WO2023122211A2

WO2023122211A2 - Coronavirus antibodies and uses thereof

Info

Publication number: WO2023122211A2
Application number: PCT/US2022/053713
Authority: WO
Inventors: Kevin SAUNDERS; Barton F. Haynes; Bryan KRAFT; David Montefiori
Original assignee: Duke University
Priority date: 2021-12-21
Filing date: 2022-12-21
Publication date: 2023-06-29
Also published as: WO2023122211A3

Abstract

Recombinant monoclonal antibodies (mAbs) and fragments that bind specifically to coronavirus spike protein. Herein, monoclonal antibodies were recombinantly derived from isolated B cell from coronavirus infected individuals. Such antibodies bind various epitopes on the coronavirus spike protein and are neutralizing. The invention provides methods for using the inventive antibodies in prophylactic and/or therapeutic methods to prevent or treat coronavirus infection.

Description

CORONAVIRUS ANTIBODIES AND USES THEREOF

[0001] This application is an International Application, which claims the benefit of priority from U.S. provisional patent application no. 63/292,233, filed on December 21, 2021, the entire contents of which are incorporated herein by reference in its entirety.

[0002] All patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

SEQUENCE LISTING

[0004] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [ ], is named [ ] and is [ ] bytes in size.

TECHNICAL FIELD

[0005] The invention relates to human antibodies binding to and/or neutralizing Coronaviruses, including but not limited to SARS Coronavirus 2 (SARS-CoV-2) virus and their uses.

BACKGROUND

[0006] Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is related to SARS- CoV and several SARS-like bat CoVs (F. Wu, et al., A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020)). CoV entry into host cells is mediated by the viral S glycoprotein, which forms trimeric spikes on the viral surface (F. Li, Structure, function, and evolution of coronavirus spike proteins. Annu. Rev. Virol. 3, 237-261 (2016)).

Each monomer in the trimeric S assembly is a heterodimer of SI and S2 subunits. The SI subunit is composed of four domains: an N-terminal domain (NTD), a C-terminal domain (CTD), and subdomains I and II (A. C. Walls, et al., Cryo-electron microscopy structure of a coronavirus spike glycoprotein trimer. Nature 531, 114-117 (2016); M. A. Tortorici, D. Veesler, Structural insights into coronavirus entry. Adv. Virus Res. 105, 93-116 (2019); D. Wrapp, et al., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263 (2020)). The CTD of both SARS-CoV and SARS-CoV-2 functions as the receptor-binding domain (RBD) for the shared entry receptor, human angiotensin converting enzyme 2 (hACE2 or ACE-2) (W. Song et al., Cryo-EM structure of the SARS coronavirus spike glycoprotein in complex with its host cell receptor ACE2. PLOS Pathog. 14, el007236 (2018); J. Lan et al., Structure of the SARS-CoV-2 spike receptor-binding domain bound to the ACE2 receptor.

Nature 581, 215-220 (2020); M. Hoffmann et al., SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and is blocked by a clinically proven protease inhibitor. Cell 181, 271-280. e8 (2020); Q. Wang et al., Structural and functional basis of SARS-CoV-2 entry by using human ACE2. Cell 181, 894-904. e9 (2020); F. Li, Receptor recognition mechanisms of coronaviruses: A decade of structural studies. J. Virol. 89, 1954-1964 (2015)). The S2 subunit contains the fusion peptide, heptad repeat 1 and 2, and a transmembrane domain, all of which are required for fusion of the viral and host cell membranes.

[0007] The S glycoprotein of HCoVs is the primary target for neutralizing antibodies (nAbs) (S. Jiang, et al., Neutralizing antibodies against SARS-CoV-2 and other human coronaviruses. Trends Immunol. 41, 355-359 (2020)). SARS-CoV and SARS-CoV-2 share 76% amino acid identity in their S proteins, raising the possibility of conserved immunogenic surfaces on these antigens. Studies of convalescent sera and a limited number of monoclonal antibodies (mAbs) have revealed limited to no cross-neutralizing activity, demonstrating that conserved antigenic sites are rarely targeted by nAbs (D. Wrapp, et al., Cryo-EM structure of the 2019-nCoV spike in the prefusion conformation. Science 367, 1260-1263 (2020); Q. Wang et al., Structural and functional basis of SARS-CoV-2 entry by using human ACE2. Cell 181, 894-904. e9 (2020); X. Ou et al., Characterization of spike glycoprotein of SARS-CoV-2 on virus entry and its immune cross-reactivity with SARS-CoV. Nat. Commun. 11, 1620 (2020); H. Lv et al., Cross-reactive antibody response between SARS-CoV-2 and SARS-CoV infections. Cell Rep. 31, 107725 (2020)). However, the frequencies, specificities, and functional activities of cross-reactive antibodies induced by natural SARS-CoV and SARS-CoV-2 infection remain poorly defined. SUMMARY OF THE INVENTION

[0008] In certain aspects the present invention provides monoclonal antibodies (mAbs) and fragments thereof that bind to alpha, beta, delta or gamma coronavirus antigens, including without limitation SARS-CoV-2 antigens. In certain embodiments, these are neutralizing antibodies that bind to SARS-CoV-2 spike protein. In other embodiments, these are antibody dependent cellular cytotoxicity or other anti-viral Fc-Receptor mediating antibodies. As used herein, the term “antibody” is used broadly, and can refer to proteins and/or nucleic acids of a full-length antibody, a fragment, or synthetic forms thereof.

[0009] In certain aspects the present invention provides recombinant coronavirus antibodies, or antigen binding fragments thereof, wherein in certain non-limiting embodiments the antibody or fragment thereof binds to a coronavirus. A recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds to coronavirus spike protein and comprises a variable heavy (VH) domain and a variable light (VL) domain that have amino acid sequences that have an overall 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH and VL domains of an antibody described herein, for example listed in Tables 3, or wherein the VH domain and VL domain each have at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to the VH and VL domains, respectively, of an antibody an antibody described herein, for example listed in Table 3. In certain embodiments the antibodies are neutralizing antibodies. In certain embodiments, the antibody specifically binds to RBD of the coronavirus spike protein. In certain embodiments, the RBD binding antibodies block ACE2 receptor interaction. In certain embodiments, the antibody is specific for CoV2. In certain embodiments the antibody is cross- reactive with other coronaviruses, e.g. without limitation SARS-CoV-1, MERS-CoV, 229E, NL63, HKU1, OC43, bat coronavirus, and/or pangolin coronavirus. In certain embodiments, the antibody, or the antigen-binding fragment thereof, wherein the concentration of the antibody, or antigen-binding fragment thereof, required for 50% neutralization of coronavirus, e.g but not limited to CoV2 virus, (IC50) is as described in Figures 1-5. In certain embodiments, IC50 is up to about 1 mcg/ml, up to about 500 ng/ml, up to about 250 ng/ml, up to about 100 ng/ml or up to about 50 ng/ml. In certain embodiments, the antibody or antigen binding fragment thereof, binds to the surface of coronavirus-infected cells and mediates either clearance, complement lysis or NK or CD8 killing of coronavirus-infected cells.

[0010] All embodiments described herein that refer to Table 3 may also encompass variations of these antibody sequences.

[0011] In certain aspects, the antibody binds to CoV-2 spike protein. In certain aspects, the antibody binds to the CoV-2 spike protein and may not be cross-reactive to SARS-CoV spike protein. In certain aspects, the antibody binds CoV-2 spike protein and is cross-reactive to other human or animal-derived coronavirus spike proteins, for example but not limited to SARS-CoV- 1 spike protein. In certain aspects, the antibody can neutralize or inhibit binding of SARS CoV-2 to the human or animal ACE2 receptor. In certain aspects, the antibody has a binding affinity to SARS-CoV-2 spike protein that is stronger than the binding affinity between SARS-CoV-2 spike protein and the human ACE2 receptor. Herein, monoclonal antibodies were recombinantly produced from B cell receptor cells isolated from patients infected with SARS-CoV2. The invention provides methods for using such antibodies for passive immunotherapy and/or diagnostic purposes.

[0012] In certain aspects, provided are antibodies and fragments comprising VH and VL (as used herein, VH and VL can also be referred to as VH or VL and VH or VL, respectively) sequences of the antibodies described in Table 3, the Examples and Figures disclosing amino acid and nucleic acid sequences. Figures disclose complete heavy and light nucleotide sequences, and the VH and VL domains are readily determined in these sequences. For example, VH and VL are readily derived from the nucleotide sequences or amino acid sequences by any suitable method, such as but not limited by IMGT and other online tools as cited herein, which tools not only provide amino acid translations, but also variable domains of the heavy and light chains, predicted CDR and framework boundaries. See, e.g., http://www.imgt.org/ll\/IGT_vquest/. More specifically, for example, a nucleotide sequence for a VH or VL domain can be input at http://www.imgt.org/ll\/IGT_vquest/input and results include “V-REGION translation” that provides the nucleotide sequence and amino acid translation along with the framework and CDR boundaries according to the IMGT scheme.

[0013] In certain aspects, the antibodies or fragments have a binding specificity as described in the accompanying Examples and Figures. In certain embodiments, the antibody is any one of DH1284, DH1286, DH1287, DH1288, DH1289, DH1290 DH1291, DH1292, DH1293, DH1294, DH1295, DH1296, or DH1297.

[0014] In certain aspects, the antibodies are recombinant antibodies having an IgG or IgM Fc domain, or a portion thereof.

[0015] In certain aspects, provided are recombinant antibodies and fragments comprising HCDR1-3 and LCDR1-3 (as used herein, the VH CDRs can be referred to as HCDR1-3 or CDRH1-3; likewise the VL CDRs can be referred to as LCDR1-3 or CDRL1-3) from the pairs of VH and VL sequences as described in Table 3, and accompanying Figures. In one aspect, the antibody comprises HCDR1-3 and LCDR1-3 of antibody DH1284.

[0016] In certain aspects, an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Table 3 is affinity matured by testing mutations in one or more of the CDRs. In one aspect, an antibody that comprises HCDR1-3 and LCDR1-3 of an antibody of Tables 3 is affinity matured by testing mutations only in HCDR3. In certain aspects, mutations are favorable when the antibody maintains binding specificity but improves affinity or avidity for the antigen.

[0017] In certain aspects, the invention provides a pharmaceutical composition comprising the recombinant antibodies of the invention.

[0018] In certain aspects, the invention provides nucleic acids comprising sequences encoding antibodies comprising VH and VL sequences of the inventive antibodies, e.g. from Tables 3. In certain embodiments, the nucleic acids are DNAs. In certain embodiments, the nucleic acids are mRNAs. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention.

[0019] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive antibodies. In certain embodiments, these are optionally formulated in lipid nanoparticles (LNPs). In certain embodiments, the mRNAs are modified. Modifications include without limitations modified ribonucleotides, poly-A tail, 5 ’cap.

[0020] In certain aspects, the invention provides a kit comprising: a composition comprising an antibody of the invention, a syringe, needle, or applicator for administration of the antibody to a subject; and instructions for use.

[0021] In certain aspects, the invention provides prophylactic methods comprising administering the pharmaceutical composition of the invention. [0022] In certain aspects, the invention provides methods of treatment comprising administering the pharmaceutical composition of the invention. The methods are applicable to diseases or conditions, e.g. but not limited to prophylaxis, suspected or diagnosed coronavirus infection, that would benefit from passive immunization with coronavirus targeting antibodies or combinations thereof.

[0023] In certain aspects, provided are antibodies and fragments comprising VH and VL sequences as described, e.g. Table 3.

[0024] In certain aspects, the invention provides a pharmaceutical composition comprising the recombinant antibodies of the invention.

[0025] In certain aspects, the invention provides nucleic acids comprising sequences encoding coronavirus antibodies comprising VL and VH sequences of the invention. In certain embodiments, the nucleic acids are DNAs. In certain embodiments, the nucleic acids are mRNAs. In certain aspects, the invention provides expression vectors comprising the nucleic acids of the invention.

[0026] In certain aspects, the invention provides a pharmaceutical composition comprising mRNAs encoding the inventive antibodies. In certain embodiments, these are optionally formulated in lipid nanoparticles (LNPs). In certain embodiments, the mRNAs are modified. Modifications include without limitations modified ribonucleotides, poly-A tail, 5 ’cap.

[0027] In certain aspects, the invention provides prophylactic methods comprising administering the pharmaceutical composition of the invention. In certain embodiments, the methods lead to protection from infection, disease, reduced severity of disease, including but not limited to reduced severity of symptoms and/or reduced duration of coronavirus infection and disease. [0028] In non-limiting embodiments, the coronavirus infection is caused by SARS-CoV-2. In non-limiting embodiments the disease is COVID19.

[0029] In certain aspects, the invention provides methods of treatment comprising administering the pharmaceutical composition of the invention. The pharmaceutical composition comprises at least one antibody of the invention formulated as a recombinant protein, a nucleic acid encoding the antibody or a combination thereof.

[0030] In certain embodiments, the methods comprise administering additional therapeutic or prophylactic agents, including but not limited to additional coronavirus neutralizing antibodies, small molecule therapeutics, or any other suitable agent. In certain embodiments, the coronavirus antibodies have different specificities.

[0031] In certain aspects, the invention provides a kit comprising: a composition comprising an antibody of the invention, a syringe, needle, or applicator for administration of the antibody to a subject; and instructions for use.

[0032] In certain aspects the invention provides a method of treating a subject, the method comprising steps of: administering to a subject suffering from or susceptible to coronavirus infection therapeutically effective amount of an antibody of the invention. In certain embodiments the antibody is administered as a therapeutic and/or prophylactic measure. Prophylactic methods contemplate pre-exposure administration. In some embodiments, the methods comprise administering an antibody of the invention as recombinant protein. In some embodiments, the methods comprise administering an antibody of the invention as a nucleic acid.

[0033] In some embodiments, the methods comprise administering a combination treatment with antibodies, wherein the combination comprises at least one inventive antibody. In some embodiments, at least one of the antibodies in a combination treatment is a neutralizing antibody. Without wishing to be bound by theory, a treatment method comprising a combination of antibodies targeting different epitopes can reduce viral neutralization escape.

[0034] In some embodiments, at least one or more of the antibodies has RBD specificity. In some embodiments, at least one or more of the antibodies is RBD/ACE2 blocking. In some embodiments, where for example two antibodies are administered, the antibodies have different epitopes.

[0035] Different epitopes could be located in the same area of the spike protein or different area of the spike protein, e.g. but not limited to the RBD, NTD, S2 etc.

[0036] In certain embodiments, the antibody used in the methods of the invention is any one of the antibodies from Table 3. In certain embodiments, the antibody is any one of DH1284, DH1286, DH1287, DH1288, DH1289, DH1290 DH1291, DH1292, DH1293, DH1294, DH1295, DH1296, DH1297, a fragment thereof, a variant thereof or a combination thereof. In certain embodiments, the methods comprise administering a combination, wherein at least one of the antibodies, a fragment thereof, a variant thereof in a combination is selected from Table 3. In some embodiments the combination comprises RBD/ACE2 blocking antibody, e.g. any of the antibodies of Table 3, or NTD binding antibodies, or a combination thereof. Non-crossblocking antibodies targeting RBD could be combined with an antibody targeting any other epitope, e.g., without limitation NTD targeting antibodies.

[0037] In certain embodiments, the antibody or antigen binding fragment preferentially or specifically binds to coronavirus spike protein. In certain embodiments, the VH domain and VL domain each have at least 90% sequence identity to the entire VH and VL domains, the CDRs, and or the framework, respectively, of an antibody listed in Table 3.

[0038] In certain embodiments, (a) VL domain CDRL1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRL1-3 regions of an antibody listed in Table 3, and (b) VH domain CDRH1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRH1-3 regions of an antibody listed in Table 3. [0039] In certain embodiments, wherein the antibody or fragment is human or fully-human.

[0040] In certain embodiments, the VH domain and VL domain of the antibody or fragment comprises framework regions that each have no more than 20, or 10 or 5 amino acid variations as compared to the corresponding framework regions of a human antibody listed in Tables 3. In certain embodiments, the VH domain and VL domain of the antibody or fragment comprises framework regions that each have each have at least 90% sequence identity as compared to the corresponding framework regions of a human antibody listed in Tables 3.

