may comprise the VH and VL of the RQ43-GL-H-LO1 antibody, and the second antibody or antigen-binding fragment thereof may comprise the VH and VL of the celgavir monoclonal antibody. In some aspects, 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.

[0188] The first antibody or antigen binding fragment thereof may comprise the VH and VL of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the VH and VL of RQ29 or RQ29-GL-LH antibody. The first antibody or antigen binding fragment thereof may comprise the VH and VL of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the VH and VL of 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 VH and VL of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the VH and VL of RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the VH and VL of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the VH and VL of RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1 or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof may comprise the VH and VL of RQ20, RQ20-GL-LH or RQ20-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise the VH and VL of RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH or RQ43-GL-LH-LO1 antibody.

[0189] The first antibody or antigen binding fragment thereof may comprise the VH and VL of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise 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 may comprise the VH and VL of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise the VH and VL of the RQ40 or RQ40-GL-LH antibody. The first antibody or antigen binding fragment thereof may comprise the VH and VL of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen binding fragment thereof may comprise 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 may comprise the VH and VL of the RQ29 or RQ29-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may 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.

[0190] The first antibody or antigen-binding fragment thereof may comprise the VH and VL of 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 VH and VL of RQ40 or RQ40-GL-LH antibody. The first antibody or antigen-binding fragment thereof may comprise the VH and VL of 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 VH and VL of RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1 or RQ41-LO1 antibody. The first antibody or antigen-binding fragment thereof may 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 may 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.

[0191] The first antibody or antigen-binding fragment thereof may comprise the VH and VL of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may comprise 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 may comprise the VH and VL of the RQ40 or RQ40-GL-LH antibody, and the second antibody or antigen-binding fragment thereof may 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.

[0192] The first antibody or its antigen-binding fragment may comprise the VH and VL of RQ41, RQ41-GL-LH, RQ41-GL-LH-LO1 or RQ41-LO1 antibody, and the second antibody or its antigen-binding fragment may comprise the VH and VL of RQ43, RQ43-GL-H, RQ43-GL-H-LO1, RQ43-GL-L, RQ43-GL-LH or RQ43-GL-LH-LO1 antibody.

[0193] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof competitively inhibits binding of 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 to the spike protein of SARS-CoV-2, and the second antibody or antigen-binding fragment thereof competitively inhibits binding of 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 to the spike protein of SARS-CoV-2. In some aspects, 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 aspects, 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 aspects, 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.

[0194] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment 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 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.

[0195] In some aspects 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 having 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 having SEQ ID NOs: 69, 70, 71, 73, 74, and 75, respectively.

[0196] In some aspects 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 aspects, 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.

[0197] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof competitively inhibits binding 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 to the spike protein of SARS-CoV-2, and the second antibody or antigen-binding fragment thereof competitively inhibits binding of 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 to the spike protein of SARS-CoV-2. In some aspects, 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 aspects, 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.

[0198] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment 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 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: 63 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 61.

[0199] In some aspects 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 having 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 having SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively.

[0200] In some aspects 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.

[0201] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment thereof competitively inhibits binding 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 to the spike protein of SARS-CoV-2, and the second antibody or antigen-binding fragment thereof competitively inhibits binding 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 to the spike protein of SARS-CoV-2. In some aspects, 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 aspects, 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.

[0202] In some aspects of the combinations, compositions, or methods provided herein, the first antibody or antigen-binding fragment 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 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: 52 and a variable light chain (VL) comprising the amino acid sequence of SEQ ID NO: 53.

[0203] In some aspects 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 having 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 having SEQ ID NOs: 20, 21, 22, 23, 67, and 24, respectively.

[0204] In some aspects 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.

[0205] In some aspects of the combination, composition or method provided herein, the combination or composition neutralizes SARS-CoV-2 pseudovirus. In some aspects, the combination or composition neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1, SARS-CoV-2 BA.2, SARS-CoV-2δ and / or SARS-CoV-2 D614G pseudovirus, and its EC50 is 75ng / mL or lower (for example, EC50 is about 1ng / mL to about 75ng / mL or about 2.5ng / mL to about 75ng / mL). In some aspects, the combination or composition neutralizes SARS-CoV-2 BA.1, SARS-CoV-2 BA1.1 and / or SARS-CoV-2 BA.2 pseudovirus, and its EC50 is 25ng / mL or lower (for example, EC50 is about 1ng / mL to about 25ng / mL or about 2.5ng / mL to about 25ng / mL). In some aspects, the combination or composition neutralizes SARS-CoV-2δ and / or SARS-CoV-2 D614G pseudovirus 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 aspects, 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-2δ 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).

[0206] In some aspects of the combinations, compositions or methods provided herein, the combination or composition neutralizes the SARS-CoV-2 virus. In some aspects, the combination or composition neutralizes SARS-CoV-2 D614G, SARS-CoV-2α, SARS-CoV-2δ+T51I+T95I, SARS-CoV-2 BA.1, SARS-CoV-2 BA.1.1, SARS-CoV-2 BA.2, SARS-CoV-2BA.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 aspects, the combination or composition neutralizes SARS-CoV-2 D614G, SARS-CoV-2α, SARS-CoV-2δ+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).

[0207] In some aspects of the combinations, compositions or methods provided herein, the combination or composition neutralizes the SARS-CoV-2 BA.2.12.1 virus. In some aspects 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 aspects 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 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).

[0208] In some aspects of the combinations and methods provided herein, the first and second antibodies or antigen-binding fragments thereof are in the same composition. In some aspects of the combinations and methods provided herein, the first and second antibodies or antigen-binding fragments thereof are in separate compositions.

[0209] 7.4 Antibody Production

[0210] 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, such as by chemical synthesis or by recombinant expression techniques. Unless otherwise indicated, the methods described herein employ conventional techniques of molecular biology, microbiology, genetic analysis, recombinant DNA, organic chemistry, biochemistry, PCR, oligonucleotide synthesis and modification, nucleic acid hybridization, and related fields within the technical scope of the art. These techniques are described, for example, in the references cited herein and are fully explained 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 annual updates); Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); Gait (ed.) (1984) Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein (ed.) (1991) Oligonucleotides and Analogues: A Practical Approach, IRL Press; Birren J et al., Current Protocols in Immunology, John Wiley & Sons (1987 and annual updates); B et al. (eds.) (1999) Genome Analysis: A Laboratory Manual. Cold Spring Harbor Laboratory Press.

[0211] In some aspects, provided herein is a method for preparing an antibody or antigen binding fragment that immunospecifically binds to the spike protein of SARS-CoV-2, the method comprising culturing a cell or host cell as described herein. In some aspects, provided herein is a method for preparing an antibody or antigen binding fragment that immunospecifically binds to the spike protein of SARS-CoV-2, the method comprising expressing (e.g., recombinantly expressing) the antibody or antigen binding fragment using a cell or host cell as described herein (e.g., a cell or host cell comprising a polynucleotide encoding an antibody or antigen binding fragment as described herein). In some aspects, the cell is a separated cell. In some aspects, an exogenous polynucleotide has been introduced into a cell. In some aspects, the method further comprises a step of separating or purifying an antibody or antigen binding fragment obtained from a cell, host cell, or culture.

[0212] Methods for producing polyclonal antibodies are known in the art (see, e.g., Chapter 11: Short Protocols in Molecular Biology, (2002) 5th edition, Ausubel FM et al., eds., John Wiley and Sons, New York).

[0213] Monoclonal antibodies or antigen-binding fragments thereof can be prepared using a variety of techniques known in the art, including the use of hybridomas, recombinant and phage display techniques, yeast-based presentation techniques, or a combination thereof. For example, monoclonal antibodies or antigen-binding fragments thereof can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow E and Lane D, Antibodies: A Laboratory Manual [antibodies: Laboratory Manual], (Cold Spring Harbor Laboratory Press, 2nd edition, 1988); Hammerling GJ et al., in: Monoclonal Antibodies and T-Cell Hybridomas [monoclonal antibodies and T cell hybridomas] 563681 (Elsevier, NY, 1981), or as described in Kohler G and Milstein C (1975) Nature [nature] 256: 495. Examples of yeast-based presentation methods that can be used to select and generate the antibodies described herein include, for example, those disclosed in WO 2009 / 036379A2, WO 2010 / 105256, and WO 2012 / 009568, each of which is incorporated herein by reference in its entirety.