[0041] In certain embodiments, (a) VL domain CDRL1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRL1-3 regions of an antibody listed in Tables 3, (b) VH domain CDRH1-3 regions together have no more than 10 amino acid variations as compared to the corresponding CDRH1-3 regions of the antibody listed in Table 3, and (c) the VL domain and VH domain framework regions each have no more than 10 or 5 amino acid variations as compared to the corresponding framework regions of the human antibody listed in Table 3.

[0042] In certain embodiments, the antibody, or the antigen-binding fragment thereof comprises an Fc moiety. In certain embodiments, wherein the antibody, or antigen-binding fragment thereof, comprises a mutation(s) in the Fc moiety that reduces binding of the antibody to an Fc receptor and/or increases the half-life of the antibody.

[0043] In certain embodiments, the antibody, or the antigen binding fragment thereof, is a purified antibody, a single chain antibody, Fab, Fab', F(ab')2, Fv or scFv. [0044] In certain embodiments, the antibody is of any isotype.

[0045] In certain aspect the invention provide antibody, or the antigen-binding fragment thereof, for use as a medicament.

[0046] In certain aspect the invention provide a nucleic acid molecule comprising a polynucleotide encoding an antibody of the invention or the antigen-binding fragment thereof. [0047] In certain embodiments, the polynucleotide sequence comprises, consists essentially of or consists of a nucleic acid sequence according to any one of the sequences in Figure 7 or 8; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments, the functional variation is 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0048] In certain aspects the invention provides a vector comprising a nucleic acid molecule encoding an antibody of the invention or antigen binding fragment thereof.

[0049] In certain aspects the invention provides a cell expressing an antibody of the invention, or the antigen binding fragment thereof.

[0050] In certain aspects the invention provides pharmaceutical composition comprising an antibody of the invention, a combination of antibodies comprising at least one antibody of the invention or the antigen binding fragment thereof, a nucleic acid encoding an antibody of the invention or a fragment thereof and/or a cell expressing an antibody of the invention, or the antigen binding fragment thereof, and optionally a pharmaceutically acceptable carrier. In certain embodiments, the composition is suitable for pharmaceutical use.

[0051] In certain aspect, the composition further comprises a pharmaceutically acceptable excipient, diluent or carrier.

[0052] In certain aspects, the invention provides an in vitro transcription system to synthesize ribonucleic acids (RNAs) encoding antibodies of the invention, comprising: a reaction vessel, a DNA vector template comprising nucleic acid sequence encoding an antibody of the invention as described in Tables 3, and reagents for carrying out an in vitro transcription reaction that produces mRNA encoding an antibody or fragment thereof of the invention. In certain embodiments the mRNA is modified mRNA. [0053] In certain aspects, the invention provides a method for manufacturing an mRNA encoding an antibody or antigen binding fragment thereof, comprising: a. providing an in vitro transcription reaction vessel comprising a DNA template encoding an antibody or fragment thereof according to any of the preceding claims and reagents under conditions suitable for in vitro transcription of the nucleic acid template, thereby producing an mRNA template encoding an antibody or fragment thereof, and b. isolating the mRNA by any suitable method of purification and separating reaction reagents, the DNA template, and/or mRNA product related impurities. In certain embodiments, the mRNA comprises modified nucleotides. In certain embodiments, the mRNA comprises 5’ -CAP, and/or any other suitable modification.

[0054] In certain aspects, the invention provides a method for manufacturing an antibody or antigen binding fragment thereof, comprising culturing a host cell comprising a nucleic acid encoding an antibody of the invention under conditions suitable for expression of the antibody or fragment thereof and isolating said antibody or antigen binding fragment thereof.

[0055] In certain embodiments, the antibodies can be combined in one trispecific antibody with each arm of the antibody expressing a fragment of one of the antibodies in Table 3. See Ling Xu, et al. Science 06 Oct 2017: Vol. 358, Issue 6359, pp. 85-90 DOI: 10.1126/science.aan8630. [0056] In certain embodiments, the antibodies can be combined in various forms of bi-specific antibodies such as DARTS or other bispecific designs. See e.g. J Clin Invest 2015 Nov 2;125(l l):4077-90, doi: 10.1172/JCI82314. Epub 2015 Sep 28, Bispecific Antibodies Targeting Different Epitopes on the HIV-1 Envelope Exhibit Broad and Potent Neutralization. Asokan M, Rudicell RS, Louder M, McKee K, O'Dell S, Stewart-Jones G, Wang K, Xu L, Chen X, Choe M, Chuang G, Georgiev IS, Joyce MG, Kirys T, Ko S, Pegu A, Shi W, Todd JP, Yang Z, Bailer RT, Rao S, Kwong PD, Nabel GJ, Mascola JR.J Virol. 2015 Dec;89(24): 12501-12. doi: 10.1128/JVI.02097-15. Epub 2015 Oct 7.PMID: 26446600.

[0057] In certain aspect the invention provides a recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds to coronavirus spike protein and comprises a variable heavy (VH) domain and a variable light (VL) domain that have amino acid sequences that have an overall 80% sequence identity to the VH and VL domains of an antibody listed in Table 3, or wherein the VH domain and VL domain each have at least 80% sequence identity to the VH and VL domains, respectively, of an antibody listed in Table 3. In certain embodiments the antibodies are neutralizing antibodies. In certain embodiments, the antibody specifically binds to RBD of the coronavirus spike protein. In certain embodiments, the RBD binding antibodies block ACE2 receptor interaction. In certain embodiments, the antibody is specific for CoV2. In certain embodiments the antibody is crossreactive with other coronaviruses, e.g.SARS-CoV-1, MERS-CoV, 229E, NL63, HKU1, OC43, bat coronavirus, and/or pangolin coronavirus. In certain embodiments, the antibody, or the antigen-binding fragment thereof, wherein the concentration of the antibody, or antigen-binding fragment thereof, required for 50% neutralization of coronavirus, e.g but not limited to CoV2 virus, (IC50) is as described in Figures 4 and 5. In certain embodiments, IC50 is up to about 1 microg/ml, up to about 500 ng/ml, up to about 250 ng/ml, up to about 100 ng/ml or up to about 50 ng/ml.

[0058] In non-limiting embodiments, the antibody, or antigen binding fragment thereof binds to coronavirus domain RBD, NTD, or S2. In some embodiments, the antibody or antigen binding fragment can bind to the SI or S2 subunits of the coronavirus spike. In some embodiments, the antibody or antigen binding fragment can bind to the RBD, S2 helix, fusion domain, and the like of the coronavirus spike. In certain embodiments the binding specificity is as described in Example 5. In some embodiments, the DH1294 antibody can bind to a fusion domain of the coronavirus spike protein. In some embodiments, the DH1284 antibody can bind to an RBD domain of the coronavirus spike protein.

[0059] The antibody, or the antigen-binding fragment thereof, wherein the antibody, or antigenbinding fragment thereof, is a recombinant human monoclonal antibody.

[0060] In certain aspects the invention provides recombinant coronavirus antibody or antigen binding fragment thereof, as described in Table 3, e.g. without limitation DH1284, DH1286, DH1287, DH1288, DH1289, DH1290 DH1291, DH1292, DH1293, DH1294, DH1295, DH1296, or DH1297.

[0061] In certain embodiments the antibody or antigen binding fragment thereof, comprises a heavy chain (VH) comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain (VL) comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, comprises, consists essentially of or consists of an amino acid sequence according to any of the sequences listed in Figures 7 or 8, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments, the functional sequence variant has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. In certain embodiments, the sequence identity is within the CDRs. In certain embodiments, the sequence identity is within the framework regions.

[0062] In certain embodiments the antibody or antigen binding fragment thereof, comprises a heavy chain (VH) comprising CDRH1, CDRH2 and CDRH3 and a light chain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein the CDR, comprises, consists essentially of or consists of an amino acid sequence according to any of the sequences listed in Figures 7 or 8, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments, the functional sequence variant has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0063] In certain embodiments, (a) VI domain CDRL1-3 regions together have no more than 5, 6, 7 , 8, 9, or 10 amino acid variations as compared to the corresponding CDRL1-3 regions of an antibody listed in Table 3, (b) Vh domain CDRH1-3 regions together have no more than 5, 6, 7, 8, 9, or 10 amino acid variations as compared to the corresponding CDRH1-3 regions of the antibody listed in Table 3, and (c) the VI domain and Vh domain framework regions are derived from a human antibody.

[0064] In certain embodiments, (a) VI domain CDRL1-3 regions together have 1, 2, 3, 4 5, 6, 7 , 8, 9, or 10 amino acid variations as compared to the corresponding CDRL1-3 regions of an antibody listed in Table 3, (b) Vh domain CDRH1-3 regions together have 1, 2, 3, 4 5, 6, 7 , 8, 9, or 10 amino acid variations as compared to the corresponding CDRH1-3 regions of the antibody listed in Table 3, and (c) the VI domain and Vh domain framework regions are derived from a human antibody.

[0065] In certain embodiments, (a) VI domain framework regions together have 1, 2, 3, 4 5, 6, 7, 8, 9, or 10 amino acid variations as compared to the corresponding framework regions of an antibody listed in Table 3, (b) Vh domain framework regions together have 1, 2, 3, 4 5, 6, 7, 8, 9, or 10 amino acid variations as compared to the corresponding framework regions of the antibody listed in Table 3, and (c) the VI domain and Vh domain framework regions are derived from a human antibody.

[0066] In certain embodiments, the antibody or antigen binding fragment thereof, comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, comprises, consists essentially of or consists of an amino acid sequence according to any of the antibody paired Vh and VI sequences in Figure 7, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments, the functional sequence variant has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0067] In certain embodiments, the antibody or antigen binding fragment thereof, the antibody or antigen binding fragment thereof, comprises, consists essentially of or consists of paired antibody paired Vh and VI sequences in Figure 7 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments the functional sequence variant has 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0068] In certain embodiments, the antibody or antigen binding fragment thereof comprises, consists essentially of or consists of paired antibody DH1284 Vh and VI sequences in Figure 7. [0069] In certain embodiments, the antibody or antigen binding fragment thereof is any one of the antibodies from Table 3. In certain embodiments, the antibody is DH1284, DH1286, DH1287, DH1288, DH1289, DH1290 DH1291, DH1292, DH1293, DH1294, DH1295, DH1296, or DH1297.

[0070] The antibody, or the antigen binding fragment thereof, according to any of the previous paragraphs, wherein the antibody, or the antigen binding fragment thereof, is a purified antibody, a single chain antibody, Fab, Fab', F(ab')2, Fv or scFv.

[0071] In certain aspects, the invention provides an in vitro transcription system to synthesize ribonucleic acids (RNAs) encoding antibodies of the invention, comprising: a reaction vessel, a DNA vector template comprising nucleic acid sequence encoding an antibody of the invention as described in any of the preceding claims, and reagents for carrying out an in vitro transcription reaction that produces mRNA encoding an antibody or fragment thereof of the invention. In certain embodiments the mRNA is modified mRNA.

[0072] In certain aspects, the invention provides a method for manufacturing an mRNA encoding an antibody or antigen binding fragment thereof, comprising: a. providing an in vitro transcription reaction vessel comprising a DNA template encoding an antibody or fragment thereof according to any of the preceding claims and reagents under conditions suitable for in vitro transcription of the nucleic acid template, thereby producing an mRNA template encoding the antibody or fragment thereof according to any of the preceding claims, and b. isolating the mRNA by any suitable method of purification and separating reaction reagents, the DNA template, and/or mRNA product related impurities. In certain embodiments, the mRNA comprises modified nucleotides. In certain embodiments, the mRNA comprises 5' - CAP, and/or any other suitable modification.

[0073] In certain aspects, the invention provides a method for manufacturing an antibody or antigen binding fragment thereof, comprising culturing a host cell comprising a nucleic acid according to any of the preceding claims under conditions suitable for expression of the antibody or fragment thereof and isolating said antibody or antigen binding fragment thereof.

[0074] In certain aspects, the invention provides a method of treating or protecting against coronavirus infection comprising administering a composition comprising an antibody or fragment thereof comprising a Vh or VI sequence of any of the antibodies of the invention, including without limitation Vh and VI sequences comprised in a bi- or tri-specific antibody format, or multivalent antibody forms.

[0075] In certain aspects the invention provides multivalent antibodies comprising any of antibodies or antigen binding fragments described herein. In certain aspects the invention provides multispecific antibodies or antigen binding fragments thereof comprising any of the antibodies described herein. In non-limiting embodiments, the multispecific antibody comprises fragments from DH1284. In non-limiting embodiments, the multispecific antibody comprises fragments from DH1047. See WO App Number PCT/US2021/050552 for DH1047.

[0076] In certain aspects the invention provides therapeutic and/or prophylactic methods comprising administering a therapeutic and/or prophylactic amount in a subject in need thereof anyone of the antibodies or antigen binding fragments of the invention, including without limitation a purified antibody IgG antibody, a multivalent antibody, multispecific antibody, a single chain antibody, Fab, Fab', F(ab')2, Fv or scFv, or any combination thereof.

[0077] A recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds to coronavirus spike protein and comprises a variable heavy (VH) domain and a variable light (VL) domain that have amino acid sequences that have an overall 80% sequence identity to the VH and VL domains of an antibody listed in Table 3, or wherein the VH domain and VL domain each have at least 80% sequence identity to the VH and VL domains, respectively, of an antibody listed in Table 3.

[0078] In certain aspects, the invention provides a recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds coronavirus spike protein, which comprises: a. Vh domain CDRH1-3 regions from an antibody listed in Table 3; and/or b. VI domain CDRL1-3 regions from an antibody listed in Table 3, wherein the Vh and VI are from the same antibody; and c. Wherein the framework of the variable heavy (Vh) domain comprises amino acid sequences that have at least 90% sequence identity to the V gene, D gene and J gene of the Vh gene of the corresponding antibody from which the CDRs are derived and wherein the framework of the variable light (VI) domain comprises amino acid sequences that have at least 90% sequence identity to the V and J genes of the VI gene from the corresponding antibody from which the CDRs are derived. In nonlimiting embodiments, the antibody is DH1284.

[0079] In certain aspects, the invention provides a recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds coronavirus spike protein, which comprises: a. Vh domain CDRH1-3 regions from an antibody listed in Table 3; and/or b. VI domain CDRL1-3 regions from an antibody listed in Table 3, wherein the Vh and VI are from the same antibody; and c. Wherein the framework of the variable heavy (Vh) domain comprises amino acid sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V gene, D gene and J gene making up the Vh gene of the corresponding antibody from which the CDRs are derived and wherein the framework portions of the variable light (VI) domain comprises amino acid sequences that have at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V and J genes making up the VI gene from the corresponding antibody from which the CDRs are derived. In non-limiting embodiments, the antibody is DH1284.

[0080] In certain aspects, the invention provides a recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds coronavirus spike protein, which comprises: a. Vh domain CDRH1-3 regions from an antibody listed in Table 3; and/or b. VI domain CDRL1-3 regions from an antibody listed in Table 3, wherein the Vh and VI are from the same antibody; and c. Wherein the framework of the variable heavy (Vh) domain comprises amino acid sequences that have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V gene, D gene and J gene making up the Vh gene of the corresponding antibody from which the CDRs are derived and wherein the framework portions of the variable light (VI) domain comprises amino acid sequences that have 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , or 99% sequence identity to the V and J genes making up the VI gene from the corresponding antibody from which the CDRs are derived. In non-limiting embodiments, the antibody is DH1284.

[0081] In certain embodiments, the framework of the variable heavy (Vh) domain comprises, consists essentially of, consists of or has amino acid sequences derived from human IGHV1-24 and IGHJ4 Ig genes (See e.g. Table 3) and wherein the framework of the variable light (VI) domain comprises amino acid sequences derived from IGKVl-5and IGKJ2 human IgG genes (See e.g. Table 3).

[0082] In certain embodiments, the antibody or antigen binding fragment thereof, comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, comprises, consists essentially of or consists of an amino acid sequence according to any of paired antibody Vh and VI sequences in Figure 7, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments, the functional sequence variant has 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0083] In certain embodiments, the antibody or antigen binding fragment thereof, comprises, consists essentially of or consists of paired antibody Vh and VI sequences in Figure 7 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments the functional sequence variant has 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0084] In certain embodiments, the antibody, or antigen binding fragment thereof binds to coronavirus domain RBD, NTD, or S2.

[0085] In certain embodiments, the antibody, or antigen binding fragment thereof binds RBD. [0086] In certain embodiments, wherein the antibody, or antigen-binding fragment thereof, comprises an Fc moiety. In certain embodiments, wherein the antibody, or antigen-binding fragment thereof, comprises a mutation(s) in the Fc moiety, in certain embodiments the mutation reducing binding of the antibody to an Fc receptor, in certain embodiments the mutation increasing the half-life of the recombinant antibody. In certain embodiments, the Fc mutation is “LS” mutation. In certain embodiments, the Fc mutation is “4A” mutation.

[0087] In certain embodiments, the antibody, or the antigen binding fragment thereof, is a purified antibody IgG antibody, a multivalent antibody, multispecific antibody, a single chain antibody, Fab, Fab', F(ab')2, Fv or scFv. In certain embodiments, the antibody is of any isotype. [0088] In certain aspects, the invention provides an antibody, or the antigen-binding fragment thereof for use as a medicament. In certain embodiments, the use is in the prevention and/or treatment of coronavirus infection. In certain embodiments the coronavirus is CoV2.

[0089] In certain aspects, the invention provides a nucleic acid molecule comprising a polynucleotide encoding the antibody, or the antigen-binding fragment thereof, according to any of the preceding paragraphs. [0090] In certain embodiments, the polynucleotide sequence comprises, consists essentially of or consists of a nucleic acid sequence according to any one of the sequences in Figures 20-26, 46, 38 and 45; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments, the functional variation is 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0091] In certain embodiments the nucleic acid is a ribonucleic acids (RNA). In certain embodiments the RNA is mRNA which suitable for use and delivery as a therapeutic mRNA. In certain embodiment, the mRNA comprises a 5'-terminal CAP modification. In certain embodiments the mRNA comprises modified nucleotides. In certain embodiments, the mRNA is formulated in a lipid nanoparticle.

[0092] In certain aspects, the invention provides a vector comprising the nucleic acid molecule according to any of the preceding paragraphs.

[0093] In certain aspects, the invention provides a cell expressing the antibody, or the antigen binding fragment thereof, according to any of the preceding claims; or comprising a vector of the invention.

[0094] In certain aspects, the invention provides a pharmaceutical composition comprising the antibody, or the antigen binding fragment thereof, a nucleic acid encoding the same, a vector comprising the nucleic acid and/or a cell comprising the nucleic acid or vector, and optionally a pharmaceutically acceptable carrier. In certain embodiments the antibody is DH1284, or a combination of DH1284 with any other suitable coronavirus antibody. Non-limiting example of additional coronavirus antibodies are described in WO App Number PCT/US2021/050552. Additional coronavirus are also known in the art, e.g. without limitation antibodies generated by Regeneron, Vir Pharmaceuticals, Eli Lilly, the Vaccine Research Institute, etc.

[0095] In certain aspects, the invention provides a pharmaceutical composition comprising the antibody, or the antigen binding fragment thereof, wherein the antibody, or the antigen binding fragment thereof, comprise a Vh and VI sequence of any of the preceding claims, wherein the Vh and VI sequences form a multivalent or multispecific antibody. [0096] In certain aspects, the invention provides a pharmaceutical composition comprising at least one RBD binding antibody of the invention and at least one NTD binding antibody. Nonlimiting examples of NTD antibodies are described in WO App Number PCT/US2021/050552. [0097] In certain aspects, the invention provides a pharmaceutical composition comprising two RBD binding antibodies or antigen binding fragments thereof, wherein the two antibodies or antigen binding fragments thereof have non-overlapping epitopes.

[0098] In certain embodiments the compositions further comprises a pharmaceutically acceptable excipient, diluent, adjuvant or carrier.

[0099] In certain aspects, the invention provides a method of treating or preventing coronavirus infection in a subject in need thereof, comprising administering the antibody, or the antigen binding fragment thereof, a nucleic acid encoding the same, a vector comprising the nucleic acid and/or a cell comprising the nucleic acid or vector, or a pharmaceutical composition of the invention in an amount suitable to effect treatment or prevention of coronavirus infection.

[0100] In certain embodiments, the antibody is administered prior to coronavirus exposure or at the same time as coronavirus exposure.

[0101] In certain aspects, the invention provides a method of treating or protecting against coronavirus infection comprising administering therapeutic or prophylactic amount of a composition comprising an antibody or antigen binding fragment thereof comprising a Vh and VI sequence of any of the preceding paragraphs, wherein the Vh and VI sequences are comprised in a bi- or tri-specific antibody format and/or in a multivalent format. In certain aspects the invention provides, nucleic acid sequences encoding these bi- or tri-specific antibody formats and/or in a multivalent formats, including modified mRNAs suitable for pharmaceutical use and delivery.

[0102] Use of the multispecific or multivalent antibodies or fragments thereof, administer as recombinant proteins, nucleic acids, including without limitation mRNAs, or a combination thereof in prophylactic or therapeutic methods. The nucleic acids and recombinant proteins could be formulated in any suitable formulation, and optionally comprise an adjuvant. In some embodiments, the adjuvant is LNP. In some embodiments, the formulation comprises LNPs. BRIEF DESCRIPTION OF DRAWINGS

[0103] Figure 1. Serum neutralization of SARS-CoV-2 D614G pseudovirus infection in 293T/ACE2 cells. Fifty percent inhibitory dilution (ID50) and eighty percent inhibitory dilution (ID80) titers neutralization titers are shown as reciprocal serum dilution. Potent neutralization and modest neutralization are indicated to the right of the figure..

[0104] Figure 2. ELISA binding profile of SARS-CoV-2 RBD monoclonal antibodies isolated from PTID002. Darker shading indicates stronger binding. Binding titer is shown as area under the log-transformed curve (log AUC).

[0105] Figure 3. DH1284 and four other mAbs compete with ACE2 and other RBD antibodies for binding to SARS-CoV-2 Spike. Each graph represents an individual antibody. DH1284 IC50 was <0.045 mcg/mL.

[0106] Figure 4. DH1284 is the most potent neutralizing antibody isolated from Subject 002 in this study. Neutralization titer is shown as mcg/mL against SARS-CoV-2 Wuhan-1 D614G, B.1.351, and B.1.617.2

[0107] Figure 5. Human RBD antibody neutralization of SARS-CoV-2 ancestral and variant strains. Neutralization titer is shown as mcg/mL. DH1047 is a cross-neutralizing RBD antibody and DH1041 is a potent SARS-CoV-2 neutralizing RBD antibody whose epitope encompasses most of the ACE2 binding site.

[0108] Figure 6. Structure of DH1284 bound to SARS-CoV-2 spike ectodomain. The Fab of DH1284 was complexed to SARS-CoV-2 Spike HexaPro. The structure of the complex was determined by imaging 214,970 particles by negative stain electron microscopy. A 3D reconstruction of 21,494 of the particles was refined to give the final.

[0109] Figure 7 shows amino acid sequences of Vh and VI sequences of antibodies listed in Table 3. CDRs are underlined.

[0110] Figure 8 shows non-limiting embodiments of nucleic acid sequences encoding the Vh and VI sequences shown in Figure 7.

[0111] Figure 9 shows DH1047 and DH1284 Cross-reactive and potent Group 2b BetaCoronavirus Antibodies. DH1284- cross-reacts with many group 2b CoV and potently neutralizes all example CoV-2 variants.

[0112] Figure 10 shows Omicron Neutralization By Cross-reactive Neutralizing Antibodies including DH1284 in a Pseudovirus Assay. [0113] Figure 11 shows binding of DH1294 and DH1284 antibodies to various viral targets (higher numbers indicate better binding).

[0114] Figure 12 shows blocking of DH1284 and DH 1294 binding to an RBD target by Angiotensin-converting enzyme 2 (ACE-2).

[0115] Figure 13 shows IC50 titers (ng/ml) for antibody DH1294 and DH1284 binding to various viral targets.

[0116] Figure 14A-D shows a viral challenge study using DH1284 antibody. Twelve-months old BALB/c mice were infected with IxlO⁴ PFU RsSHC014-MA15 virus at day 0. In the prophylactic study, 300 ug of antibody was administered intraperitoneally 12 hours prior to infection. In the therapeutic study, the same amount of antibody was administered at the time of viral infection. A CH65 control was used. Figure 14A shows weight loss in the mice over time (days). Figure 14B shows virus titer in the mice at the end of the experiment. Figure 14C shows lung hemorrhage scores in the mice at the end of the experiment. Figure 14D shows survival of the mice over time.

[0117] Figure 15 shows results from live virus neutralization assays using DH1284 and DH1294 antibodies. ID50 neutralization titers are shown.

[0118] Figure 16 shows results from pseudotyped virus neutralization assays using DH1284 and DH1294 antibodies. ID50 neutralization titers are shown.

[0119] Figure 17 shows results from neutralization assays in 293T/ACE2 cells of various spike pseudotyped viruses transfected into 293T/17 cells. IC50 and IC80 neutralization titers are shown.

DETAILED DESCRIPTION

[0120] The present invention relates to antibodies and antigen binding fragments thereof, including recombinant and/or derivative forms that bind to coronavirus spike protein. In some embodiments, the antibodies or fragments bind specifically to epitopes on spike protein. In nonlimiting embodiments, the antibodies are RBD binding antibodies. In some non-limiting embodiments the RBD binding antibodies are ACE-2 blocking neutralizing antibodies. In some non-limiting embodiments the RBD binding antibodies are non- ACE-2 blocking neutralizing antibodies. In some non-limiting embodiments the RBD binding antibodies are SARS-CoV-2 neutralizing antibodies. In some non-limiting embodiments the RBD binding antibodies are cross reactive with SARS-CoV-1. In other non-limiting embodiments, the antibodies are NTD binding antibodies. In some non-limiting embodiments, the NTD antibodies are neutralizing antibodies.

RECOMBINANT ANTIBODIES

[0121] Recombinant antibodies of the invention include antibodies derived from rearranged VDJ variable heavy chain (VH) and/or rearranged VJ variable light chain (VL) sequences from individual or clonal cells that express an antibody that specifically binds to coronavirus spike protein, and optionally are neutralizing. Antibodies are described in the accompanying examples, figures, and tables, and the invention includes antibodies comprising CDR sequences contained with the VH and VL amino acid sequences described herein. In certain embodiments, the invention provides monoclonal antibodies. In certain embodiments the monoclonal antibodies are produced by a clone of B -lymphocytes. In certain embodiments the monoclonal antibody is recombinant and is produced by a host cell into which an expression vector(s) encoding the antibody, or fragment thereof, has been transfected.

[0122] Methods for obtaining rearranged heavy and light chain sequences are well known in the art and often involve amplification-based-cloning and sequencing. Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies or antibody fragments of the present invention. Desired DNA sequences may be synthesized completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate.

[0123] The invention encompasses antibodies which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 75% identical to the VH and/or VL variable domain amino acid sequences of the antibodies described herein in the Figures or Table 3. Further, the invention encompasses variants having one or mutations (99% et seq. per above) as compared to the sequences of the antibodies of the Figures or Table 3 with one or more of the additional requirements: (1) the variant maintains antigen binding specificity to the spike protein , and in some embodiments, maintains the ability to specifically bind an epitope that includes RBD, NTD, or S2, (2) the variant does not have a decrease in binding affinity or avidity that is more than 10-fold, 5-fold, 2-fold, or 1-fold than the corresponding antibody of the Figures or Table 3, (3) the variant has a binding affinity or avidity that is an improvement of more than 100-fold, 10-fold, 5-fold, 2-fold, or 1-fold more than the corresponding antibody of the Figures or Table 3, (4) the variant does not have a decrease in promoting neutralization that is more than 10-fold, 5-fold, 2-fold, or 1-fold as compared to the corresponding antibody of the Figures or Table 3 , (5) the variant has an increase in neutralization that is 10-fold, 5-fold, 2-fold, or 1-fold more as compared to the corresponding antibody of the Figures or Tables, and (6) as compared to the antibodies listed in the Figures or Table 3 , the variant has a V region with shared mutations compared to the germline, identical VDJ or VJ gene family usage, identical or the same or similar HCDR3 length, and the same VL and JLgene family usage. For example, one or more of these six requirements are applicable to any antibody, including fragments (see below, Fab, Fv, et al.) or portions (VH, VL, one or more CDRs from a VH/VL pair) thereof, derived from the antibodies listed in Table 3 or the Figures. Various figure provide non-limiting embodiments of nucleic acids and plasmids for expression of mRNA encoded antibodies.

[0124] With respect to the antibodies listed in Table 3, their corresponding sequences are provided in at least Figures 7 and 8, where VH and VL portions are indicated. CDR regions are determined by any suitable method, e.g. but not limited by IMGT

(http .//w w.im uest/). The boundaries of these CDR regions can be modified or

substituted according to other CDR conventions as known in the art and as described herein. [0125] Binding specificity can be determined by any suitable assay in the art, for example but not limited competition binding assays, epitope mapping, structural studies of antibodies, or fragments thereof, bound to target envelopes, etc.

[0126] Affinity can be measured, for example, by surface plasmon resonance. It is well-known in the art how to conduct SPR for measuring antibody affinity to an antigen. SPR affinity measurements can provide the affinity constant KD of an antibody, which is based on the association rate constant k_on divided by the disassociation rate constant k_Off. Thus, in certain embodiments, comparing affinity between a variant and an antibody of the invention (e.g. Table 3) is based on KD. In other embodiments, the comparison is based only on k_Off. When comparing affinity between antibodies, the antibodies should have the same valency, i.e., Fab vs. Fab, scFv vs. scFv, IgG v. IgG, IgM v. IgM, etc. Thus, when the comparison is between antibodies that are not monovalent, then affinity is a measure of functional affinity. In the art, functional affinity covers the binding strength of a bi- or polyvalent antibody to antigens that present more than one copy of an epitope, because they are multimeric or conjugated in multiple copies to a solid phase, thus allowing cross-linking by the antibody. It is known in the art that a monovalent antibody fragment (e.g., Fab) provides a measure of intrinsic affinity irrespective of the density of antigens (SPR often immobilizes antigen on a solid substrate and the antibody is flowed over the substrate thereby allowing kinetic measurements of antibody association and disassociation rates).

[0127] Avidity can also be measured by SPR. Avidity can be quantitatively expressed, for example, by the ratio of KD for a Fab over the multivalent form, e.g., IgG, IgM..

[0128] Potency can be measured, for example, by a virus inhibition pseudovirus assay or authentic virus Plaque Reduction Neutralization Test).

[0129] In certain embodiments, the invention provides antibodies with CDR amino acid sequences that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to the CDR1, 2, and/or 3 of VH (also referred to as CDRH1, CDRH2, and CDRH3) and/or CDR1, 2, and/or 3 of VL (also referred to as CDRL1, CDRL2, and CDRL3) amino acid sequences of the antibodies of Table 3.

[0130] In certain embodiments, the invention provides antibodies with CDR amino acid sequences that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80% identical to CDRs to an antibody of Table 3, where each CDR can have a different percent identity. For example, the antibody has at least 99%, 98%, 97%, 96%, or 95% identity for all CDRs as compared to the CDRs of an antibody listed in Table 3-6 except HCDR3 and LCDR3, which can allow for a lower percent identity, for example, 99% to 80%, 99% to 85%, 99% to 90%, or 99% to 95%.

[0131] In certain embodiments, the invention provides antibodies which can tolerate a larger percent variation in the sequences outside of the VH and/VL sequences of the antibodies. In certain embodiments, the invention provides antibodies which can tolerate a larger percent variation in the sequences outside of the CDRs sequences, e.g. within the framework, of the antibodies. In certain embodiments, the invention provides antibodies which are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65% identical, wherein the identity is outside of the VH or VL regions, or the CDRs of the VH or VL chains of the antibodies described herein. [0132] In some aspects, the antibody or antigen binding fragment thereof, comprises a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3 and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR, comprises, consists essentially of or consists of an amino acid sequence according to any of the sequences of the instant antibodies, or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments, the functional variation is 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity. As stated herein, the person of ordinary skill in the art can select from one or more CDR conventions to identify the boundaries of the CDR regions.