[0214] In some aspects, a 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), wherein the antibody or antigen-binding fragment immunospecifically binds to the spike protein of SARS-CoV-2, for example as determined by ELISA or other antigen-binding assays known in the art. In some aspects, the monoclonal antibody or antigen-binding fragment thereof can be a human antibody or antigen-binding fragment thereof. In some aspects, the monoclonal antibody or antigen-binding fragment thereof can be a Fab fragment or a F(ab')2 fragment. The monoclonal antibodies or antigen-binding fragments thereof described herein can be prepared, for example, by the hybridoma method as described in Kohler G and Milstein C (1975) Nature 256:495, or can be isolated from a phage library, for example using techniques as described herein. Other methods for preparing clonal cell lines and monoclonal antibodies and antigen-binding fragments thereof expressed thereby are well known in the art (see, e.g., Chapter 11 of Short Protocols in Molecular Biology, 5th ed. (2002), Ausubel FM et al., supra).

[0215] Antigen-binding fragments of the antibodies described herein can be generated by any technique known to those skilled in the art. For example, Fab and F(ab')2 fragments described herein can be generated by proteolytic cleavage of immunoglobulin molecules using enzymes such as papain (for producing Fab fragments) or pepsin (for producing F(ab')2 fragments). The 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 the heavy chain. The F(ab')2 fragment contains two antigen-binding arms of a tetrameric antibody molecule, which are connected by a disulfide bond in the hinge region.

[0216] In addition, various phage display and / or yeast-based presentation methods known in the art can also be used to generate antibodies or antigen-binding fragments thereof described herein. In phage display methods, proteins are displayed on the surface of phage particles, which carry polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of infected tissues). DNA encoding these VH and VL domains is recombined with scFv linkers by PCR and cloned into phagemid vectors. The vector is transferred to E. coli by electroporation, and the E. coli is infected with helper phage. The phages used in these methods are typically filamentous phages including fd and M13, and the VH and VL domains are usually recombinantly fused to phage gene III or gene VIII. Phages expressing antibodies or antigen-binding fragments thereof that bind to specific antigens can be selected or identified with antigens, for example, using labeled antigens or antigens bound or captured on solid surfaces or beads. Examples of phage display methods that can be used to prepare the antibodies or fragments described herein include those disclosed 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 and Barbas CF (1994) Advan Immunol 57:191-280; PCT Application No. PCT / GB9I / 001134; International Publication Nos. WO 90 / 02809, WO 91 / 10737, WO 92 / 01047, WO 92 / 18619, WO 93 / 11236, WO 95 / 15982, WO 95 / 20401, and WO 97 / 13844; and U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 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.

[0217] 7.4.1 Polynucleotides

[0218] In some aspects, provided herein are polynucleotides comprising nucleotide sequences encoding antibodies or antigen-binding fragments thereof described herein that immunospecifically bind to the spike protein of SARS-CoV-2, or domains thereof (e.g., variable light chain regions and / or variable heavy chain regions), and vectors, e.g., vectors comprising such polynucleotides for recombinant expression in host cells (e.g., Escherichia coli (E. coli) and mammalian cells).

[0219] In some aspects, provided herein are polynucleotides comprising a nucleotide sequence encoding an antibody or antigen-binding fragment thereof that immunospecifically binds to the spike protein of SARS-CoV-2 and comprises an amino acid sequence as described herein, as well as antibodies or antigen-binding fragments that compete with such an antibody or antigen-binding fragment for binding to SARS-CoV-2 (e.g., in a dose-dependent manner) or bind to the same epitope as such an antibody or antigen-binding fragment.

[0220] Also provided herein are polynucleotides encoding antibodies or antigen-binding fragments thereof described herein that specifically bind to the spike protein of SARS-CoV-2, which are optimized, for example, by codon / RNA optimization, heterologous signal sequence substitution, and elimination of mRNA instability elements. The method of generating an optimized nucleic acid encoding an antibody or antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2 or its domain (e.g., heavy chain, light chain, VH domain, or VL domain) for recombinant expression by introducing codon changes (e.g., codon changes that encode the same amino acid due to the degeneracy of the genetic code) and / or eliminating inhibitory regions in mRNA for recombinant expression can be performed by adopting, for example, the optimization methods described in U.S. Pat. Nos. 5,965,726; 6,174,666, 6,291,664, 6,414,132, and 6,794,498 accordingly.

[0221] Polynucleotides encoding antibodies or antigen-binding fragments thereof described herein or their domains can be generated from nucleic acids from suitable sources (e.g., hybridomas) using methods well known in the art (e.g., PCR and other molecular cloning methods). For example, PCR amplification using synthetic primers that can hybridize to the 3' and 5' ends of known sequences can be performed using genomic DNA obtained from hybridoma cells that produce target antibodies. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding light chains and / or heavy chains of antibodies or their antigen-binding fragments. Such PCR amplification methods can be used to obtain nucleic acids containing sequences encoding variable light chain regions and / or variable heavy chain regions of antibodies or their antigen-binding fragments. The amplified nucleic acids can be cloned into vectors for expression in host cells and further cloned, for example, to generate chimeric antibodies and humanized antibodies or their antigen-binding fragments.

[0222] The polynucleotides provided herein can be, for example, in the form of RNA or in the form of DNA. DNA includes cDNA, genomic DNA and synthetic DNA, and the DNA can be double-stranded or single-stranded. If it is single-stranded, the DNA can be a coding strand or a non-coding (antisense) strand. In some aspects, the polynucleotide is a cDNA or DNA lacking one or more endogenous introns. In some aspects, the polynucleotide can be a non-naturally occurring polynucleotide. In some aspects, the polynucleotide is recombinantly produced. In some aspects, the polynucleotide is separated. In some aspects, the polynucleotide is substantially pure. In some aspects, the polynucleotide is purified from natural components.

[0223] 7.4.2 Cells and vectors

[0224] In some aspects, provided herein are vectors (e.g., expression vectors) comprising polynucleotides comprising nucleotide sequences encoding antibodies and antigen-binding fragments thereof or domains thereof that bind to the spike protein of SARS-CoV-2 for recombinant expression in a host cell, such as a mammalian cell. Also provided herein are cells, such as host cells, that contain such vectors to recombinantly express antibodies or antigen-binding fragments thereof described herein that bind to the spike protein of SARS-CoV-2 (e.g., human antibodies or antigen-binding fragments thereof). In a specific aspect, provided herein are methods for producing antibodies or antigen-binding fragments thereof described herein, comprising expressing such antibodies or antigen-binding fragments thereof in a host cell.

[0225] In some aspects, the recombinant expression of an antibody or antigen-binding fragment thereof described herein that specifically binds to the spike protein of SARS-CoV-2 or its domain (e.g., a heavy chain or light chain described herein) involves the construction of an expression vector containing a polynucleotide encoding the antibody or its antigen-binding fragment or its domain. Once a polynucleotide encoding an antibody or its antigen-binding fragment or its domain (e.g., a heavy chain or light chain variable domain) as described herein is obtained, a vector for producing the antibody or its antigen-binding fragment can be produced by recombinant DNA technology using techniques well known in the art. Therefore, a method for preparing a protein by expressing a polynucleotide containing a nucleotide sequence encoding an antibody or its antigen-binding fragment or its domain (e.g., a light chain or a heavy chain) is described herein. Methods well known to those skilled in the art can be used to construct an expression vector containing a coding sequence of an antibody or its antigen-binding fragment or its domain (e.g., a light chain or a heavy chain) and appropriate transcription and translation control signals. These methods include, for example, in vitro recombinant DNA technology, synthetic techniques, and in vivo genetic recombination. Replicable vectors are also provided, and these vectors include nucleotide sequences of coding antibodies as described herein or their Fab, heavy chain or light chain, heavy chain or light chain variable domains or heavy chain or light chain CDRs operably connected to promoters. Such vectors can, for example, include nucleotide sequences of the constant regions encoding antibodies or their Fab (see, for example, International Publication Nos. WO 86 / 05807 and WO 89 / 01036; and U.S. Patent No. 5,122,464), and the variable domains of antibodies or their Fab can be cloned into such vectors for expression of whole heavy chain, whole light chain or whole heavy chain and light chain.