[0133] In some aspects, the antibody or antigen binding fragment thereof, comprises, consists essentially of or consists of a VH amino acid sequence or a VL amino acid sequence in Figure 7 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. In certain embodiments the functional variation is 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

[0134] In some aspects, the antibody or antigen-binding fragment thereof, comprises, consists essentially of or consists of a VH amino acid sequence and/or a VL amino acid sequence according to Figure 7.

[0135] Furthermore, the invention provides antibodies that are affinity matured in vitro. The affinity of an antibody to its antigen target can be modulated by identifying mutations introduced into the variable region or into targeted sub-regions. For example, it is known in the art that one can sequentially introduce mutations through each of the CDRs, optimizing one at a time, or to focus on CDRH3 and CDRL3, or CDRH3 alone, because it often forms the majority of antigen contacts. Alternatively, it is known in the art how to simultaneously mutagenize all six CDRs by generating large-scale, high-throughput expression and screening assays, such as by antibody phage display. Antibody-antigen complex structural information can also be used to focus affinity maturation to a small number of residues in the antibody binding site. [0136] Various algorithms for sequence alignment are known in the art. For example, the NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403, 1990) is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Md.) and on the internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. A description of how to determine sequence identity using this program is available on the NCBI website on the internet.

[0137] The similarity between amino acid sequences is expressed in terms of the similarity between the sequences, otherwise referred to as sequence identity. Sequence identity is frequently measured in terms of percentage identity (or similarity or homology); the higher the percentage, the more similar the two sequences are. Homologs or variants of a polypeptide will possess a relatively high degree of sequence identity when aligned using standard methods.

[0138] CDRs and Frameworks

[0139] In some embodiments, the CDRs of the antibodies and fragments of the invention are defined according to the IMGT scheme. IMGT-defmed CDR regions have been highlighted/underlined in the nucleotide and amino acid sequences for each of the VH and VL variable regions of the antibodies of Table 3. (See Figures 20, 22.) IMGT sequence analysis tools will identify CDR and framework regions in the nucleotide sequence and translated amino acid sequence. See http://www.imgt.org/IMGT_vquest/anaiysis

[0140] In some embodiments, CDR and framework regions can be identified based on other classical variable region numbering and definition schemes or conventions, including the Kabat, Chothia, Martin, and Aho schemes. The ANARCI (Antigen receptor Numbering And Receptor Classification; see http://opig.stats.ox.ac.uk/webapps/newsabdab/sabpred/anarci/) online tool allows one to input amino acid sequences and to select an output with the IMGT, Kabat, Chothia, Martin, or AHo numbering scheme. With these numbering schemes, CDR and framework regions within the amino acid sequence can be identified. The person of ordinary skill is able to ascertain CDR and framework boundaries using one or more of several publicly available tools and guides.

[0141] Different methods of identifying CDRs are well known and described in the art (e.g. Kabat, Chothia, IMGT). Delineating CDRs by any one of these methods would result in CDRs with specific boundaries within a VH or VL sequence as listed herein. CDRs identified by any one of the methods are specific and well defined. See, for example, Martin, A.C..R, "Chapter3: Protein Sequence and Structure Analysis of Antibody Variable Domains," Antibody Engineering, vol. 2 (2nd ed.), Springer-Verlag, Berlin Heidelberg pp. 33-51 (2010) (describing inter alia Kabat, Chothia, IMGT); and Munshaw, S. and Kepler, T.B., "SoDA2: a Hidden Markov Model approach for identification of immunoglobulin rearrangements," Bioinformatics, vol. 26, No. 7, pp. 867-872 (Feb. 2010) (describing SoDA2). Any of these methods for identifying CDRs may be used with the instant technology.

[0142] For example, provides below is a general, not limiting guide, for the CDR regions as based on different numbering schemes (see http://www.bioinf.org.Uk/abs/info.html#cdrid). In the Table, any of the numbering schemes can be used for these CDR definitions, except the Contact CDR definition uses the Chothia or Martin (Enhanced Chothia) numbering.

[0143] Table 1. General Guide of Selected CDR Definitions

CDR Loop Kabat CDR AbM CDR Chothia Contact IMGT CDR Definition Definition CDR CDR Definition Definition Definition

CDRL1 L24-L34 L24-L34 L24-L34 L30-L36 L27-L32 CDRL2 L50-L56 L50-L56 L50-L56 L46-L55 L50-L51 CDRL3 L89-L97 L89-L97 L89-L97 L89-L96 L89-L97 CDRH1 H31-H35B H26-H35B H26- H30-H35B H26-H35B (Kabat H32..H34 numbering) CDRH1 H31-H35 H26-H35 H26-H32 H30-H35 H26-H33 (Chothia numbering)

CDRH2 H50-H65 H50-H58 H52-H56 H47-H58 H51-H56 CDRH3 H95-H102 H95-H102 H95-H102 H93-H101 H93-H102

[0144] In the table above, for CDRH1 Kabat numbering using the Chothia CDR definition, the boundary is H26 to H32 or H34 because the Kabat numbering convention varies between H32 and H34 depending on the length of the loop. This is because the Kabat numbering scheme places insertions at H35A and H35B. If neither H35A nor H35B is present, CDRH2 ends at H32. If only H35A is present, the loop ends at H33. If both H35A and H35B are present, the loop ends at H34.

[0145] Alternatively or in combination, one can examine amino acid sequences and identify CDR and framework regions according to the following alternative general guideline of Table AB below, which is non-limiting. 1 [0146] Table 2. General Guide for Demarcating CDR Boundaries

CDR Region Guidelines

LCDR1 Start: approx, residue 24

Residue before: always a Cys

Residue after: always a Trp. Typically Trp-Tyr-Gln, but also, Trp-Leu-Gln, Trp-Phe-Gln, Trp-Tyr-Leu

Length: 10 to 17 residues

LCDR2 Start: always 16 residues after the end of LI. Residues before generally Ile- Tyr, but also, Val-Tyr, Ile-Lys, Ile-Phe

Length: always 7 residues (except NEW (7FAB) which has a deletion in this region)

LCDR3 Start: always 33 residues after end of L2 (except NEW (7FAB) which has the deletion at the end of CDR-L2). Residue before always Cys. Residues after always Phe-Gly-XXX-Gly Length 7 to 11 residues

HCDR1 Start

Approx, residue 26 (always 4 after a Cys) [Chothia / AbM definition];

Kabat definition starts 5 residues later Residues before always Cys-XXX-XXX-XXX

Residues after always a Trp. Typically Trp-Val, but also, Trp-Ile, Trp-Ala

Length

10 to 12 residues [AbM definition];

Chothia definition excludes the last 4 residues

HCDR2 Start always 15 residues after the end of Kabat / AbM definition) of CDR-H1 Residues before typically Leu-Glu-Trp-Ile-Gly, but a number of variations Residues after

Ly s/ Arg-Leu/Il e/ V al/Phe/Thr/ Al a-Thr/Ser/Il e/ Al a Length

Kabat definition 16 to 19 residues;

AbM (and recent Chothia) definition ends 7 residues earlier

HCDR3 Start always 33 residues after end of CDR-H2 (always 2 after a Cys) Residues before always Cys-XXX-XXX (typically Cys- Ala-Arg)

Residues after always Trp-Gly-XXX-Gly Length

3 to 25 residues

[0147] In Figures showing sequences of the inventive antibodies, CDR boundaries can be defined according to any method, e.g. the IMGT scheme. Because framework (FR) regions constitute all of the variable domain sequence outside of the CDRs, once CDR boundaries are identified, framework regions are necessarily identified. The convention within the art is to label the framework regions as FR1 (sequence before CDR1), FR2 (sequence between CDR1 and CDR2), FR3 (sequence between CDR2 and CDR3), and FR4 (sequence after CDR3).

[0148] CDR and framework regions can also be demarcated using other numbering schemes and CDR definitions. The ABnum tool numbers the amino acid sequences of variable domains according to a large and regularly updated database called Ahysis, which takes into account insertions of variable lengths and integrates sequences from Kabat, IMGT, and the PDB databases. The Honneger scheme is based on structural alignments of the 3D structures of immunoglobulin variable regions and allows one to define structurally conserved Ca positions and deduction of appropriate framework regions and CDR lengths (Honegger and Pliickthun, J. Mol. Biol., 2001, 309:657-70). Similarly, Ofran et al. used a multiple structural alignment approach to identify the antigen binding residues of the variable regions called “Antigen Binding Regions (AB Rs)” (Ofran et al., J. ImunoL, 2008, 181 :6230-5). ABRs can be identified using the Paratome online tool that identifies ABR by comparing the antibody sequence with a set of antibody-antigen structural complexes (Kunik et al., Nucleic Acids Res., 2012, 40:521-4). Another alternative tool is the proABC software, which estimates the probabilities for each residue to form an interaction with the antigen (Olimpieri et al., Bioinformatics, 2013, 29:2285- 91).

[0149] In some embodiments, the CDRs of the antibodies of the invention are defined by the scheme or tool that provides the broadest or longest CDR sequence. In some embodiments, the CDRs are defined by a combination of schemes or tools that provides the broadest/longest CDRs. For example, from the Table of CDR Definitions above, CDRL1 would be L24-L36, CDRL2 would be L46-L56, CDR3 would be L89-L97, CDRH1 would be H26-H35/H35B, CDRH2 would be H47-H65, and CDRH3 would be H93-H102. In some embodiments, the CDRs are defined by the Anticalign software, which automatically identifies all hypervariable and framework regions in experimentally elucidated antibody sequences from an algorithm based on rules from the Kabat and Chothia conventions (Jarasch et al., Proteins Struct. Funct. Bioinforma, 2017, 85:65-71). In some embodiments, the CDRs are defined by a combination of the Kabat, IMGT, and Chothia CDR definitions. In some embodiments, the CDRs are defined by the Martin scheme in combination with the Kabat and IMGT schemes. In some embodiments, the CDRs are defined by a combination of the Martin and Honneger schemes. In some embodiments, the CDRs comprise the ABR residues identified by the Paratome tool. [0150] The complete human immunoglobulin germline gene loci and alleles are available in the Immunogenetics Database (IMGT). Skilled artisan can readily determine the V, D, and/or J heavy and/or light sequences of various embodiments of antibodies of the invention of fragments there of.

[0151] Fragments and Other Engineered Antibody Forms

[0152] In certain embodiments the invention provides antibody fragments, which have the binding specificity and/or properties of the inventive antibodies. Recombinant fragments of the antibodies can be obtained by cloning and expression of part of the sequences of the heavy or light chains.

[0153] Antibody "fragments" include Fab, Fab', F(ab')2, F(ab)c, diabodies, Dabs, nanobodies, and Fv fragments. Also included are heavy or light chain monomers and dimers, single domain heavy chain antibodies, single domain light chain antibodies, (a single domain antibody, sdAb, is also referred to in the art as a nanobody) as well as single chain antibodies, e.g., single chain Fv in which the heavy and light chain variable domains are joined by a peptide linker. (See, e.g., Bird et al., Science 242:423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988; McCafferty et al., Nature 348:552-554, 1990). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.

[0154] A recombinant antibody can also comprise a heavy chain variable domain from one antibody and a light chain variable domain from a different antibody. Further, the invention encompasses chimeric antigen receptors (CARs; chimeric T cell receptors) engineered from the variable domains of antibodies. (Chow et al, Adv. Exp. Biol. Med., 2012, 746: 121-41). The Chimeric Antigen Receptor (CAR) consists of an antibody-derived targeting domain (including fragments such as scFv or Fab) fused with T-cell signaling domains that, when expressed by a T- cell, endows the T-cell with antigen specificity determined by the targeting domain of the CAR. [0155] Fc Domains

[0156] Whether full-length, or fragments engineered to have a Fc domain, (or particular constant domain portions thereof), the antibodies of the invention can be of any isotype or have any Fc (or portion thereof) of any isotype. It is well-known in the art how to engineer Fc domains or portions together with antibody fragments.

[0157] In certain embodiments, the antibodies of the invention can be used as IgGl, IgG2, IgG3, IgG4, whole IgGl or IgG3s, whole monomeric IgAs, dimeric IgAs, secretory IgAs, IgMs as monomeric, pentameric, hexameric, or other polymer forms of IgM. The class of an antibody comprising the VH and VL chains described herein can be specifically switched to a different class of antibody by methods known in the art.

[0158] In some embodiments, the nucleic acid encoding the VH and VL can encode an Fc domain (immunoadhesin). The Fc domain can be an IgA, IgM or IgG Fc domain. The Fc domain can be an optimized Fc domain, as described in U.S. Published Patent Application No. 20100093979, incorporated herein by reference. In one example, the immunoadhesin is an IgGl Fc. In one example, the immunoadhesin is an IgG3 Fc.

[0159] In one embodiment, the IgG constant region comprises the LS mutation. Additional variants of the Fc portion of the antibody are also contemplated by the invention. See Maeda et al. MAbs. 2017 Jul; 9(5): 844-853. Published online 2017 Apr 7, PMID: 28387635; see also Booth et al. MAbs. 2018 Oct; 10(7): 1098-1110. Published online 2018 Jul 26. doi: 10.1080/19420862.2018.1490119.

[0160] In certain embodiments the antibodies comprise amino acid alterations, or combinations thereof, for example in the Fc region outside of epitope binding, which alterations can improve their properties. Various Fc modifications are known in the art.

[0161] In some embodiments, the invention contemplates antibodies comprising mutations that affect neonatal Fc receptor (FcRn) binding, antibody half-life, and localization and persistence of antibodies at mucosal sites. See e.g. Ko SY et al., Nature 514: 642-45, 2014, at Figure la and citations therein; Kuo, T. and Averson, V., mAbs 3(5): 422-430, 2011, at Table 1, US Pub 20110081347 (an aspartic acid at Kabat residue 288 and/or a lysine at Kabat residue 435), US Pub 20150152183 for various Fc region mutation, incorporated by reference in their entirety.

[0162] In certain embodiments, the antibodies comprise AAAA substitution in and around the Fc region of the antibody that has been reported to enhance ADCC via NK cells (AAA mutations) containing the Fc region aa of S298A as well as E333A and K334A (Shields RI et al JBC , 276: 6591-6604, 2001) and the 4^th A (N434A) is to enhance FcR neonatal mediated transport of the IgG to mucosal sites (Shields RI et al. ibid).

[0163] Other antibody mutations have been reported to improve antibody half-life or function or both and can be incorporated in sequences of the antibodies. These include the DLE set of mutations (Romain G, et al. Blood 124: 3241, 2014), the LS mutations M428L/N434S, alone or in a combination with other Fc region mutations, (Ko SY et al. Nature 514: 642-45, 2014, at Figure la and citations therein; Zlevsky et al., Nature Biotechnology, 28(2): 157-159, 2010; US Pub 20150152183); the YTE Fc mutations (Robbie G et al Antimicrobial Agents and Chemotherapy 12: 6147-53, 2013) as well as other engineered mutations to the antibody such as QL mutations, IHH mutations (Ko SY et al. Nature 514: 642-45, 2014, at Figure la and relevant citations; See also Rudicell R et al. J. Virol 88: 12669-82, 201).

[0164] In some embodiments, modifications, such as but not limited to antibody fucosylation, may affect interaction with Fc receptors (See e.g. Moldt, et al. IVI 86(11): 66189-6196, 2012). In some embodiments, the antibodies can comprise modifications, for example but not limited to glycosylation, which reduce or eliminate polyreactivity of an antibody. See e.g. Chuang, et al. Protein Science 24: 1019-1030, 2015.

[0165] In some embodiments the antibodies can comprise modifications in the Fc domain such that the Fc domain exhibits, as compared to an unmodified Fc domain enhanced antibody dependent cell mediated cytotoxicity (ADCC); increased binding to Fc.gamma.RIIA or to Fc.gamma.RIIIA; decreased binding to Fc.gamma.RIIB; or increased binding to Fc.gamma.RIIB. See e.g. US Pub 20140328836.

[0166] Multivalent and Multispecific Antibodies

[0167] In certain embodiments the invention provides a multivalent and multispecific antibody. A multivalent antibody has at least two antigen-binding sites, i.e., at least two heavy /light chain pairs, or fragments thereof. When the heavy /light pairs of a multivalent antibody bind to different epitopes, whether on the same antigen or on different antigens, the antibody is considered to be multispecific. Antibody fragments may impart monovalent or multivalent interactions and be contained in a variety of structures as described above. For instance, monovalent scFv molecules may be synthesized to create a bivalent diabody, a trivalent "triabody" or a tetravalent "tetrabody." The scFv molecules may include a domain of the Fc region resulting in bivalent minibodies. In addition, the sequences of the invention may be a component of multispecific molecules in which the sequences of the invention target the epitopes of the invention and other regions of the molecule bind to other targets. Exemplary molecules include, but are not limited to, bispecific Fab2, trispecific Fab3, bispecific scFv, and diabodies (Holliger and Hudson, 2005, Nature Biotechnology 9: 1126-1136).

[0168] In some embodiments, multivalent but not multispecific antibodies are provided, where the multivalent antibody comprises multiple identical VH/VL pairs, or the CDRs from the VH and a VL pairs. This type of multispecific antibody will serve to improve the avidity of an antibody. For example, a tetramer can comprise four identical scFvs where the scFv is based on the VH/VL pair from an antibody of Table 3.

[0169] In some embodiments, multivalent but not multispecific antibodies comprise multiple VH/VL pairs (or the CDRs from the pairs) where each pair binds to an overlapping epitope. In some embodiments, multivalent but not multispecific antibodies comprise multiple VH/VL pairs (or the CDRs from the pairs) where each pair binds to an non-overlapping epitope. Determining overlapping epitopes can be conducted, for example, by structural analysis of the antibodies and competitive binding assays as known in the art.

[0170] In some embodiments, multispecific antibodies comprise multiple VH/VL pairs (or the CDRs from the pairs) where each pair binds to a distinct epitope (not overlapping) spike protein. [0171] In some embodiments, multispecific antibodies or fragments of the invention comprise at least a VH and a VL pair from Table 3, or the CDRs from the VH and a VL pair, in order to provide the multispecific antibody with binding specificity to the spike protein. The multispecific antibody can have one or more additional binding specificities by further comprising antibody binding site fragments from antibodies that bind to different antigens.

[0172] In certain embodiments the invention provides a bispecific antibody. A bispecific or bifunctional/dual targeting antibody is an artificial hybrid antibody having two different heavy /light chain pairs and two different binding sites (see, e.g., Romain Rouet & Daniel Christ “Bispecific antibodies with native chain structure” Nature Biotechnology 32, 136-137 (2014); Garber “Bispecific antibodies rise again” Nature Reviews Drug Discovery 13, 799-801 (2014), Figure la; Byrne et al. “A tale of two specificities: bispecific antibodies for therapeutic and diagnostic applications” Trends in Biotechnology, Volume 31, Issue 11, November 2013, Pages 621-632 Songsivilai and Lachmann, Clin. Exp. Immunol., 79:315-321 (1990); Kostelny et al., J. Immunol. 148: 1547-53 (1992) (and references therein)). In certain embodiments the bispecific antibody is a whole antibody of any isotype. In other embodiments it is a bispecific fragment, for example but not limited to F(ab)2 fragment. In some embodiments, the bispecific antibodies do not include Fc portion, which makes these diabodies relatively small in size and easy to penetrate tissues.

[0173] Non-limiting examples of multispecific antibodies also include: (1) a dual-variable- domain antibody (DVD-Ig), where each light chain and heavy chain contains two variable domains in tandem through a short peptide linkage (Wu et al., Generation and Characterization of a Dual Variable Domain Immunoglobulin (DVD-Ig. TM.) Molecule, In: Antibody Engineering, Springer Berlin Heidelberg (2010)); (2) a Tandab, which is a fusion of two single chain diabodies resulting in a tetravalent bispecific antibody that has two binding sites for each of the target antigens; (3) a flexibody, which is a combination of scFvs with a diabody resulting in a multivalent molecule; (4) a so called "dock and lock" molecule, based on the "dimerization and docking domain" in Protein Kinase A, which, when applied to Fabs, can yield a trivalent bispecific binding protein consisting of two identical Fab fragments linked to a different Fab fragment; (5) a so-called Scorpion molecule, comprising, e.g., two scFvs fused to both termini of a human Fc-region. Examples of platforms useful for preparing bispecific antibodies include but are not limited to BiTE (Micromet), DART (MacroGenics) (e.g., US Patents 8,795,667; US Publications 20090060910; 20100174053), Fcab and Mab2 (F-star), Fc-engineered IgGl (Xencor) or DuoBody (based on Fab arm exchange, Genmab).

[0174] In certain embodiments, the multispecific antibodies can include an Fc region. For example, Fc bearing DARTs are heavier, and could bind neonatal Fc receptor, increasing their circulating half-life. See Garber “Bispecific antibodies rise again” Nature Reviews Drug Discovery 13, 799-801 (2014), Figure la; See US Pub 20130295121, incorporated by reference in their entirety.

[0175] The invention also provides trispecific antibodies comprising binding specificities of the inventive antibodies. Non-limiting embodiments of trispecific format is described in Xu et al. Science 06 Oct 2017, Vol. 358, Issue 6359, pp. 85-90; US Pub 20190054182; US Pub 20200054765.

[0176] In certain embodiments, the invention encompasses multispecific molecules comprising an Fc domain or portion thereof (e.g. a CH2 domain, or CH3 domain). The Fc domain or portion thereof may be derived from any immunoglobulin isotype or allotype including, but not limited to, IgA, IgD, IgG, IgE and IgM. In some embodiments, the Fc domain (or portion thereof) is derived from IgG. In some embodiments, the IgG isotype is IgGl, IgG2, IgG3 or IgG4 or an allotype thereof.

[0177] In some embodiments, the multispecific molecule comprises an Fc domain, which Fc domain comprises a CH2 domain and CH3 domain independently selected from any immunoglobulin isotype (i.e. an Fc domain comprising the CH2 domain derived from IgG and the CH3 domain derived from IgE, or the CH2 domain derived from IgGl and the CH3 domain derived from IgG2, etc.). In some embodiments, the Fc domain may be engineered into a polypeptide chain comprising the multispecific molecule of the invention in any position relative to other domains or portions of the polypeptide chain (e.g., the Fc domain, or portion thereof, may be c-terminal to both the VL and VH domains of the polypeptide of the chain; may be n- terminal to both the VL and VH domains; or may be N-terminal to one domain and c-terminal to another (i.e., between two domains of the polypeptide chain)).

[0178] The present invention also encompasses molecules comprising a hinge domain. The hinge domain be derived from any immunoglobulin isotype or allotype including IgA, IgD, IgG, IgE and IgM. In some embodiments, the hinge domain is derived from IgG, wherein the IgG isotype is IgGl, IgG2, IgG3 or IgG4, or an allotype thereof. The hinge domain may be engineered into a polypeptide chain comprising the multispecific molecule together with an Fc domain such that the multispecific molecule comprises a hinge-Fc domain. In certain embodiments, the hinge and Fc domain are independently selected from any immunoglobulin isotype known in the art or exemplified herein. In other embodiments the hinge and Fc domain are separated by at least one other domain of the polypeptide chain, e.g., the VL domain. The hinge domain, or optionally the hinge-Fc domain, may be engineered into a polypeptide of the invention in any position relative to other domains or portions of the polypeptide chain. In certain embodiments, a polypeptide chain of the invention comprises a hinge domain, which hinge domain is at the C-terminus of the polypeptide chain, wherein the polypeptide chain does not comprise an Fc domain. In yet other embodiments, a polypeptide chain of the invention comprises a hinge-Fc domain, which hinge-Fc domain is at the C-terminus of the polypeptide chain. In further embodiments, a polypeptide chain of the invention comprises a hinge-Fc domain, which hinge-Fc domain is at the N-terminus of the polypeptide chain. [0179] In some embodiments, the invention encompasses multimers of polypeptide chains, each of which polypeptide chains comprise a VH and a VL domain, comprising CDRs as described herein. In certain embodiments, the VL and VH domains comprising each polypeptide chain have the same specificity, and the multimer molecule is bivalent and monospecific. In other embodiments, the VL and VH domains comprising each polypeptide chain have differing specificity and the multimer is bivalent and bispecific. In some embodiments, the polypeptide chains in multimers further comprise an Fc domain. Dimerization of the Fc domains leads to formation of a diabody molecule that exhibits immunoglobulin-like functionality, i.e., Fc mediated function (e.g., Fc-Fc.gamma.R interaction, complement binding, etc.).

[0180] In yet other embodiments, diabody molecules of the invention encompass tetramers of polypeptide chains, each of which polypeptide chain comprises a VH and VL domain. In certain embodiments, two polypeptide chains of the tetramer further comprise an Fc domain. The tetramer is therefore comprised of two 'heavier' polypeptide chains, each comprising a VL, VH and Fc domain, and two Tighter' polypeptide chains, comprising a VL and VH domain. Interaction of a heavier and lighter chain into a bivalent monomer coupled with dimerization of the monomers via the Fc domains of the heavier chains will lead to formation of a tetraval ent immunoglobulin-like molecule. In certain aspects the monomers are the same, and the tetravalent diabody molecule is monospecific or bispecific. In other aspects the monomers are different, and the tetravalent molecule is bispecific or tetraspecific.

[0181] Formation of a tetraspecific diabody molecule as described supra requires the interaction of four differing polypeptide chains. Such interactions are difficult to achieve with efficiency within a single cell recombinant production system, due to the many variants of chain mispairings. One solution to increase the probability of mispairings, is to engineer "knobs-into- holes" type mutations into the desired polypeptide chain pairs. Such mutations favor heterodimerization over homodimerization. For example, with respect to Fc-Fc-interactions, an amino acid substitution (preferably a substitution with an amino acid comprising a bulky side group forming a knob', e.g., tryptophan) can be introduced into the CH2 or CH3 domain such that steric interference will prevent interaction with a similarly mutated domain and will obligate the mutated domain to pair with a domain into which a complementary, or accommodating mutation has been engineered, i.e., 'the hole' (e.g., a substitution with glycine). Such sets of mutations can be engineered into any pair of polypeptides comprising the diabody molecule, and further, engineered into any portion of the polypeptides chains of the pair. Methods of protein engineering to favor heterodimerization over homodimerization are well known in the art, in particular with respect to the engineering of immunoglobulin-like molecules, and are encompassed herein (see e.g., Ridgway et al. (1996) "'Knobs-Into-Holes' Engineering Of Antibody CH3 Domains For Heavy Chain Heterodimerization," Protein Engr. 9:617-621, Atwell et al. (1997) "Stable Heterodimers From Remodeling The Domain Interface Of A Homodimer Using A Phage Display Library," J. Mol. Biol. 270: 26-35, and Xie et al. (2005) "A New Format Of Bispecific Antibody: Highly Efficient Heterodimerization, Expression And Tumor Cell Lysis," J. Immunol. Methods 296:95-101; each of which is hereby incorporated herein by reference in its entirety).

[0182] The invention also encompasses diabody molecules comprising variant Fc or variant hinge-Fc domains (or portion thereof), which variant Fc domain comprises at least one amino acid modification (e.g. substitution, insertion deletion) relative to a comparable wild-type Fc domain or hinge-Fc domain (or portion thereof). Molecules comprising variant Fc domains or hinge-Fc domains (or portion thereof) (e.g., antibodies) normally have altered phenotypes relative to molecules comprising wild-type Fc domains or hinge-Fc domains or portions thereof. The variant phenotype may be expressed as altered serum half-life, altered stability, altered susceptibility to cellular enzymes or altered effector function as assayed in an NK dependent or macrophage dependent assay. Fc domain variants identified as altering effector function are known in the art. For example International Application W004/063351, U.S. Patent Application Publications 2005/0037000 and 2005/0064514.

[0183] The bispecific diabodies of the invention can simultaneously bind two separate and distinct epitopes. In certain embodiments the two separate epitopes are on different cells. In certain embodiments, the two separate epitopes are on two different inhibitory receptors on the same cell. In certain embodiments the epitopes are from the same antigen. In other embodiments, the epitopes are from different antigens. In some embodiments, at least one epitope binding site is specific for a determinant expressed on an immune effector cell (e.g. CD3, CD 16, CD32, CD64, etc.) which are expressed on T lymphocytes, natural killer (NK) cells or other mononuclear cells. In one embodiment, the diabody molecule binds to the effector cell determinant and also activates the effector cell. In this regard, diabody molecules of the invention may exhibit Ig-like functionality independent of whether they further comprise an Fc domain (e.g., as assayed in any effector function assay known in the art or exemplified herein (e.g., ADCC assay).

[0184] In certain embodiments the bispecific antibodies engage cells for Antibody-Dependent Cell-mediated Cytotoxicity (ADCC). In certain embodiments the bispecific antibodies engage natural killer cells, neutrophil polymorphonuclear leukocytes, monocytes and macrophages. In certain embodiments the bispecific antibodies are T-cell engagers. In certain embodiments, the bispecific antibody comprises an coronavirus binding fragment and a CD3 binding fragment. Various CD3 antibodies are known in the art. See for example US Patent 8,784,821. In certain embodiments, the bispecific antibody comprises a coronavirus binding fragment and CD 16 binding fragment.

[0185] In certain embodiments the invention provides antibodies with dual targeting specificity. In certain aspects the invention provides bi-specific molecules that can localize an immune effector cell to a coronavirus expressing cell, such as a host cell or a virally infected cell, so as facilitate the killing of this cell. In this regard, bispecific antibodies bind with one "arm" to a surface antigen on target cells, and with the second "arm" to an activating, invariant component of the T cell receptor (TCR) complex or to an activating, invariant component of a different stimulatory receptor such as NKG2C on NK cells or other immune effector cells. The simultaneous binding of such an antibody to both of its targets will force a temporary interaction between target cell and effector cell, causing, for example, activation of any cytotoxic T cell or NK cell and subsequent lysis of the target cell. Hence, the immune response is re-directed to the target cells and may be independent of classical MHC class I peptide antigen presentation by the target cell or the specificity of the T cell as would be relevant for normal MHC-restricted activation of CTLs. In this context it is crucial that CTLs are only activated when a target cell is presenting the bispecific antibody to them, i.e. the immunological synapse is mimicked.

Particularly desirable are bispecific antibodies that do not require lymphocyte preconditioning or co-stimulation in order to elicit efficient lysis of target cells.

[0186] Several bispecific antibody formats have been developed and their suitability for T cell mediated immunotherapy investigated. Out of these, the so-called BiTE (bispecific T cell engager) molecules have been very well characterized and already shown some promise in the clinic (reviewed in Nagorsen and Bauerle, Exp Cell Res 317, 1255-1260 (2011)). BiTEs are tandem scFv molecules wherein two scFv molecules are fused by a flexible linker. Further bispecific formats being evaluated for T cell engagement include diabodies (Holliger et al., Prot Eng 9, 299-305 (1996)) and derivatives thereof, such as tandem diabodies (Kipriyanov et al., J Mol Biol 293, 41-66 (1999)). DART (dual affinity retargeting) molecules are based on the diabody format that separates cognate variable domains of heavy and light chains of the two antigen binding specificities on two separate polypeptide chains but feature a C-terminal disulfide bridge for additional stabilization (Moore et al., Blood 117, 4542-51 (2011)). The invention also contemplates Fc-bearing DARTs. The so-called triomabs, which are whole hybrid mouse/rat IgG molecules and also currently being evaluated in clinical trials, represent a larger sized format (reviewed in Seimetz et al., Cancer Treat Rev 36, 458-467 (2010)).

[0187] The invention also contemplates bispecific molecules with enhanced pharmacokinetic properties. In some embodiments, such molecules can have increased serum half-life. In some embodiments, these are Fc-bearing DARTs (see supra).