[0226] The expression vector can be transferred to 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 six CDRs, VH, VL, VH and VL, heavy chains, light chains, or heavy and light chains of an antibody provided in Table 1) or a domain thereof (e.g., VH, VL, VH and VL, heavy chains, or light chains of an antibody provided in Table 1). Thus, provided herein are host cells containing polynucleotides encoding an antibody or antigen-binding fragment thereof described herein (e.g., an antibody or antigen-binding fragment thereof comprising six CDRs, VH, VL, VH and VL, heavy chains, light chains, or heavy and light chains of an antibody provided in Table 1) or a domain thereof (e.g., VH, VL, VH and VL, heavy chains, or light chains of an antibody provided in Table 1), which are operably linked to a promoter to express such sequences in the host cell. In some aspects for expressing double-chain antibodies or antigen-binding fragments thereof, vectors encoding both heavy and light chains alone can be co-expressed in host cells for expressing the entire immunoglobulin, as described in detail below. In some aspects, the host cell contains a vector comprising a polynucleotide encoding 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 aspects, the host cell contains two different vectors, the first vector comprising a polynucleotide encoding the heavy chain or heavy chain variable region of an antibody or antigen-binding fragment thereof described herein, and the second vector comprising a polynucleotide encoding the light chain or light chain variable region or a domain thereof of an antibody described herein (e.g., an antibody comprising six CDRs of an antibody provided in Table 1). In some aspects, 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 six CDRs of an antibody provided in Table 1). In some aspects, the heavy chain / heavy chain variable region expressed by the first cell associates with the light chain / 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 six CDRs of an antibody provided in Table 1). In some aspects, a host cell population comprising such a first host cell and such a second host cell is provided herein.

[0227] In some aspects, provided herein are vector populations comprising: a first vector comprising a polynucleotide encoding a light chain / 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 / 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 a heavy chain polypeptide and a light chain polypeptide may be used.

[0228] 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 the coding sequences of interest can be produced and subsequently purified, and also represent cells that, when transformed or transfected with the appropriate nucleotide coding sequences, express the antibodies or antigen-binding fragments thereof described herein in situ. These include, but are not limited to, microorganisms such as bacteria (e.g., E. coli and Bacillus subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems transformed with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems (e.g., such as Chlamydomonas reinhardtii) infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences. reinhardtii);Or a mammalian cell system (e.g., COS (e.g., COS1 or COS), CHO, BHK, MDCK, HEK 293, NS0, PER.C6, VERO, CRL7030, HsS78Bst, HeLa and NIH 3T3, HEK-293T, HepG2, SP210, R1.1, BW, LM, BSC1, BSC40, YB / 20 and BMT10 cells) with a recombinant expression construct containing a promoter derived from a mammalian cell genome (e.g., a metallothionein promoter) or a promoter derived from a mammalian virus (e.g., adenovirus late promoter; vaccinia virus 7.5K promoter). In some aspects, the cell used 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) is a CHO cell, such as from a CHO GS System TM(Lonza) CHO cells. In some aspects, the cells used to express the antibodies described herein are human cells, such as human cell lines. In some aspects, the mammalian expression vector is pOptiVEC TM Or pcDNA3.3. In some aspects, bacterial cells (such as E. coli) or eukaryotic cells (e.g., mammalian cells) particularly useful for expressing complete recombinant antibody molecules are used to express recombinant antibody molecules. For example, mammalian cells such as Chinese hamster ovary (CHO) cells, combined with vectors such as the major immediate early gene promoter element from human cytomegalovirus, are effective expression systems for antibodies (Foecking MK and Hofstetter H (1986) Gene [Gene] 45: 101-105; and Cockett MI et al., (1990) Biotechnology [Sheng Technology] 8: 662-667). In some aspects, the antibodies or antigen-binding fragments thereof described herein are produced by CHO cells or NS0 cells.

[0229] In addition, host cell strains may be selected that modulate the expression of the inserted sequence, or modify and process the gene product in a desired particular manner. Such modification (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 with cellular machinery for the correct processing of the primary transcript, glycosylation and phosphorylation of the gene product may 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 murine myeloma cell line that does not endogenously produce any immunoglobulin chains), CRL703O, 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 aspects, 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.

[0230] Once the antibodies or antigen-binding fragments thereof described herein have been produced by recombinant expression, they can be purified by any method known in the art for purification of immunoglobulin molecules, for example, by chromatography (e.g., ion exchange chromatography, affinity chromatography (particularly by affinity chromatography for specific antigens after protein A purification), and size exclusion chromatography), centrifugation, differential solubility, or by any other standard technique for purification of proteins. In addition, 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.

[0231] In some aspects, the antibodies or antigen-binding fragments thereof described herein are isolated or purified. Typically, an isolated antibody or antigen-binding fragment thereof is an antibody or antigen-binding fragment thereof that is substantially free of other antibodies or antigen-binding fragments thereof having an antigenic specificity different from that of the isolated antibody or antigen-binding fragment thereof. For example, in some aspects, a preparation of an antibody or antigen-binding fragment thereof described herein is substantially free of cellular material and / or chemical precursors.

[0232] 7.5 Pharmaceutical Compositions

[0233] 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 having a desired degree of purity in a physiologically acceptable carrier, excipient or stabilizer (Remington's Pharmaceutical Sciences (1990) Mack Publishing Co., Easton, PA). The carrier, excipient or stabilizer is nontoxic to recipients at the dosages and concentrations employed.

[0234] In some aspects, 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 pharmaceutically 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 Excipients Press (1996). Press) (2000). In some aspects, the pharmaceutical compositions described herein comprise two antibodies or antigen-binding fragments that bind to the spike protein of SARS-CoV-2, for example, two antibodies or antigen-binding fragments thereof that bind to different epitopes of the spike protein of SARS-CoV-2. In some aspects, 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 aspects, 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 aspects, the pharmaceutical compositions described herein comprise two antibodies or antigen-binding fragments that can bind to SARS-CoV-2 simultaneously.

[0235] The pharmaceutical compositions described herein can be used to neutralize SARS-CoV-2.

[0236] The pharmaceutical compositions described herein can be used to prevent and / or treat SARS-CoV-2 infection in patients or one or more symptoms or complications associated with SARS-CoV-2 infection in patients. In some aspects, the patient may have been exposed to SARS-CoV-2. Examples of one or more symptoms 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, dyspnea, myalgia, chills, myalgia, chills, sore throat, loss of taste or smell, headache, chest pain, nausea, vomiting, and diarrhea. Other examples of one or more symptoms or complications associated with SARS-CoV-2 infection in patients treated according to the methods described herein include, but are not limited to, cardiac complications, respiratory complications, diabetic complications, organ failure, and blood clots. In some aspects, the pharmaceutical compositions provided herein can be used to treat or prevent SARS-CoV-2 infection or one or more symptoms or complications associated with SARS-CoV-2 infection as described herein in patients with one or more risk factors for SARS-CoV-2 infection. In some aspects, risk factors include, but are not limited to, being 65 years of age or older, being immunocompromised, and having one or more of chronic lung disease, asthma, or diabetes.

[0237] In some aspects, the pharmaceutical compositions described herein are used as medicaments. In some aspects, the pharmaceutical compositions described herein are used as diagnostic agents, for example to detect the presence of SARS-CoV-2 in a sample obtained from a patient (e.g., a human patient).

[0238] The compositions provided herein can be formulated for intramuscular (IM) administration, such as IM injection.

[0239] The compositions provided herein for in vivo administration can be sterile. This is easily achieved, for example, by filtration through sterile filtration membranes.

[0240] 7.6 Purpose and Method

[0241] 7.6.1 Therapeutic uses and methods

[0242] In some aspects, provided herein are methods 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. The method of treating or preventing SARS-CoV-2 infection may comprise administering to a patient in need (e.g., a human patient) one or more antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2.

[0243] In some aspects, provided herein are methods of reducing the likelihood of infection in a subject at risk of acquiring a SARS-CoV-2 infection. The method of reducing the likelihood of infection in a subject at risk of acquiring 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.

[0244] In some aspects, provided herein are methods for preventing and / or treating SARS-CoV-2 infection or one or more conditions or complications associated with SARS-CoV-2 infection. Conditions or complications associated with SARS-CoV-2 infection include, but are not limited to, fever, cough, fatigue, shortness of breath, dyspnea, myalgia, chills, myalgia, chills, sore throat, loss of taste or smell, headache, chest pain, nausea, vomiting, and diarrhea. In some aspects, provided herein are methods for preventing and / or treating SARS-CoV-2 infection in patients with one or more risk factors for SARS-CoV-2 infection. In some aspects, risk factors include, but are not limited to, being 65 years of age or older, having impaired immune function, suffering from one or more of chronic lung disease, asthma, or diabetes, and / or having impaired immune function. In some aspects, such methods include administering to patients in need (e.g., human patients) an antibody or its antigen-binding fragment bound to the spike protein of SARS-CoV-2 provided herein, or a pharmaceutical composition comprising an antibody or its antigen-binding fragment bound to the spike protein of SARS-CoV-2 herein.

[0245] In some aspects, such methods include administering to a patient in need (e.g., a human patient) 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.