[0188] In certain embodiments, such bispecific molecules comprise one portion which targets one portion of the spike protein and a second portion which binds a second target on the spike protein. In certain embodiments, the first portion comprises VH and VL sequences, or CDRs from the antibodies described herein. In certain embodiments, the second target could be, for example but not limited to an effector cell. In certain embodiments the second portion is a T-cell engager. In certain embodiments, the second portion comprises a sequence/paratope which targets CD3, CD16, or another suitable target. In certain embodiments, the second portion is an antigen-binding region derived from a CD3 antibody, optionally a known CD3 antibody. In certain embodiments, the anti-CD antibody induce T cell-mediated or NK-mediated killing. In certain embodiments, the bispecific antibodies are whole antibodies. In other embodiments, the dual targeting antibodies consist essentially of Fab fragments. In other embodiments, the dual targeting antibodies comprise a heavy chain constant region. In certain embodiments, the bispecific antibody does not comprise Fc region. In certain embodiments, the bispecific antibodies have improved effector function. In certain embodiments, the bispecific antibodies have improved cell killing activity. Various methods and platforms for design of bispecific antibodies are known in the art. See for example US Pub. 20140206846, US Pub. 20140170149, US Pub. 20090060910, US Pub 20130295121, US Pub. 20140099318, US Pub. 20140088295 which contents are herein incorporated by reference in their entirety. BINDING AND NEUTRALIZATION ASSAYS

[0189] In some embodiments, the antibodies of the invention are SARS CoV-2 neutralizing antibodies. SARS-CoV-2 neutralization can be determined by assays known in the art. Nonlimiting examples of neutralization assays include PRNT assay, various pseudovirus based neutralization assays are known and described in the art.

[0190] Neutralization assays measure neutralizing properties of antibodies. Neutralization properties can correlate with protection from infection and/or therapeutic benefits post infection. In non-limiting examples, neutralization is measured by plaque reduction neutralization test (PRNT). Although PRNT (neutralization) and ELISA (binding) results correlate with each other, PRNT remains the gold-standard for determining neutralization properties.

[0191] In other embodiments, fluorescence-based assay that rapidly and reliably measures neutralization of a reporter SARS-CoV-2 by antibodies provide a higher throughput neutralization assay.

[0192] In some embodiments, neutralization can be determined using a lentivirus-based pseudotyped virus with SARS-CoV-2 S protein. With such a system, neutralization can be assessed with respect to the spike protein variants of different SARS CoV-2 strains. Strain information and sequence can be obtained from the Global Initiative for Sharing All Influenza Data (GISAID) database and Nextstrain. Non-limiting methods for carrying out neutralization assays are described in the supplementary methods of Korber et al. 2020, Cell 182, 812-827. [0193] Nie et al. used the full length S gene from strain Wuhan-Hu- 1 with the vesicular stomatitis virus (VSV) pseudovirus system. (Nie et al., Establishment and validation of a pseudovirus neutralization assay for SARS-CoV-2, Emerging Microbes Infection, 2020, 9:680- 686; citing Whitt, MA., Generation of VSV pseudotypes using recombinant Del taG- VSV for studies on virus entry, identification of entry inhibitors, and immune responses to vaccines, J Virol Methods, 2010, 169(2):365-74.) Briefly, the SARS-CoV-2 spike gene can be codon- optimized and cloned into a eukaryotic expression plasmid. 293T cells can be transfected by the plasmid and later infected with a VSV pseudotyped virus (G*DG-VSV), which substitutes the VSV-G gene with luciferase expression cassettes. The culture supernatants are then harvested and filtered 24 h postinfection. The SARS-CoV-2 pseudovirus presents the SARS-CoV-2 spike protein in the surface of the VSV particle as can be confirmed by Western blot with SARS-CoV- 2 convalescent patient sera. (Id.) [0194] The SARS-CoV-pseudovirus-based neutralization assay (PBNA) tests whether antibodies are able to neutralize SARS-CoV-2 pseudovirus infection of susceptible cell-lines (e.g., Vero, Huh7, 293T, HepG2, CHO, MDCK; with Huh7 identified as the best cell substrate for the system), as indicated by inhibition curves of % reduction of RLU relative to antibody sample dilution. (Id.) The SARS-CoV-2 pseudovirus should not be neutralized by VSV-G antibodies. In one embodiment, the pseudovirus neutralization assays can be performed using Huh-7 cell lines. Huh-7 are human hepatocellular carcinoma cells that express both ACE2 and TMPRSS2. Various concentrations of mAbs (e.g., 3-fold serial dilution using DMEM, 50 mL aliquots) are mixed with the same volume of SARS-CoV-2 pseudovirus with a TCIDso of 1.3xl0⁴ in a 96 well-plate. The mixture is incubated for 1 h at 37°C, supplied with 5% CO2. Negative control wells are supplied with 100 mL DMEM (1% (v/v) antibiotics, 25 nM HEPES, 10% (v/v) FBS). Positive control wells are supplied with 100 mL DMEM. Pre-mixed Huh-7 cells (100 mL, 2 3 105 in DMEM) are added to all wells, and the 96-well plates are incubated for 24 h at 37°C supplied with 5% CO2. After the incubation, 150 mL of supernatants are removed, and 100 mL D-luciferin reagent (Invitrogen) is added to each well and incubated for 2 mins. After the incubation, every well is mixed 10 times by pipetting, and 150 mL of the mixture is used to measure luciferase activity using a microplate spectrophotometer (Perkinelmer EnSight). The inhibition rate is calculated by comparing the OD value to the negative and positive control wells. IC50 and IC80 are determined by a four-parameter logistic regression using GraphPad Prism 8.0 (GraphPad Software Inc.).

[0195] In some embodiments, neutralization is determined using a SARS CoV-2 S pseudotyped virus where the spike protein is the G614 variant. (Korber, B., et al., Tracking changes in SARS- CoV-2 Spike: evidence that D614G increases infectivity of the COVID-19 virus, Cell, Jul. 2, 2020, doi:https://doi.org/10.1016/j. cell.2020.06.043; describing the Spike D614G amino acid change is caused by an A-to-G nucleotide mutation at position 23,403 in the Wuhan reference strain.) The D614G change is almost always accompanied by three other mutations: a C-to-T mutation in the 5’ UTR (position 241 relative to the Wuhan reference sequence), a silent C-to-T mutation at position 3,037; and a C-to-T mutation at position 14,408 that results in an amino acid change in RNA-dependent RNA polymerase (RdRp P323L). The haplotype comprising these 4 genetically linked mutations is now the globally dominant form. (Id.) In some embodiments, neutralization is determined using a SARS CoV-2 S pseudotyped virus where the spike protein is from strain Wuhan-Hu-1. (GenBank: MN908947.) In some embodiments, a SARS CoV-2 S pseudotyped virus uses the VSV pseudovirus or a murine leukemia virus (MLV) pseudotype system. In some embodiments, neutralization assays use authentic SARS-CoV-2.

PRODUCTION OF ANTIBODIES

[0196] Antibodies, such as monoclonal antibodies, according to the invention can be made by any method known in the art.

[0197] In certain embodiments, plasma cells are cultured in limited numbers, or as single plasma cells in microwell culture plates. Antibodies can be isolated from the plasma cell cultures. VH and VL can be isolated from single cell sorted plasma cells. From the plasma cell, RNA can be extracted and PCR can be performed using methods known in the art. The VH and VL regions of the antibodies can be amplified by RT-PCR (reverse transcriptase PCR), sequenced and cloned into intermediate vectors for further engineering or into an expression vector that is then transfected into HEK293T cells or other host cells as described below or known in the art. The cloning of nucleic acid in intermediate vectors, expression vectors, the transfection of host cells, the culture of the transfected host cells and the isolation of the produced antibody can be done using any methods known to one of skill in the art. Antibody isolation and purification techniques are known in the art, which can include filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography. Techniques for purification of antibodies, e.g., monoclonal antibodies, including techniques for producing pharmaceuticalgrade antibodies (and at a sufficiently high concentration or titer for therapeutic use), are well known in the art.

[0198] In some embodiments, the antibodies or fragments of the invention have an IgM Fc region or constant domains thereof. It is established that IgM can assume both pentameric and hexameric configurations, depending on the substitution of the J-chain with an additional Fab(2) monomer, which increases the number of Fabs on a single IgM from 10 to 12 (Hiramoto et al Sci. Adv. 2018; 4: eaaul l99; Moh ES et al J Am Soc Mass Spectrom. 2016 Jul;27(7):l 143-55). Methods for the recombinant production of polymeric IgM (both with and without J chain) have been described (Chromikova et al., Cytotechnology, 2015, 67:343-356; Gilmour et al. Transfusion Medicine, 2008. 18: 167-174; Hennicke et al., PLoS ONE, 2020, 15(3):e0229992). Stable or transient IgM producing cells lines can be generated as described in Chromikova et al., 2015 and Hennicke et al., 2020, where two different pIRES expression vectors are used, one to express the heavy chain and the other to express the light chain and the J-chain sequence. IgM antibodies can be purified according to standard methods in the art, including IgM specific resins for use in affinity chromatography (e.g., POROS Capture Select IgM Affinity Matrix by ThermoFisherScientific.) Transmission electron microscopy (TEM) can be used to confirm pentameric and hexameric forms of IgM.

[0199] In addition, besides expression constructs that encode antibody fragments or elements, protein fragments of antibodies can be obtained by methods that include digestion with enzymes, such as pepsin or papain, and/or by cleavage of disulfide bonds by chemical reduction.

[0200] Any suitable host cell/vector system may be used for expression of the DNA sequences encoding the antibody molecules of the present invention or fragments thereof. Bacterial, for example E. coli, and other microbial systems may be used, in part, for expression of antibody fragments such as Fab and F(ab')2 fragments, and especially Fv fragments and single chain antibody fragments, for example, single chain Fvs. eukaryotic, e.g., mammalian, host cell expression systems may be used for production of larger antibody molecules, including complete antibody molecules. Suitable mammalian host cells include, but are not limited to, CHO, HEK293T, PER.C6, NS0, myeloma or hybridoma cells. Mammalian cell lines suitable for expression of therapeutic antibodies are well known in the art.

[0201] The antibody molecule may comprise only a heavy or light chain polypeptide, in which case only a heavy chain or light chain polypeptide coding sequence needs to be used to transfect the host cells. For production of products comprising both heavy and light chains, the cell line may be transfected with two vectors, a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector may be used, the vector including sequences encoding light chain and heavy chain polypeptides. Alternatively, antibodies according to the invention may be produced by (i) expressing a nucleic acid sequence according to the invention in a host cell, e.g. by use of a vector according to the present invention, and (ii) isolating the expressed antibody product. Additionally, the method may include (iii) purifying the isolated antibody. Transformed B cells and cultured plasma cells may be screened for those producing antibodies of the desired specificity or function.

[0202] The screening step may be carried out by any immunoassay, e.g., ELISA, by staining of tissues or cells (including transfected cells), by neutralization assay or by one of a number of other methods known in the art for identifying desired specificity or function. The assay may select on the basis of simple recognition of one or more antigens, or may select on the additional basis of a desired function e.g., to select neutralizing antibodies rather than just antigen-binding antibodies, to select antibodies that can change characteristics of targeted cells, such as their signaling cascades, their shape, their growth rate, their capability of influencing other cells, their response to the influence by other cells or by other reagents or by a change in conditions, their differentiation status, etc.

[0203] Individual transformed B cell clones may then be produced from the positive transformed B cell culture. The cloning step for separating individual clones from the mixture of positive cells may be carried out using limiting dilution, micromanipulation, single cell deposition by cell sorting or another method known in the art.

[0204] Nucleic acid from the cultured plasma cells can be isolated, cloned and expressed in HEK293T cells or other known host cells using methods known in the art.

[0205] B cell clones or transfected host-cells of the invention can be used in various ways e.g., as a source of monoclonal antibodies, as a source of nucleic acid (DNA or mRNA) encoding a monoclonal antibody of interest, for research, etc.

[0206] Expression from recombinant sources is common for pharmaceutical purposes than expression from B cells or hybridomas e.g., for reasons of stability, reproducibility, culture ease, etc.

[0207] Thus the invention also provides a method for preparing a recombinant cell, comprising the steps of: (i) obtaining one or more nucleic acids (e.g., heavy and/or light chain mRNAs) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; (ii) inserting the nucleic acid into an expression vector and (iii) transfecting the vector into a host cell in order to permit expression of the antibody of interest in that host cell.

[0208] Similarly, the invention provides a method for preparing a recombinant cell, comprising the steps of: (i) sequencing nucleic acid(s) from the B cell clone or the cultured plasma cells that encodes the antibody of interest; and (ii) using the sequence information from step (i) to prepare nucleic acid(s) for insertion into a host cell in order to permit expression of the antibody of interest in that host cell. The nucleic acid may, but need not, be manipulated between steps (i) and (ii) to introduce restriction sites, to change codon usage, and/or to optimize transcription and/or translation regulatory sequences. [0209] Furthermore, the invention also provides a method of preparing a transfected host cell, comprising the step of transfecting a host cell with one or more nucleic acids that encode an antibody of interest, wherein the nucleic acids are nucleic acids that were derived from a cell sorted B cell or a cultured plasma cell of the invention.

[0210] These recombinant cells of the invention can then be used for expression and culture purposes. They are particularly useful for expression of antibodies for large-scale pharmaceutical production. They can also be used as the active ingredient of a pharmaceutical composition. Any suitable culture technique can be used, including but not limited to static culture, roller bottle culture, ascites fluid, hollow-fiber type bioreactor cartridge, modular minifermenter, stirred tank, microcarrier culture, ceramic core perfusion, etc.

[0211] Any suitable host cells could be used for transfection and production of the antibodies of the invention. The transfected host cell may be a eukaryotic cell, including yeast and animal cells, particularly mammalian cells (e.g., CHO cells, NS0 cells, human cells such as PER.C6 or HKB-11 cells, myeloma cells, or a human liver cell), as well as plant cells. In certain embodiments, expression hosts can glycosylate the antibody of the invention, particularly with carbohydrate structures that are not themselves immunogenic in humans. In one embodiment, the transfected host cell may be able to grow in serum-free media. In a further embodiment, the transfected host cell may be able to grow in culture without the presence of animal-derived products. The transfected host cell may also be cultured to give a cell line.

[0212] In general, protein therapeutics are produced from mammalian cells. The most widely used host mammalian cells are Chinese hamster ovary (CHO) cells and mouse myeloma cells, including NS0 and Sp2/0 cells. Two derivatives of the CHO cell line, CHO-K1 and CHO pro-3, gave rise to the two most commonly used cell lines in large scale production, DUKX-X11 and DG44. (Kim, J., et al., AppL Microbiol. BiotechnoL, 2012, 93:917-30.) Other mammalian cell lines for recombinant antibody expression include, but are not limited to, COS, HeLa, HEK293T, U2OS, A549, HT1080, CAD, P19, NIH 3T3, L929, N2a, HEK 293, MCF-7, Y79, SO-Rb50, HepG2, J558L, and BHK. If the aim is large-scale production, the most currently used cells for this application are CHO cells. Guidelines to cell engineering for mAbs production were also reported. (Costa et al., Eur J Pharm Biopharm, 2010, 74: 127-38.) Using heterologous promoters, enhancers and amplifiable genetic markers, the yields of antibody and antibody fragments can be increased. Thus, in certain embodiments, the invention provides an antibody, or antibody fragment, that is recombinantly produced from a mammalian cell-line, including a CHO cell-line. In certain embodiments, the invention provides a composition comprising an antibody, or antibody fragment, wherein the antibody or antibody fragment was recombinantly produced in a mammalian cell-line, and wherein the antibody or antibody fragment is present in the composition at a concentration of at least 1, 10, 100, 1000 micrograms/mL, or at a concentration of at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or 100 milligrams/mL.