[0246] In some aspects, such methods include administering to a patient in need thereof (e.g., a human patient) a composition comprising one or more antibodies or antigen-binding fragments thereof that bind to a spike protein of SARS-CoV-2 as described herein. In some aspects, the patient has risk factors including, but not limited to, being 65 years of age or older, having impaired immunosuppression, and having one or more of chronic lung disease, asthma, or diabetes.

[0247] In some aspects, one or more antibodies or antigen-binding fragments thereof, or pharmaceutical compositions that bind to the spike protein of SARS-CoV-2 are administered to a subject (e.g., a human subject) at risk of acquiring SARS-CoV-2.

[0248] In some aspects, one or more antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 are administered intramuscularly, for example by intramuscular injection.

[0249] Typically, the patient is a human, but non-human mammals, including transgenic mammals, may also be treated.

[0250] In some aspects, the present invention relates to an antibody or antigen binding fragment thereof, a combination of an antibody or antigen binding fragment 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 an antibody or antigen binding fragment thereof, or a pharmaceutical composition provided herein for use in a method for preventing or treating SARS-CoV-2 infection. In some aspects, the present invention relates to an antibody or antigen binding fragment thereof, a combination of an antibody or antigen binding fragment thereof, or a pharmaceutical composition provided herein for use in a method for treating SARS-CoV-2 infection in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen binding fragment thereof, a combination of an antibody or antigen binding fragment thereof, or a pharmaceutical composition provided herein.

[0251] The amount of the antibody or antigen-binding fragment thereof or composition that will be effective in the treatment of a condition will depend on the nature of the disease. The precise dose employed in the composition will also depend on the route of administration and the severity of the disease.

[0252] 7.6.2 Detection and diagnostic purposes

[0253] The antibodies or antigen-binding fragments thereof described herein that bind to the spike protein of SARS-CoV-2 (see, e.g., Section 7.2) can be used to determine the level of SARS-CoV-2 protein or SARS-CoV-2 in a biological sample using classical methods known to those skilled 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; radioactive isotopes, such as iodine ( 125 I. 121 I), carbon ( 14 C) Sulfur ( 35 S), tritium ( 3 H), Indium ( 121 In) and technetium ( 99Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin. 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 and used in combination 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.

[0254] Determining 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, directly (e.g., by measuring or estimating absolute protein levels) or relatively (e.g., by comparing to the level of a disease-associated polypeptide in a second biological sample). The level of SARS-CoV-2 protein expression in a first biological sample can be measured or estimated and compared to a standard SARS-CoV-2 protein level, which is taken from a second biological sample obtained from an individual without the disease, or determined by averaging the levels from a population of individuals without the disease.

[0255] As used herein, the term "biological sample" refers to any biological sample obtained from a subject, cell line, tissue, or other cell 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.

[0256] The antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 described herein can carry a detectable or functional label. When a fluorescent label is used, currently available microscopy and fluorescence-activated cell sorting analysis (FACS) or a combination of two procedures known in the art can be used to identify and quantify specific binding members. The antibodies or antigen-binding fragments thereof that bind to the spike protein of SARS-CoV-2 described herein can carry a fluorescent label. Exemplary fluorescent labels include, for example, reactive probes and conjugated probes, for example, aminocoumarin, fluorescein and Texas red, Alexa Fluor dyes, Cy dyes, and DyLight dyes. The antibodies or antigen-binding fragments thereof that specifically bind to the spike protein of SARS-CoV-2 can carry a radioactive label, such as an isotope. 3 H. 14 C. 32 P. 35 S. 36 Cl, 51 Cr, 57 Co. 58 Co. 59Fe, 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 Re. When a radioactive label is used, the specific binding of an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 can be identified and quantified using currently available counting procedures known in the art. In the case where the label is an enzyme, detection can be accomplished by any of the currently utilized colorimetric, spectrophotometric, fluorescence spectrophotometric, amperometric, or gas quantitation techniques known in the art. This can be achieved by contacting a sample or a 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 substance and the spike protein of SARS-CoV-2. Any complex formed between the antibody or antigen-binding fragment and the spike protein of SARS-CoV-2 can be detected in the sample and compared (optionally compared to a control). In view of the specific binding of the antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2 described herein to SARS-CoV-2, these antibodies or antigen-binding fragments thereof can be used to specifically detect SARS-CoV-2 (e.g., in a subject).

[0257] Also included herein is an assay system that can be prepared in the form of a test kit for quantitative analysis of, for example, the presence of a SARS-CoV-2 spike protein. The system or test kit can include labeled components, such as labeled antibodies or antigen-binding fragments, and one or more other immunochemical reagents. For more information about the kit, see, for example, Section 7.7 below.

[0258] In some aspects, 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 an antigen-binding fragment thereof. In some aspects, provided herein is the use of an antibody or an antigen-binding fragment thereof provided herein for detecting SARS-CoV-2 spike protein in a sample in vitro. On the one hand, provided herein is an antibody or an antigen-binding fragment thereof or a pharmaceutical composition used in detecting SARS-CoV-2 spike protein in a subject or a sample obtained from a subject. On the one hand, provided herein is an antibody or an antigen-binding fragment thereof or a pharmaceutical composition provided herein for use as a diagnostic agent. In some aspects, the antibody comprises a detectable label. In some aspects, the subject is a human.

[0259] 7.7 Kit

[0260] Provided herein is a kit comprising one or more antibodies or their Fab as described herein. In some respects, a pharmaceutical package or kit is provided herein, which includes a component filled with a pharmaceutical composition as described herein, such as one or more antibodies or their Fab as provided herein, or one or more containers. Optionally, associated with such one or more containers may be an announcement in the form of regulations of a government agency for the manufacture, use or sale of a drug or biological product, which reflects that the agency is administered for human use, to permission for manufacture, use or sale.

[0261] Also provided herein is a kit that can be used in a diagnostic method. In some aspects, the kit comprises an antibody or antigen-binding fragment thereof described herein in one or more containers, such as a purified antibody or antigen-binding fragment thereof. In some aspects, the kit described herein contains a substantially separated SARS-CoV-2 spike protein antigen that can be used as a control. In some aspects, the kit described herein further includes a control antibody or antigen-binding fragment thereof that does not react with the SARS-CoV-2 spike protein antigen. In some aspects, the kit described herein contains one or more elements for detecting the binding of an antibody or its antigen-binding fragment to a SARS-CoV-2 spike protein antigen (e.g., an antibody or its antigen-binding fragment can be conjugated with a detectable substrate (such as a fluorescent compound, an enzyme substrate, a radioactive compound, or a luminescent compound) or a second antibody or its antigen-binding fragment that recognizes a first antibody or its antigen-binding fragment can be conjugated with a detectable substrate). In some aspects, the kit 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 attached to a solid support. In some aspects, the detection device of the above-described kit includes a solid support to which the SARS-CoV-2 spike protein antigen is attached. Such a kit may also include an unattached reporter-labeled anti-human antibody or antigen-binding fragment thereof or an anti-mouse / rat antibody or antigen-binding fragment thereof. In this regard, 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 can be detected by binding of the reporter-labeled antibody or antigen-binding fragment thereof.

[0262] The following examples are offered by way of illustration and not by way of limitation.

[0263] 8. Examples

[0264] The examples in this Examples section (ie, Section 8) are provided by way of illustration and not by way of limitation.

[0265] 8.1 Example 1: Antibody and protein production

[0266] Antibody VH and VL DNA fragments are cloned into CMV-driven mammalian expression plasmids containing human IgG1 Fc with YTE serum half-life extension modification and TM effector function elimination modification. Plasmids are sequence verified by Sanger sequencing. According to the manufacturer's instructions, antibodies are transiently expressed in HEK293F cells using 293Fectin (Gibco#12347019) and grown for 6 days in Freestyle culture medium (Gibco#12338018). On the 6th day, the culture medium is clarified by centrifugation, filtered, and purified using MabSelect SuRe resin (Cytiva#11-0034-94). As described below, HP-SEC is used to measure antibody purity, and the correct quality of the antibody is verified by mass spectrometry. The sequence of exemplary antibodies is provided in Table 1.

[0267] SARS-CoV-2 RBD (residues 334-526) was cloned with an N-terminal CD33 leader sequence and a C-terminal GSSG linker, AviTag, GSSG linker, and 8xHisTag, expressed in FreeStyle 293 cells (ThermoFisher), 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 ensure purity and appropriate molecular weight. Charles River Endotoxin levels were measured using a TRAIL-400 cartridge (Charles River) and found to be less than 1 EU / mg.