[0213] Furthermore, large-scale production of therapeutic-grade antibodies are much different than those for laboratory scale. There are extreme purity requirements for therapeutic-grade. Large-scale production of therapeutic-grade antibodies requires multiples steps, including product recovery for cell-culture harvest (removal of cells and cell debris), one or more chromatography steps for antibody purification, and formulation (often by tangential filtration). Because mammalian cell culture and purification steps can introduce antibody variants that are unique to the recombinant production process (i.e., antibody aggregates, N- and C- terminal variants, acidic variants, basic variants, different glycosylation profiles), there are recognized approaches in the art for analyzing and controlling these variants. (See, Fahrner, et al., Industrial purification of pharmaceutical antibodies: Development, operation, and validation of chromatography processes, Biotech. Gen. Eng. Rev., 2001, 18:301-327.) In certain embodiments of the invention, the antibody composition comprises less than 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 50, or 100 nanograms of host cell protein (i.e., proteins from the cell-line used to recombinantly produce the antibody)). In other embodiments, the antibody composition comprises less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or 25 ng of protein A per milligram of antibody or antibody fragment (i.e., protein A is a standard approach for purifying antibodies from recombinant cell culture, but steps should be done to limit the amount of protein A in the composition, as it may be immunogenic). (See, e.g., U.S. Patent No. 7,458,704, Reduced protein A leaching during protein A affinity chromatography; which is hereby incorporated-by- reference.)

[0214] In certain aspects the invention provides nucleic acids encoding the inventive antibodies. In non-limiting embodiments, the nucleic acids are mRNA, modified or unmodified, suitable for use any use, e.g. but not limited to use as pharmaceutical compositions. In certain embodiments, the nucleic acids are formulated in lipid, such as but not limited to LNPs. PHARMACEUTICAL COMPOSITIONS

[0215] The present invention also provides a pharmaceutical composition comprising one or more of: (i) the antibody, or the antibody fragment thereof, according to the present invention; (ii) the nucleic acid encoding the antibody, or antibody fragments according to the present invention; (iii) the vector comprising the nucleic acid according to the present invention; and/or (iv) the cell expressing the antibody according to the present invention or comprising the vector according to the present invention.

[0216] In certain aspects, the invention provides a pharmaceutical composition comprising the antibody, or the antigen binding fragment thereof, according to the present invention, the nucleic acid according to the present invention, the vector according to the present invention and/or the cell according to the present invention.

[0217] The pharmaceutical composition may also contain a pharmaceutically acceptable carrier, diluent and/or excipient. Although the carrier or excipient may facilitate administration, it should not itself induce the production of antibodies harmful to the individual receiving the composition. Nor should it be toxic. Suitable carriers may be large, slowly metabolized macromolecules such as proteins, polypeptides, liposomes, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers and inactive virus particles. In general, pharmaceutically acceptable carriers in a pharmaceutical composition according to the present invention may be active components or inactive components. In certain embodiments the pharmaceutically acceptable carrier in a pharmaceutical composition according to the present invention is not an active component in respect to coronavirus infection.

[0218] Pharmaceutically acceptable salts can be used, for example mineral acid salts, such as hydrochlorides, hydrobromides, phosphates and sulphates, or salts of organic acids, such as acetates, propionates, malonates and benzoates.

[0219] Pharmaceutically acceptable carriers in a pharmaceutical composition may additionally contain liquids such as water, saline, glycerol and ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents or pH buffering substances, may be present in such compositions. Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries and suspensions, for ingestion by the subject.

[0220] Pharmaceutical compositions of the invention may be prepared in various forms. For example, the compositions may be prepared as injectables, either as liquid solutions or suspensions. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared (e.g., a lyophilized composition, similar to Synagis. TM. and Herceptin. TM., for reconstitution with sterile water containing a preservative). The composition may be prepared for topical administration e.g., as an ointment, cream or powder. The composition may be prepared for oral administration e.g., as a tablet or capsule, as a spray, or as a syrup (optionally flavored). The composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray. The composition may be prepared as a suppository or pessary. The composition may be prepared for nasal, aural or ocular administration e.g., as drops. The composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a subject. For example, a lyophilized antibody may be provided in kit form with sterile water or a sterile buffer.

[0221] A thorough discussion of pharmaceutically acceptable carriers is available in Gennaro (2000) Remington: The Science and Practice of Pharmacy, 20th edition, ISBN: 0683306472. [0222] Pharmaceutical compositions of the invention have a pH between 5.5 and 8.5, in some embodiments this may be between 6 and 8, and in other embodiments about 7. The pH may be maintained by the use of a buffer. The composition may be sterile and/or pyrogen free. The composition may be isotonic with respect to humans. In one embodiment pharmaceutical compositions of the invention are supplied in hermetically-sealed containers.

[0223] Within the scope of the invention are compositions present in several forms of administration; the forms include, but are not limited to, those forms suitable for parenteral administration, e.g., by injection or infusion, for example by bolus injection or continuous infusion. Where the product is for injection or infusion, it may take the form of a suspension, solution or emulsion in an oily or aqueous vehicle and it may contain formulatory agents, such as suspending, preservative, stabilizing and/or dispersing agents. Alternatively, the antibody molecule may be in dry form, for reconstitution before use with an appropriate sterile liquid. A vehicle can be a material that is suitable for storing, transporting, and/or administering a compound, such as a pharmaceutically active compound, in particular the antibodies according to the present invention. For example, the vehicle may be a physiologically acceptable liquid, which is suitable for storing, transporting, and/or administering a pharmaceutically active compound, in particular the antibodies according to the present invention. Once formulated, the compositions of the invention can be administered directly to the subject. In one embodiment the compositions are adapted for administration to mammalian, e.g., human subjects.

[0224] The pharmaceutical compositions of this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intraperitoneal, intrathecal, intraventricular, transdermal, transcutaneous, topical, subcutaneous, intranasal, enteral, sublingual, intravaginal or rectal routes. Hyposprays or nebulizers may also be used to administer the pharmaceutical compositions of the invention. In certain embodiments, the pharmaceutical composition may be prepared for oral administration, e.g. as tablets, capsules and the like, for topical administration, or as injectable, e.g. as liquid solutions or suspensions. In certain embodiments, the pharmaceutical composition is an injectable. Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection are also contemplated, e.g. that the pharmaceutical composition is in lyophilized form.

[0225] For injection, e.g. intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient could be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer's Injection, Lactated Ringer's Injection. Preservatives, stabilizers, buffers, antioxidants and/or other additives may be included, as required. Whether it is a polypeptide, peptide, or nucleic acid molecule, other pharmaceutically useful compound according to the present invention that is to be given to an individual, administration is in a "prophylactically effective amount" or a "therapeutically effective amount" , this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. For injection, the pharmaceutical composition according to the present invention may be provided for example in a pre-filled syringe.

[0226] The inventive pharmaceutical composition as defined above may also be administered orally in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also added. For oral administration in a capsule form, useful diluents include lactose and dried cornstarch. When aqueous suspensions are required for oral use, the active ingredient, i.e. the inventive transporter cargo conjugate molecule as defined above, is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added.

[0227] The inventive pharmaceutical composition may also be administered topically, especially when the target of treatment includes areas or organs readily accessible by topical application, e.g. including diseases of the skin or of any other accessible epithelial tissue. Suitable topical formulations are readily prepared for each of these areas or organs. For topical applications, the inventive pharmaceutical composition may be formulated in a suitable ointment, containing the inventive pharmaceutical composition, particularly its components as defined above, suspended or dissolved in one or more carriers. Carriers for topical administration include, but are not limited to, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene, polyoxypropylene compound, emulsifying wax and water. Alternatively, the inventive pharmaceutical composition can be formulated in a suitable lotion or cream. In the context of the present invention, suitable carriers include, but are not limited to, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

[0228] Suitable dose ranges can depend on the antibody (or fragment) and on the nature of the formulation and route of administration. For example, doses of antibodies in the range of 0.1-50 mg/kg, 1-50 mg/kg, 1-10 mg/kg, 1, 1.25, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5 or 10 mg/kg of antibody can be used. If antibody fragments are administered, then less antibody can be used (e.g., from 5 mg/kg to 0.01 mg/kg). In other embodiments, the antibodies of the invention can be administered at a suitable fixed dose, regardless of body size or weight. See Bai et al. Clinical Pharmacokinetics February 2012, Volume 51, Issue 2, pp 119-135.

[0229] Dosage also depends on whether the composition is administered as a recombinant protein or a nucleic acid.

[0230] Dosage treatment may be a single dose schedule or a multiple dose schedule. In particular, the pharmaceutical composition may be provided as single-dose product. In certain embodiments, the amount of the antibody in the pharmaceutical composition— in particular if provided as single-dose product— does not exceed 200 mg. In certain embodiments, the amount does not exceed 100 mg, and in certain embodiments, the amount does not exceed 50 mg. [0231] In non-limiting embodiments, the antibodies of the invention could be used for non- therapeutic uses, such as but not limited to diagnostic assays. In non-limiting embodiments, the antibodies could be used for serology testing in any suitable assay or format, including without limitation sandwich ELISA based detection.

ADMINISTRATION OF ANTIBODY ENCODING NUCLEIC ACID SEQUENCES

[0232] In some embodiments the antibodies are administered as nucleic acids, including but not limited to mRNAs which could be modified and/or unmodified. See US Pub 20180028645A1, , US Pub 20090286852, US Pub 20130111615, US Pub 20130197068, US Pub 20130261172, US Pub 20150038558, US Pub 20160032316, US Pub 20170043037, US Pub 20170327842, US Patent 10,006,007, US Patent 9,371,511, US Patent 9,012,219, US Pub 20180265848, US Pub 20170327842, US Pub 20180344838 Al at least at paragraphs [0260] -[0281], WO/2017/182524 for non-limiting embodiments of chemical modifications, wherein each content is incorporated by reference in its entirety.

[0233] mRNAs delivered in LNP formulations have advantages over non-LNPs formulations. See US Pub 20180028645 Al, WO/2018/081638, WO/2016/ 176330, wherein each content is incorporated by reference in its entirety.

[0234] In certain embodiments the nucleic acid encoding an envelope is operably linked to a promoter inserted an expression vector. In certain aspects the compositions comprise a suitable carrier. In certain aspects the compositions comprise a suitable adjuvant.

[0235] In certain aspects the invention provides an expression vector comprising any of the nucleic acid sequences of the invention, wherein the nucleic acid is operably linked to a promoter. In certain aspects the invention provides an expression vector comprising a nucleic acid sequence encoding any of the polypeptides of the invention, wherein the nucleic acid is operably linked to a promoter. In certain embodiments, the nucleic acids are codon optimized for expression in a mammalian cell, in vivo or in vitro. In certain aspects the invention provides nucleic acids comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting essentially of any one of the nucleic acid sequences of invention. In certain aspects the invention provides nucleic acids consisting of any one of the nucleic acid sequences of invention. In certain embodiments the nucleic acid of the invention, is operably linked to a promoter and is inserted in an expression vector. In certain aspects the invention provides an immunogenic composition comprising the expression vector. [0236] In certain aspects the invention provides a composition comprising at least one of the nucleic acid sequences of the invention. In certain aspects the invention provides a composition comprising any one of the nucleic acid sequences of invention. In certain aspects the invention provides a composition comprising at least one nucleic acid sequence encoding any one of the polypeptides of the invention.

[0237] In one embodiment, the nucleic acid is an RNA molecule. In one embodiment, the RNA molecule is transcribed from a DNA sequence described herein. In some embodiments, the RNA molecule is encoded by one of the inventive sequences. In another embodiment, the nucleotide sequence comprises an RNA sequence transcribed by a DNA sequence encoding the polypeptide sequence of the sequences in in the instant application, or a variant thereof or a fragment thereof. Accordingly, in one embodiment, the invention provides an RNA molecule encoding one or more of inventive antibodies. The RNA may be plus-stranded. Accordingly, in some embodiments, the RNA molecule can be translated by cells without needing any intervening replication steps such as reverse transcription.

[0238] In some embodiments, a RNA molecule of the invention may have a 5' cap (e.g. but not limited to a 7-methylguanosine, 7mG(5')ppp(5')NlmpNp). This cap can enhance in vivo translation of the RNA. The 5' nucleotide of an RNA molecule useful with the invention may have a 5' triphosphate group. In a capped RNA this may be linked to a 7-methylguanosine via a 5'-to-5' bridge. A RNA molecule may have a 3' poly-A tail. It may also include a poly-A polymerase recognition sequence (e.g. AAUAAA) near its 3' end. In some embodiments, a RNA molecule useful with the invention may be single-stranded. In some embodiments, a RNA molecule useful with the invention may comprise synthetic RNA.

[0239] The recombinant nucleic acid sequence can be an optimized nucleic acid sequence. Such optimization can increase or alter the immunogenicity of the antibody. Optimization can also improve transcription and/or translation. Optimization can include one or more of the following: low GC content leader sequence to increase transcription; mRNA stability and codon optimization; addition of a kozak sequence (e.g., GCC ACC) for increased translation; addition of an immunoglobulin (Ig) leader sequence encoding a signal peptide; and eliminating to the extent possible cis-acting sequence motifs (i.e., internal TATA boxes). [0240] In certain aspects, the invention provides an in vitro transcription system to synthesize ribonucleic acids (RNAs) encoding antibodies of the invention, comprising: a reaction vessel, a DNA vector template comprising nucleic acid sequence encoding an antibody of the invention as described in Table 3, and reagents for carrying out an in vitro transcription reaction that produces mRNA encoding an antibody or fragment thereof of the invention. In certain embodiments the mRNA is modified mRNA.

[0241] In certain aspects, the invention provides an in vitro transcription system to synthesize ribonucleic acids (RNAs) encoding antibodies of the invention, comprising: a reaction vessel, a DNA vector template comprising nucleic acid sequence encoding an antibody of the invention as described in Table 3, and reagents for carrying out an in vitro transcription reaction that produces mRNA encoding an antibody or fragment thereof of the invention. Methods to purify mRNA and assay its purity for therapeutic use are known in the art. In certain embodiments the mRNA is modified mRNA. A skilled artisan can readily scale up the in vitro transcription reaction methods, purification and analytical methods for large scale mRNA batches.

THERAPEUTIC METHODS

[0242] In certain aspects, the invention provides prophylactic and/or therapeutic methods comprising administering the antibodies of the invention in an amount suitable to effect prophylaxis or treatment. In certain embodiments, the methods of administering antibodies lead to protection from acquiring of infection, or reducing severity of infection or disease by binding coronavirus spike protein and neutralizing coronavirus. Therapeutic doses depend on the mode of delivery and whether the antibody is delivered as a recombinant protein or a nucleic acid.

DIAGNOSTIC METHODS

[0243] In certain aspects the invention provides methods for detecting coronavrus virus in a sample suspected of containing said coronavirus virus, comprising (1) contacting the sample with a first antibody or antigen-binding fragment thereof binding coronavirus, and assaying binding of the antibody with said sample for formation of first antibody-coronavirus sample, wherein in some embodiments the first antibody is immobilized on a suitable surface or the first antibody-coronavirus complex is immobilzed, (2) removing unbound sample and/or first coronavirus antibody, (3) contacting the immbolized first antibody-coronavirus sample complex with a second antibody, and assaying binding of the second coronavirus antibody to the first antibody-coronavirus complex, wherein the second antibody has a different binding epitope from the first antibody, and wherein the first and/or the second antibody is any one of the antibodies of the invention, e.g. Table 3. Techniques to assay and quantitate binding between antibody and target epitope in a sample are known in the art. In certain embodiments, the second antibody is conjugated for direct detection. In non-limiting embodiments, the antibody-coronavirs complex is indirectly detected by a detection reagent which binds the second coronavirus antibody. In non-limiting embodiments, the detection reagent could be another antibody conjugated to comprises any suitable imaging agent including without limitation color detection, a fluorophore, a magnetic nano-particle, or a radionuclide. Non-limiting examples include any variation of ELISA sandwich -based immunoassay.

[0244] In certain embodiments, the sample is any suitable sample including but not limited to respiratory tract secretions, saliva, nasal swabs, etc.

[0245] In certain aspects, the invention provides kits comprising the inventive antibodies, reagents and instructions for therapeutic or diagnostic use.

[0246] CDRs can be identified by any method known in the art. In some embodiment, CDRs as identified are identified using IMGT.

[0247]

[0248] Table 3 Summary of antibodies (includes DH nomenclature and gene information). See also Figures 7 and 8XX for sequences.

[0249] The amino acid sequence and the nucleic acid sequence of the monoclonal SARS- CoV2 antibodies are provided below. The amino acid sequences of the heavy and light chain complementary determining regions (CDRs) of the COVID-19 antibodies are underlined (CDR1). underlined and bolded (CDR2). or underlined, italiciz.ed, and bolded (CDR3Y.