[0268] 8.2 Example 2: HP-SEC

[0269] HP-SEC was used to analyze the antibody samples to determine the levels of aggregates, monomers and fragments. Samples (100 μg in PBS buffer) were injected into an Agilent 1200 series high performance liquid chromatography (HPLC) instrument and separated using a TSKgelG3000SWxl 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 carried out at 280 nm. The results are shown in Table 2.

[0270] 8.3 Example 3: Baculovirus ELISA

[0271] Baculovirus particle (BVP) ELISA was performed essentially as described previously (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), but with some modifications. Briefly, a 1% baculovirus (BV) suspension in 50 mM sodium carbonate buffer (pH 9.6) was used to coat half of a 96-well ELISA plate (Nunc Maxisorp) at 4°C overnight, while the other half of the ELISA plate was left uncoated to bind the test antibody. All subsequent steps were performed at room temperature. The next day, the wells were washed with Dulbecco's PBS (DPBS) and then incubated with blocking buffer (Dulbecco's PBS containing 0.5% BSA) for 1 h, followed by three washes with DPBS. Next, 100nM and 10nM antibody in blocking buffer was added to BVP coated and uncoated wells and incubated for 1h, then washed three 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 1h, then washed three times with DPBS. Finally, TMB substrate (SeraCare #5120-0075) was added to each well and incubated for 2 minutes. The reaction was terminated by adding an equal volume of 0.2M sulfuric acid to each well. The absorbance was read at 450nm. The BVP score and plate binding were determined by normalizing the absorbance to the control wells without the test antibody. The results are shown in Table 2.

[0272] 8.4 Example 4: HEK binding assay

[0273] Nonspecific HEK cell binding was measured using a Mirrorball fluorescence cytometer (SPT Labtech). First, 10uL of Alexa Fluor647 goat anti-human IgG (H+L) antibody (Invitrogen #A-21445) diluted to 16nM in Mirrorball buffer (Hanks balanced salt solution containing 0.5% BSA) was added to the wells of a 384-well transparent bottom plate. Next, 10uL of test antibodies serially diluted in Mirrorball buffer were added to the wells. Finally, 20uL of HEK293f cells diluted to 250,000 cells / mL in Mirrorball buffer were added to the wells. The plate was incubated at room temperature for 2h, and the fluorescence of each well was measured using a Mirrorball fluorescence cytometer. The results are shown in Table 2.

[0274] 8.5 Example 5: AC-SINS

[0275] AC-SINS was performed essentially as described previously (Geng SB, Wu J, Alam ME, Schultz JS, Dickinson CD, Seminer CR, et al. Facile Preparation of Stable Antibody-Gold Conjugates and Application to Affinity-Capture Self-Interaction Nanoparticle Spectroscopy. Bioconjug Chem 2016; 27: 2287-300) with some modifications. Briefly, whole 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 then conjugated to 20 nm gold nanoparticles (Innova Biosciences #3201-0100) at a 3:2 ratio of capture antibody:non-capture antibody. Antibodies and gold nanoparticles were incubated at room temperature for 1 h at a ratio of 9:1 and then blocked for 1 h by adding 0.1 μM poly-(ethylene glycol) methyl ether thiol (2000 MW, Sigma-Aldrich #729140). The coated and blocked nanoparticles were concentrated 12.5-fold by centrifugation and stored at 4°C. To evaluate self-association, 5 μL of nanoparticles were mixed with 45 μL of purified antibodies at 50 ug / mL in PBS, pH 7.2 or buffer in a 384-well plate. Nanoparticles were mixed with buffer only (no antibody) as a control. The absorbance from 490 to 700 nm was measured on a SPECTROstar Nano UV / vis plate reader. The wavelength of peak absorbance was calculated in the MARS data analysis software and used to determine the wavelength shift compared to the nanoparticle control alone. The results are shown in Table 2.

[0276] 8.6 Example 6: Accelerated Stability Heat Stress Study

[0277] For accelerated stability testing, samples were diluted to 1 mg / mL in PBS (pH 7.2) and incubated at 4°C or 45°C for 2 weeks. Then, samples on days 0, 7 and 14 were analyzed by HP-SEC peptide mapping and DELFIA binding assays as described herein. The monomer, aggregate and fragment percentages of each sample were calculated based on curve integration using HPLC ChemStation software (Agilent). The changes in monomer, aggregate and fragment content were calculated by the difference between each sample incubated at 45°C and 4°C. The results are shown in Table 3.

[0278] 8.7 Example 7: Photostability determination

[0279] For photostability testing, antibodies were prepared at 2.5 mg / mL in PBS (pH 7.2), loaded into 1 cc Schott glass vials, stoppered / sealed, and placed in an ICH-compliant photostability chamber (Caron Model 6545-2). Samples were exposed to 3000 lux of cool white light for 1 week, with a total exposure time of approximately 500,000 lux hours. Samples were analyzed by HP-SEC and peptide mapping as described in those sections. The results are shown in Table 3.

[0280] 8.8 Example 8: Serum stability assay

[0281] The antibodies were diluted to 0.5 mg / ml in human serum (Sigma) and incubated at 37°C for 14 days. The day 0 control sample was taken and frozen at -80°C until testing. At 14 days, the incubated samples were also frozen at -80°C until all samples could be tested. As described herein, the binding of stressed and non-stressed samples was assessed by DELFIA binding assay for RBD. The results are shown in Table 3.

[0282] 8.9 Example 9: DSC

[0283] Thermal melt transitions were determined using a Microcal VP differential scanning calorimetry system (Malvern, PA). The monoclonal antibody solution was diluted to 1 mg / ml in the final buffer and the change in heat capacity (Cp) was measured when the sample was heated from 20°C to 95°C using a temperature gradient of 90°C / h. Normalized heat capacity data were obtained after subtracting the buffer blank and normalizing to the monoclonal antibody concentration. Data were analyzed using Microcal LLC origin software to calculate the thermal melt transitions associated with the expansion of different domains. The results are shown in Table 2.

[0284] 8.10 Example 10: Viscosity

[0285] The concentrated antibody solution was diluted to 100 mg / mL in the formulation. Each sample was subjected to a rotational shear stress of 1000 s-1 on a MCR301 rheometer using a CP20-1 cone-plate system (Anton Parr, part 3274) at 23°C. During data collection, 5 measurements were taken per minute and averaged to provide viscosity values. The results are shown in Table 2.

[0286] 8.11 Example 11: DLS

[0287] The antibody group was reformulated into an alternative buffer. The Z-average apparent diffusion coefficient was measured using a Dynapro plate reader (Wyatttechnology, Santabarbara, CA) equipped with a laser source 833nm wavelength. Scattered light was collected in backscattering mode at an angle of 153 °. For each antibody, 40ul of each concentration of 2, 4, 6, 8 and 10mg / ml was distributed in triplicate to a low volume 384-well plate (Corning, Tewskbury, MA, Tewskbury, Massachusetts), covered with paraffin film, and rotated at 3000RPM for 1 minute to remove any bubbles. Prior to measurement, the sample chamber was balanced for 1h, and 10 times of 10 seconds of data were collected and averaged for each hole. The Z-average translational diffusion coefficient was determined by the cumulant analysis of the autocorrelation function and modeled as:

[0288] D = D0(1 + kDC) to obtain the "kD" diffusion virus coefficient (interaction parameter).

[0289] The results are shown in Table 2.

[0290] 8.12 Example 12: Peptide Mapping

[0291] Reduction tryptic peptide mapping was performed based on manufacturing recommendations. In short, 100 μg antibody samples were denatured and reduced with dithiothreitol (DTT, 20291, Thermo Scientific) at 37 ° C in guanidine hydrochloride buffer for 30 minutes. The samples were then alkylated with iodoacetamide (IAM, 786-078, G-Biosciences) at room temperature in the dark for 30 minutes. Subsequently, the sample mixture was dialyzed into 6M urea and then diluted with Tris pH 7.5 buffer for trypsin digestion. Trypsin (V5280, Promega) was added with a 1: 20 protease: protein ratio and incubated at 37 ° C for 4h. The reaction was quenched by adding trifluoroacetic acid (TFA, T6508, Sigma-Aldrich). The digests of the samples were analyzed using a Fusion Orbitrap mass spectrometer (ThermoFisher Scientific, Waltham, MA, USA) connected to an AQUITY ultra-high performance liquid chromatograph (UPLC; Waters). Separation was performed using an AQUITY UPLC BEH300 C18 column (1.7 μm, 2.1×150 mm; Waters). 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 0-35% linear gradient, and the chromatogram was monitored by using UV absorbance at 220 nm and MS. MS data were processed by BiopharmaFinder 3.0 (Thermo Fisher Scientific). The results are shown in Table 3.

[0292] 8.13 Example 13: DELFIA Binding Assay

[0293] use (PerkinElmer) method measures antibody binding to the receptor binding domain (RBD) of the SARS-CoV-2 spike protein before and after heat stress. TM 96-well plates were coated with purified RBD Omicron protein overnight. The assay plates were then washed with 1X 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 by Europium-N1 anti-human IgG (PerkinElmer) and signals were quantified via time-resolved fluorescence on an Envision plate reader (PerkinElmer). IC was calculated by four-parameter logistic (4PL) curves using GraphPad Prism v8.0 50 . The IC of the stressed sample 50 Comparisons were made with reference samples to generate relative potency percentages.

[0294] 8.14 Example 14: FcRn affinity chromatography

[0295] About 40 μg / 40 μL of antibody was loaded onto a 1 ml huFcRN-coupled agarose affinity column using an Agilent HPLC, followed by a 3CV 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 18CV linear gradient from 40% to 100% buffer B. The experiment was performed at room temperature at a flow rate of 0.5 ml / min, and the A280 of the elution curve was measured using an Agilent-DAD. The retention time was normalized to the relative retention time using the following formula and using the well-performing control antibody NIP228 in WT and YTE formats:

[0296] Relative RT = ([sample RT - NIP228 RT] / [NIP228-YTE RT - NIP228 RT]). The results are shown in Table 3.

[0297] 8.15 Example 15: Octet Binding Competition

[0298] Binding competition was performed using an Octet model QK384 biolayer interferometry instrument (FortéBio). The streptavidin tip was first soaked in 1x kinetic buffer for 10 minutes, followed by a baseline signal measurement in 1x kinetic buffer for 60 seconds. The biotinylated SARS-CoV-2 spike RBD diluted to 4nM in kinetic buffer was then loaded onto the tip for 300 seconds. The antigen-containing tip was then added to a well containing 1μM of the first mAb (mAb1) as the mAb1 reference antibody, and the association was measured for 300 seconds. The tip was then immersed in a well containing 1μM of the second mAb (mAb2), and the second association was measured for 300 seconds. The binding signal at the end of each step was averaged based on the data points for the last 10 seconds at equilibrium to determine the change in signal. If the signal increase after the addition of mAb2 was greater than 0.5nm, it was determined that the mAb did not compete. The results are shown in Table 4 and indicate that RQ40 and RQ43 do not compete with RQ33.

[0299] 8.16 Example 16: Binding to RBD with point mutations

[0300] The kinetic rates (k) of mAb binding to SARS-CoV-2 spike RBD (wt and point mutants) were assessed using an Octet model QK 384 biolayer interferometry instrument (FortéBio). a , k d and K D ). The anti-Penta-His (HIS1K) tip was first soaked in 1x kinetic buffer for 10 minutes, followed by a baseline signal measurement in 1x kinetic buffer for 60 seconds. The His-tagged SARS-CoV-2 spike RBD diluted to 5ug / ml in kinetic buffer was then loaded onto the tip for 240 seconds. The antigen-containing tip was then added to the well containing the mAb at a starting concentration of 200nM, followed by a 2-fold dilution, and the association was measured for 300 seconds. The dissociation was then measured in 1x kinetic buffer for 300 seconds. The data were subtracted from the reference value and fitted to a 1:1 binding model using Octet data analysis software 12.0. (The collected data were fitted to a single-site binding equation using Biacore software to obtain binding kinetic measurements.) The results are shown in Table 5 and indicate that RQ33 and RQ43 are insensitive to the K444A and F486ARBD mutations.

[0301] 8.17 Example 17: Pseudovirus Neutralization Assay

[0302] Freestyle 293X cells were inoculated and transfected with a third generation human immunodeficiency virus (HIV)-based lentiviral vector expressing luciferase together with a packaging plasmid encoding the following: SARS-CoV-2 spike protein with a C-terminal 19 amino acid deletion, Rev and Gag-pol. Viral supernatant was harvested after 48 hours. Cell debris was removed by low speed centrifugation and the supernatant was passed through a 0.45 μM filter device. The supernatant was concentrated 100 times by ultracentrifugation to produce a stock virus for the assay.

[0303] Serial dilutions of mAb were prepared in 384-well microtiter plates and pre-incubated with pseudovirus for 1 hour at 37°C, to which Ad293 cells stably expressing human ACE2 were added. The plates were returned to the 37°C incubator for 48 hours and the plates were analyzed using Bright-Glo TMLuciferase activity was measured using the luciferase assay system (Promega) according to the manufacturer's recommendations. Percent inhibition was calculated relative to pseudovirus alone control, and potency (IC) was determined by nonlinear regression using GraphPad Prism software version 8.1.0. 50 The average IC of each mAb was determined from at least 2 independent experiments. 50 The results of the pseudovirus neutralization assay are shown in Figure 1 and 2 AZD7442 was used as a control in the neutralization assays testing the mAb combination. AZD7442 is a combination of two antibodies, tesagvirimab (AZD8895) and segavirimab (AZD1061), derived from B cells donated by patients who have recovered from SARS-CoV-2 infection.

[0304]

[0305]

[0306]

[0307]

[0308] 8.18 Example 18: Divergent Neutralization Curve of RQ33

[0309] RQ33, RQ43-GL-H-LO1, and the combination (RQ33+RQ43-GL-H-LO1) were evaluated in a neutralization assay to measure their antiviral activity against SARS-CoV-2 variants of concern (VOCs). Antiviral activity was observed in a dose-dependent manner, resulting in an S-shaped relationship between antibody concentration and percent neutralization for most VOCs. However, when neutralization of the BA.2.12.1 variant of the authentic SARS-CoV-2 virus was measured, negative neutralization (i.e., infection enhancement) was observed at low concentrations of RQ33 ( Figure 3 This observation is in contrast to the observations for RQ43-GL-H-LO1 and the combination (RQ33 + RQ43-GL-H-LO1), where low concentrations of antibody resulted in low or no neutralization, but not negative neutralization (i.e., infection enhancement). This observation of infection enhancement was only observed when the authentic BA.2.12.1 virus was assayed, and not when the pseudovirus ( Figure 4 ) and real viruses from other variants were not observed.

[0310] Enhanced infection with authentic BA.2.12.1 virus was observed at low concentrations of RQ33, which is inconsistent with the target product profile of a monoclonal antibody product that should prevent SARS-CoV-2 disease in a pre-exposure prophylaxis setting. Therefore, segavir (instead of RQ33) was evaluated in combination with RQ43-GL-H-LO1. Segavir and RQ43-GL-H-LO1 are biochemically compatible, as RQ43-GL-H-LO1 did not compete with segavir by Octet competition assay using BA.2RBD ( Figure 5 By comparing the binding footprints of RQ43 (the parent of RQ43-GL-H-LO1) and cilgavir on the RBD surface ( Figure 6 ) also demonstrated this. In addition, celegavir and RQ43-GL-H-LO1 together provided broad and effective coverage of all tested variants of concern (Table 6).

[0311] Table 6: SARS-CoV-2 antiviral activity of RQ43-GL-H-LO1 and celegavir as determined by real virus neutralization

[0312]

[0313] Note: The IC50 values ​​in the table are calculated from the average of 2 independent replicates, each with two replicates.

[0314] IC50, half-maximal inhibitory concentration; mAb, monoclonal antibody; SARS-CoV-2, severe acute respiratory syndrome coronavirus 2.

[0315] ***

[0316] The scope of the present invention is not limited by the aspects described herein. In fact, in addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art based on the foregoing description and the accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

[0317] All references (e.g., publications or patents or patent applications) cited herein are incorporated by reference in their entirety and 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.

[0318] Some aspects are within the scope of the following claims.

Claims

1. A composition or combination comprising: 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 comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35, and 36, respectively; 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 thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NO: 69, 70, 71, 73, 74 and 75, respectively.

2. A composition or combination comprising: A first antibody or an antigen-binding fragment thereof that specifically binds to a spike protein of SARS-CoV-2, wherein the first antibody or the antigen-binding fragment thereof competitively inhibits binding of 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 to the spike protein of SARS-CoV-2; and A second antibody or an antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or the antigen-binding fragment thereof competitively inhibits the binding of 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 to the spike protein of SARS-CoV-2.

3. A composition or combination comprising: A first antibody or an antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or the 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 an antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or the 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.

4. The composition or combination of claim 2 or 3, wherein the first antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35 and 36, respectively.

5. The composition or combination of claim 4, wherein 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.

6. The composition or combination of any one of claims 2-5, wherein the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 69, 70, 71, 73, 74 and 74, respectively.

7. A composition or combination as described in claim 6, wherein the second antibody or its antigen-binding fragment 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, optionally wherein 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.

8. The composition or combination of any one of claims 1-7, wherein 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.

9. A composition or combination as described in any one of claims 1-8, wherein the second antibody or its antigen-binding fragment 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, optionally wherein 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.

10. A composition or combination as described in any one of claims 1-9, wherein the composition or combination neutralizes the SARS-CoV-2 virus, optionally wherein the composition or combination neutralizes SARS-CoV-2BA.2.12.1, and its IC50 is 25ng / mL or less, or its IC50 is 20ng / mL or less.

11. A composition or combination as described in any one of claims 26-38, wherein the composition or combination neutralizes SARS-CoV-2D614G, SARS-CoV-2α, SARS-CoV-2δ+T51I+T95I, SARS-CoV-2BA.1, SARS-CoV-2BA.1.1, SARS-CoV-2BA.2, SARS-CoV-2BA.2.12.1 and / or SARS-CoV-2BA.5 viruses with an IC50 of 75 ng / mL or less, or an IC50 of less than 60 ng / mL or less.

12. The composition or combination of any one of claims 1-11, wherein the first antibody or antigen binding fragment is fully human and / or the second antibody or antigen binding fragment is fully human.

13. The composition or combination of any one of claims 1-12, wherein 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.

14. A composition or combination as described in any one of claims 1-13, wherein 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.

15. The composition or combination of any one of claims 1-14, wherein 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.

16. A composition or combination as described in any one of claims 1-15, wherein 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 wherein 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 wherein the heavy chain constant region is human IgG1 heavy chain constant region.

17. A composition or combination as described in any one of claims 1-16, wherein 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.

18. A composition or combination as described in any one of claims 1-17, wherein 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.

19. A composition or combination as described in any one of claims 1-18, wherein 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 IgGκ 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.

20. The composition or combination of any one of claims 1-19, wherein 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.

21. The composition or combination of any one of claims 1-20, wherein 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.

22. The composition or combination of any one of claims 1-20, wherein the first antibody or antigen binding fragment is an antigen binding fragment and / or wherein the second antibody or antigen binding fragment is an antigen binding fragment.

23. A composition or combination as described in claim 22, wherein the first antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domains, IgNar, IgGΔCH2, miniantibodies, F(ab')3, tetrabodies, triabodies, diabodies, single domain antibodies, (scFv)2 or scFv-Fc and / or the second antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domains, IgNar, IgGΔCH2, miniantibodies, F(ab')3, tetrabodies, triabodies, diabodies, single domain antibodies, (scFv)2 or scFv-Fc.

24. The composition or combination of any one of claims 1-23, wherein the first antibody or antigen binding fragment is isolated and / or the second antibody or antigen binding fragment is isolated.

25. The composition or combination of any one of claims 1-24, wherein the first antibody or antigen binding fragment is monoclonal and / or the second antibody or antigen binding fragment is monoclonal.

26. The composition or combination of any one of claims 1-25, wherein the first antibody or antigen binding fragment is recombinant and / or the second antibody or antigen binding fragment is recombinant.

27. A composition or combination comprising: A first human IgG1 antibody or an 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) 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 a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation; and A second human IgG1 antibody or an 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) 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 a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation.

28. A composition or combination as described in claim 27, wherein (i) 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 second human IgG1 antibody or antigen-binding fragment comprises the amino acid sequence of SEQ ID NO: 66, or (ii) 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, the second human IgG1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 76, and the second human IgG1 antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:

77.

29. The composition of any one of claims 1-28, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

30. The combination of any one of claims 1-28, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in the same composition, optionally wherein the composition is a pharmaceutical composition.

31. The combination of any one of claims 1-28, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in separate compositions, optionally wherein the first antibody or antigen-binding fragment thereof and / or the second antibody or antigen-binding fragment thereof are in a pharmaceutical composition.

32. 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 having the following sequences, respectively: (i) SEQ ID NOs: 31, 32, 37, 34, 35 and 36; (ii) SEQ ID NOs: 25, 26, 30, 28, 67 and 29; or (iii) SEQ ID NOs: 13, 14, 19, 16, 17 and 18.

33. 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: (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; or (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.

34. An antibody or antigen-binding fragment thereof as described in claim 32 or 33, wherein the antibody or antigen-binding fragment neutralizes SARS-CoV-2 pseudovirus, optionally wherein the antibody or antigen-binding fragment neutralizes SARS-CoV-2BA.1, SARS-CoV-2BA1.1, SARS-CoV-2BA.2, SARS-CoV-2δ and / or SARS-CoV-2D614G pseudovirus with an EC50 of 100 ng / mL or less.

35. The antibody or antigen-binding fragment thereof of any one of claims 32-34, wherein the antibody or antigen-binding fragment is fully human.

36. The antibody or antigen-binding fragment thereof of any one of claims 32-35, wherein the antibody or antigen-binding fragment comprises a light chain constant region.

37. The antibody or antigen-binding fragment thereof of claim 36, wherein 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.

38. The antibody or antigen-binding fragment thereof of any one of claims 32-37, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region.

39. The antibody or antigen-binding fragment thereof of claim 38, wherein 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, optionally wherein the heavy chain constant region is a human IgG1 heavy chain constant region.

40. The antibody or antigen-binding fragment thereof of any one of claims 32-39, wherein the antibody or antigen-binding fragment comprises (i) a human IgG1 heavy chain constant region and (ii) a human IgGκ light chain constant region.

41. The antibody or antigen-binding fragment thereof of any one of claims 32-40, wherein the 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.

42. The antibody or antigen-binding fragment thereof of any one of claims 32-41, wherein the 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.

43. The antibody or antigen-binding fragment thereof of any one of claims 32-42, wherein the antibody or antigen-binding fragment comprises a heavy chain constant region comprising the amino acid sequence of SEQ ID NO:

66.

44. The antibody or antigen-binding fragment thereof of any one of claims 32-43, which is a full-length antibody.

45. The antibody or antigen-binding fragment thereof of any one of claims 32-43, which is an antigen-binding fragment.

46. ​​The antigen-binding fragment of claim 45, wherein the antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domain, IgNar, IgGΔCH2, miniantibody, F(ab')3, tetrabody, triabody, diabody, single domain antibody, (scFv)2 or scFv-Fc.

47. The antibody or antigen-binding fragment thereof of any one of claims 32-46, wherein the antibody or antigen-binding fragment is isolated.

48. The antibody or antigen-binding fragment thereof of any one of claims 32-47, wherein the antibody or antigen-binding fragment is monoclonal.

49. The antibody or antigen-binding fragment thereof of any one of claims 32-48, wherein the antibody or antigen-binding fragment is recombinant.

50. The antibody or antigen-binding fragment thereof of any one of claims 32-49, further comprising a detectable label.

51. An isolated polynucleotide comprising a nucleic acid molecule encoding a heavy chain variable region of an antibody or antigen-binding fragment thereof as described in any one of claims 32-49 and / or a nucleic acid molecule encoding a light chain variable region.

52. An isolated vector comprising the polynucleotide of claim 51.

53. A host cell comprising a polynucleotide as described in claim 51, a vector as described in claim 52, or a first vector and a second vector, wherein the first vector comprises a nucleic acid molecule encoding the heavy chain variable region of the antibody or its antigen-binding fragment as described in any one of claims 32-49, and the second vector comprises a nucleic acid molecule encoding the light chain variable region of the antibody or its antigen-binding fragment as described in any one of claims 32-49.

54. A method for producing an antibody or antigen-binding fragment thereof that binds to the spike protein of SARS-CoV-2, the method comprising culturing the host cell of claim 53 so that the nucleic acid molecule is expressed and the antibody or antigen-binding fragment thereof is produced, optionally wherein the method further comprises isolating the antibody or antigen-binding fragment.

55. An antibody or antigen-binding fragment thereof produced by the method of claim 54.

56. A composition comprising the antibody or antigen-binding fragment thereof as described in any one of claims 32-50 and 55, optionally wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable excipient.

57. A composition or combination comprising: A first antibody or an antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or the antigen-binding fragment thereof competitively inhibits the binding 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 to the spike protein of SARS-CoV-2 and A second antibody or an antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or the antigen-binding fragment thereof competitively inhibits the binding of 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 to the spike protein of SARS-CoV-2.

58. A composition or combination comprising: A first antibody or an antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the first antibody or the 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 an antigen-binding fragment thereof that specifically binds to the spike protein of SARS-CoV-2, wherein the second antibody or the antigen-binding fragment thereof binds 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.

59. The composition or combination of claim 57 or 58, wherein the first antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 13, 14, 15, 16, 17 and 18, respectively.

60. The composition or combination of claim 59, wherein 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.

61. The composition or combination of any one of claims 57-60, wherein the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35 and 36, respectively.

62. A composition or combination as described in claim 61, wherein the second antibody or its antigen-binding fragment 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.

63. The composition or combination of any one of claims 57-60, wherein the second antibody or antigen-binding fragment thereof comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 20, 21, 22, 23, 67 and 24, respectively.

64. A composition or combination as described in claim 63, wherein the second antibody or its antigen-binding fragment 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.

65. A composition or combination comprising: 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 comprises VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2, and VL-CDR3 having 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, wherein the second antibody or antigen-binding fragment thereof comprises (i) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 31, 32, 37, 34, 35 and 36, respectively, or (ii) VH-CDR1, VH-CDR2, VH-CDR3, VL-CDR1, VL-CDR2 and VL-CDR3 having SEQ ID NOs: 20, 21, 22, 23, 67 and 24, respectively.

66. The composition or combination of any one of claims 57-65, wherein 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.

67. A composition or combination as described in any one of claims 57-62, 65 and 66, wherein 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.

68. The composition or combination of any one of claims 57-60 and 63-66, wherein 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.

69. A composition or combination as described in any of claims 57-68, wherein the composition or combination neutralizes SARS-CoV-2 pseudovirus, optionally wherein the composition or combination neutralizes SARS-CoV-2BA.1, SARS-CoV-2BA1.1, SARS-CoV-2BA.2, SARS-CoV-2δ and / or SARS-CoV-2D614G pseudovirus with an EC50 of 75 ng / mL or less.

70. A composition or combination as described in any one of claims 57-69, wherein the composition or combination (i) neutralizes SARS-CoV-2BA.1, SARS-CoV-2BA1.1 and / or SARS-CoV-2BA.2 pseudovirus with an EC50 of 25 ng / mL or less, and / or (ii) neutralizes SARS-CoV-2δ and / or SARS-CoV-2D614G pseudovirus with an EC50 of 75 ng / mL or less.

71. The composition or combination of any one of claims 57-70, wherein the first antibody or antigen binding fragment is fully human and / or the second antibody or antigen binding fragment is fully human.

72. The composition or combination of any one of claims 57-71, wherein 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.

73. A composition or combination as described in any one of claims 57-72, wherein 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.

74. The composition or combination of any one of claims 57-73, wherein 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.

75. A composition or combination as described in any of claims 57-74, wherein 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 wherein 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 wherein the heavy chain constant region is human IgG1 heavy chain constant region.

76. A composition or combination as described in any one of claims 57-75, wherein 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.

77. A composition or combination as described in any of claims 57-76, wherein 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.

78. A composition or combination as described in any of claims 57-77, wherein 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 IgGκ 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.

79. A composition or combination as described in any of claims 57-78, wherein 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.

80. The composition or combination of any one of claims 57-79, wherein 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.

81. The composition or combination of any one of claims 57-79, wherein the first antibody or antigen binding fragment is an antigen binding fragment and / or wherein the second antibody or antigen binding fragment is an antigen binding fragment.

82. The composition or combination of claim 81, wherein the first antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domains, IgNar, IgGΔCH2, miniantibodies, F(ab')3, tetrabodies, triabodies, diabodies, single domain antibodies, (scFv)2, or scFv-Fc and / or the second antigen-binding fragment comprises Fab, Fab', F(ab')2, single-chain Fv (scFv), disulfide-linked Fv, V-NAR domains, IgNar, IgGΔCH2, miniantibodies, F(ab')3, tetrabodies, triabodies, diabodies, single domain antibodies, (scFv)2, or scFv-Fc.

83. The composition or combination of any one of claims 57-82, wherein the first antibody or antigen binding fragment is isolated and / or the second antibody or antigen binding fragment is isolated.

84. The composition or combination of any one of claims 57-83, wherein the first antibody or antigen binding fragment is monoclonal and / or the second antibody or antigen binding fragment is monoclonal.

85. The composition or combination of any one of claims 57-84, wherein the first antibody or antigen binding fragment is recombinant and / or the second antibody or antigen binding fragment is recombinant.

86. A composition or combination comprising: A first human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation; and A second human IgG1 antibody or an 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) 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 a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation.

87. A composition or combination comprising A first human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation; and A second human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation.

88. A composition or combination comprising A first human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation; and A second human IgG1 antibody or an 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) 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, and a heavy chain constant region and a light chain constant region comprising a YTE mutation and a TM mutation.

89. The composition or combination of any one of claims 86-88, wherein 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.

90. The composition of any one of claims 57-89, wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

91. The combination of any one of claims 57-89, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in the same composition, optionally wherein the composition is a pharmaceutical composition.

92. The combination of any one of claims 57-89, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are in separate compositions, optionally wherein the first antibody or antigen-binding fragment thereof and / or the second antibody or antigen-binding fragment thereof are in a pharmaceutical composition.

93. A method for neutralizing SARS-CoV-2, the method comprising contacting the SARS-CoV-2 with an antibody or antigen-binding fragment thereof as described in any one of claims 32-50 and 55, or a composition or combination as described in any one of claims 1-31 and 57-92.

94. The method of claim 93, wherein the contacting is in vitro.

95. The method of claim 93, wherein the contacting is in a subject.

96. A method for providing pre-exposure prophylaxis of SARS-CoV-2 in a subject, the method comprising administering to the subject an effective amount of a composition or combination as described in any one of claims 1-31 and 57-92.

97. The method of claim 96, wherein the subject is not currently infected with SARS-CoV-2.

98. The method of claim 96 or 97, wherein the subject is not known to have had recent exposure to an individual infected with SARS-CoV-2.

99. The method of any one of claims 96-98, wherein the subject has moderate to severe immunocompromise.

100. The method of claim 99, wherein the moderate to severe immunocompromise is due to a medical condition or receipt of immunosuppressive drugs or treatments.

101. The method of any one of claims 96-100, wherein the subject may not develop an adequate immune response to COVID-19 vaccination.

102. The method of any one of claims 96-101, wherein the subject is not recommended for COVID-19 vaccination.

103. The method of claim 102, wherein COVID-19 vaccination is not recommended due to a history of serious adverse reactions to the COVID-19 vaccine and / or COVID-19 vaccine components.

104. The method of any one of claims 96-103, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are administered sequentially.

105. A method of treating or preventing SARS-CoV-2 infection in a subject, the method comprising administering to the subject an effective amount of a combination as described in any one of claims 1-32, 57-89 and 92, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof are administered sequentially.

106. The method of claim 104 or 105, wherein the first antibody or antigen-binding fragment thereof is administered prior to administering the second antibody or antigen-binding fragment thereof.

107. The method of claim 104 or 105, wherein the first antibody or antigen-binding fragment thereof is administered after administration of the second antibody or antigen-binding fragment thereof.

108. The method of any one of claims 104-107, wherein the first antibody or antigen-binding fragment is administered intramuscularly and / or wherein the second antibody or antigen-binding fragment thereof is injected intramuscularly.

109. A method for providing pre-exposure prophylaxis of SARS-CoV-2 in a subject, the method comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof as described in any one of claims 32-49 and 55.

110. A method of treating or preventing SARS-CoV-2 infection in a subject, the method comprising administering to the subject an effective amount of the antibody or antigen-binding fragment thereof as described in any one of claims 32-49 and 55.

111. The method of claim 109 or 110, wherein the administration is intramuscular.

112. The method of any one of claims 105-108, 110, and 111, wherein the subject has been exposed to or is at risk of being exposed to SARS-CoV-2.

113. The method of any one of claims 95-112, wherein the subject is a human.

114. The method of any one of claims 93-113, wherein the SARS-CoV-2 is SARS-CoV-2BA.1, SARS-CoV-2BA1.1, SARS-CoV-2BA.2, SARS-CoV-2β, SARS-CoV-2δ and / or SARS-CoV-2D614G.

115. The method of any one of claims 93-113, wherein the SARS-CoV-2 is SARS-CoV-2BA.2.12.

1.

116. A method for detecting SARS-CoV-2 in a sample, the method comprising contacting the sample with an antibody or antigen-binding fragment thereof as described in any one of claims 32-49 and 55, or a composition or combination as described in any one of claims 1-31 and 56-92.

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