EXAMPLES

Example 1 - Antibody Isolation

[0250] Antibodies were isolated and lineages were identified as previously published.

[0251] 1. Liao HX, Levesque MC, Nagel A, et al. High-throughput isolation of immunoglobulin genes from single human B cells and expression as monoclonal antibodies. J Virol Methods. 2009;158(l-2): 171-179.

[0252] 2. Kepler TB, Munshaw S, Wiehe K, et al. Reconstructing a B-Cell Clonal Lineage. II. Mutation, Selection, and Affinity Maturation. Front Immunol. 2014;5:170.

[0253] Figures 20-25 and Examples DH1041 and DH043 show non-limiting embodiments of nucleic acids encoding antibodies of the invention.

[0254] In one embodiment, the IgG constant region comprises the LS mutation. Additional variants of the Fc portion of the antibody are also contemplated by the invention. See Maeda et al. MAbs. 2017 Jul; 9(5): 844-853. Published online 2017 Apr 7, PMID: 28387635.

[0255] For LS mutation See e.g. Gaudinski MR, Coates EE, Houser KV, Chen GL, Yamshchikov G, Saunders JG, et al. (2018) Safety and pharmacokinetics of the Fc-modified HIV-1 human monoclonal antibody VRC01LS: A Phase 1 open-label clinical trial in healthy adults. PLoS Med 15(1): el002493. https://doi.org/10.1371/joumal.pmed.100249.

Example 2 -Binding and neutralization assays

[0256] ELISA binding assays were characterized essentially as described in Edwards et al. Cold sensitivity of the SARS-CoV-2 spike ectodomain, July 13, 2020 doi: 10.1101/2020.07.12.199588, published on biorxiv.org— content/10.1101/2020.07.12.199588vl).

[0257] In some embodiments of the PRNT assay, Vero cells were used. In some embodiments of the pseudovirus assay, 293T/ACE2 (293T that overexpressed human ACE2) were used.

Example 3 - Cross blocking and cross reactivity to other coronaviruses

[0258] Antibodies can be tested for crossblocking of any other coronavirus antibodies e.g.by surface plasmon resonance assays. Binding and cross blocking analysis can be conducted by standard SPR methods, where a first antibody is immobilized on the surface, an antigen is floated over and second antibody is floated over the complex.

[0259] RBD antibodies that do not cross-block each other can be combined in a therapeutic combination, the RBD and NTD antibodies that do not block each other can be . be used in combinations of neutralizing antibodies for either prevention of SARS-CoV-2 infection or for treatment of COVID-1 disease. Other antibodies listed in Table 3 can also be combined for prevention or treatment by performing the same types of blocking experiments with surface plasmon resonance.

Example 4 - Animal studies

[0260] Any other suitable coronavirus animal model could be used to characterize the antibodies of the invention. In non-limiting embodiments, a mouse, hamster, or NHP animal model is used to characterize the antibodies.

[0261] Animal studies maybe designed using either recombinant protein or mRNA in composition suitable for mRNA delivery.

[0262] Methods for pharmacokinetic evaluation of an antibody in vivo are well known in the art.

Example 5— Isolation and characterization of coronavirus antibodies

[0263] Potent neutralizing antibodies against SARS-CoV-2 can protect against infection in animal models and have been used to treat infection in humans¹'⁵. Throughout the course of the SARS-CoV-2 pandemic the Spike protein has mutated to evade neutralizing antibody recognition⁶'⁹. Thus, neutralizing antibodies that recognize most or all of the predominant variant SARS-CoV-2 viruses spreading globally are needed. To find broadly-reactive and potent neutralizing antibodies we identified a SARS-CoV-2 infected individual with potent serum neutralization of SARS-CoV-2 D614G pseudovirus (Figure 1).

[0264] The serum from this individual exhibited neutralizing antibody fifty percent inhibitory dilution (ID50) and eighty percent inhibitory dilution (ID80) titers of 1:24,948 and 1:5,109 respectively. These ID50 and ID80 titers were 16 and 12-fold higher than the geometric mean titer for the entire cohort (Cohort geometric mean ID50 = 1560 and ID80 = 427; N = 25). We sought to identify monoclonal antibodies that recapitulated the potent serum neutralizing activity from this individual. B cells that bound to SARS-CoV-2 spike ectodomain and receptor-binding domain (RBD) were sorted into single wells and their respective antibodies were cloned. ELISA binding assays identified 9 receptor-binding domain antibodies, 2 S2 antibodies, and one NTD antibody. The binding of seven of the RBD antibodies was affected by mutations at 417, 484 or 501. Such mutations occur in the Beta variant of SARS-CoV-2⁷'¹⁰. However, two of the RBD antibodies (DH1284 and DH1286) exhibited strong binding to RBD or Spike ectodomain with mutations at these sites. Moreover, these two RBD antibodies showed low levels of cross-reactivity with SARS-related pangolin (GXP4L) and Bat (RsSHC014 and RaTG13) coronavirus RBD or Spike ectodomains. Thus, DH1284 and DH1286 were of note since they were cross-reactive antibodies that were moderately affected or unaffected by the presence of natural resistance mutations in Spike.

[0265] Next, we examined each antibody’s ability to block ACE2 binding to SARS-CoV-2 Spike ectodomain, since blocking activity correlates with neutralization potency^{1 11}. DH1286 did not block RBD binding to ACE2, but DH1284 and four other antibodies blocked ACE2 binding to Spike. DH1284 was the most potent ACE2 blocking antibody, exhibiting a fifty percent inhibitory concentration of <0.045 mcg/mL (Figure 3). DH1284 also competed with neutralizing RBD antibodies DH1042, DH1047, and to a lesser extent DH1041 (Figure 3). DH1042, DH1041 and DH1047 are described in WO App Number PCT/US2021/050552 and Li et al . Voksm ; 84, Issue 16, 5 August 20 1 , Pages 4203-4219. e32.

[0266] Monoclonal antibody (mAb) DH1284 was derived from IGHV1-24 and VK1-5 heavy chain and light chain gene segments (Table 3), thus it was distinct from class I RBD antibodies known to potently block ACE2 binding to RBD. DH1284 was examined for neutralization of SARS-CoV-2 D614G and compared to the other mAbs isolated from the same subject. DH1284 was the most potent neutralizing antibody of the nine antibodies isolated. DH1284 neutralized the D614G and the B.1.617.2 Delta variant with an IC50 of 2 and 0.6 ng/mL respectively in this pseudovirus assay (Figure 4). Also, of all 12 monoclonal antibodies tested from subject 002, DH1284 was the only antibody capable of neutralizing B.1.351 (Figure 4). Next, we assessed DH1284 neutralization of the WA-1 strain as well as an expanded panel of SARS-CoV-2 variants of concern or interest. DH1284 potently neutralized the ancestral WA-1 strain of SARS-CoV-2 with an IC50 of 3.4 ng/mL (Figure 5). SARS-CoV-2 variants B.1.1.7, B.1.427, B.1.526, B.1.617.1, and B.1.617.2 (Delta variant) were also potently neutralized with IC50 values ranging from 3.4 to 6.7 ng/mL (Figure 5). The B.1.351 and P.l variants were slightly more resistant to DH1284 neutralization, but were still potently neutralized at 24.9 and 53 ng/mL (Figure 5). The neutralization titers for DH1284 were more potent than broadly neutralizing CoV antibody DH1047 (Figure 5). Neutralizing RBD antibody DH1041 was similarly potent against the WA-1 strain but was unable to neutralize 4 of the 7 variants of SARS-CoV-2 (Figure 5). Thus, DH1284 exhibited extraordinary SARS-CoV-2 neutralization potency and breadth.

[0267] RBD antibodies can be classified based on their structures and immunogenetics¹². To determine the binding mode of DH1284, we solved the structure of DH1284 in complex with SARS-CoV-2 HexaPro Spike (Figure 6). Immunogenetics are shown in Table 3. In the class of molecules used for the 3D reconstruction, DH1284 bound to the spike with a three fab to one trimer stoichiometric ratio. DH1284 bound to the vertical apex of the RBD with an approach angle that was parallel to the trimer axis (Figure 6). Only RBDs in the up conformation were observed bound to DH1284. Fitting the structure of Spike with three RBDs in the up conformation into the density of the 3D reconstruction showed that the DH1284 Fab bound at the same location as ACE2 (Figure 6). The overlap in binding site explained the potent ACE2 blocking and provides a potential mechanism for the potent neutralization activity (Figure 3). Interestingly, other antibodies such as DH1041 that bind to the ACE2 binding site on RBD lose binding in the presence of 417, 484, and 501 mutations^7,9. However, DH1284 accommodated mutations at those sites while still binding at the ACE2 binding site. [0268] Antibodies from this Example will be tested for neutralization of any other variant, including without limitation omicron variant.

[0269] References for Example 5

[0270] 1 Zost, S. J. et al. Potently neutralizing and protective human antibodies against SARS-CoV-2. Nature 584, 443-449, doi:10.1038/s41586-020-2548-6 (2020).

[0271] 2 Li, D. et al. In vitro and in vivo functions of SARS-CoV-2 infection-enhancing and neutralizing antibodies. Cell 184, 4203-4219. e4232, doi: 10.1016/j . cell.2021.06.021 (2021).

[0272] 3 Baum, A. et al. REGN-COV2 antibodies prevent and treat SARS-CoV-2 infection in rhesus macaques and hamsters. Science 370, 1110-1115, doi: 10.1126/science.abe2402 (2020).

[0273] 4 Chen, P. et al. SARS-CoV-2 Neutralizing Antibody LY-CoV555 in Outpatients with Covid-19. N Engl JMed384, 229-237, doi:10.1056/NEJMoa2029849 (2021).

[0274] 5 Lundgren, J. D. et al. A Neutralizing Monoclonal Antibody for Hospitalized Patients with Covid-19. N Engl J Med 384, 905-914, doi: 10.1056/NEJMoa2033130 (2021).

[0275] 6 Dejnirattisai, W. et al. Antibody evasion by the P.l strain of SARS-CoV-2. Cell 184, 2939-2954. e2939, doi:10.1016/j.cell.2021.03.055 (2021).

[0276] 7 Wang, P. etal. Antibody resistance of SARS-CoV-2 variants B.1.351 and B.1.1.7. Nature 593, 130-135, doi:10.1038/s41586-021-03398-2 (2021). [0277] 8 Shen, X. et al. Neutralization of SARS-CoV-2 Variants B.1.429 and B.1.351. N Engl J Med 384, 2352-2354, doi:10.1056/NEJMc2103740 (2021).

[0278] 9 Hoffmann, M. et al. SARS-CoV-2 variants B.1.351 and P.l escape from neutralizing antibodies. Cell 184, 2384-2393.e2312, doi:10.1016/j.cell.2021.03.036 (2021).

[0279] 10 Tegally, H. et al. Detection of a SARS-CoV-2 variant of concern in South Africa. Nature 592, 438-443, doi:10.1038/s41586-021-03402-9 (2021).

[0280] 11 Abe, K. T. et al. A simple protein-based surrogate neutralization assay for SARS-CoV-2. JCI Insight 5, doi: 10.1172/j ci. insight.142362 (2020).

[0281] 12 Barnes, C. O. et al. SARS-CoV-2 neutralizing antibody structures inform therapeutic strategies. Nature 588, 682-687, doi:10.1038/s41586-020-2852-l (2020).

Example 6— Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, neutralization and antigenicity

[0282] Antibodies from Example 5 will be further analyzed to determine epitope binding, and ability to neutralize mutations in SARS-CoV-2 on spike.

Example 7 - DH1293 and DH1284 antibodies

[0283] The antibodies DH1284 and DH1294 were used in additional studies as described herein. Figure 11 shows binding of the two antibodies to various viral targets. Figure 12 shows blocking of DH1284 binding to the receptor binding domain (RBD) of the spike protein by ACE-2. The data show that DH1294 binding to the spike RBD is not blocked by ACE-2. Figure 13 shows IC50 titers of DH1294 and DH1284 binding to various viral targets. Figure 14A-D shows data using DH1284 antibody as a prophylactic and therapeutic in mice infected with RsSHC014-MA15 virus. Figure 16 shows viral neutralization data using DH1284 and DH1294 against various viruses. Figure 17 shows neutralization data against various pseudotyped viruses using DH1284 and DH1294.

Claims

What is claimed is:

1. A recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds to coronavirus spike protein and comprises a variable heavy (VH) domain and a variable light (VL) domain that have amino acid sequences that have an overall 80% sequence identity to the VH and VL domains of an antibody listed in Table 3, or wherein the VH domain and VL domain each have at least 80% sequence identity to the VH and VL domains, respectively, of an antibody listed in Table 3.

2. The recombinant coronavirus monoclonal antibody, or the antigen binding fragment thereof of claim 1, wherein there is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity within a framework region of the VH and/or VL domain.

3. The recombinant coronavirus monoclonal antibody, or the antigen binding fragment thereof of claim 1, wherein there is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity is within a CDR region of the VH and/or VL domain.

4. A recombinant coronavirus monoclonal antibody, or an antigen binding fragment thereof, which binds coronavirus spike protein, the antibody or antigen binding fragment thereof, comprising: a. Vh domain CDRH1-3 regions from an antibody listed in Table 3; and/or b. VI domain CDRL1-3 regions from an antibody listed in Table 3, wherein the Vh and VI are from the same antibody; and c. wherein a framework of the variable heavy (Vh) domain comprises amino acid sequences that have at least 90% sequence identity to the V gene, D gene and J gene of the Vh gene of the corresponding antibody from which the CDRs are derived and wherein a framework of the variable light (VI) domain comprises amino acid sequences that have at least 90% sequence identity to the V and J genes of the VI gene from the corresponding antibody from which the CDRs are derived.

5. The recombinant coronavirus monoclonal antibody, or the antigen binding fragment thereof of claim 4, wherein, the Vh domain CDRH1-3 regions are at least 90% identical to the CDRH1-3 to an antibody listed in Table 3; and/or the VI domain CDRL1-3 regions

72 are at least 90% identical to an antibody listed in Table 3, wherein the Vh and VI are from the same antibody. The recombinant coronavirus antibody, or the antigen binding fragment thereof of any one of claims 1 or 4, wherein a framework region of the variable heavy (Vh) domain comprises amino acid sequences derived from human IGHV1-24 and IGHJ4 Ig genes (Table 3) and wherein a framework region of the variable light (VI) domain comprises amino acid sequences derived from IGKVl-5and IGKJ2 human IgG genes (Table 3). The recombinant coronavirus antibody, or the antigen binding fragment thereof of to claim 1, , comprising a heavy chain comprising at least one CDRH1, at least one CDRH2 and at least one CDRH3, and a light chain comprising at least one CDRL1, at least one CDRL2 and at least one CDRL3, wherein at least one CDR comprises an amino acid sequence according to any of paired Vh and VI sequences of antibody DH1284, DH1286, DH1287, DH1288, DH1289, DH1290 DH1291, DH1292, DH1293, DH1294, DH1295, DH1296, or DH1297 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. The recombinant coronavirus antibody, or the antigen binding fragment thereof of claim 7, comprising paired Vh and VI sequences of antibody DH1284, DH1286, DH1287, DH1288, DH1289, DH1290 DH1291, DH1292, DH1293, DH1294, DH1295, DH1296, or DH1297 or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity. The recombinant coronavirus antibody, or the antigen binding fragment thereof of any one of claims 7 or 8, comprising DH1284. The recombinant coronavirus antibody, or the antigen binding fragment thereof of any one of claims 7 or 8, comprising DH1294. The recombinant antibody, or the antigen binding fragment thereof of any one of claims 1, 4, 7 or 8, wherein the antibody or antigen binding fragment thereof binds to a receptor binding domain (RBD) of a coronavirus spike protein. The recombinant coronavirus antibody, or the antigen binding fragement thereof of any one of claims 1, 4, 7 or 8, wherein the antibody or antigen binding fragment thereof binds to a fusion domain of the coronavirus spike protein.

73 The recombinant coronavirus antibody, or the antigen-binding fragment thereof of any one of claims 1 or 4, comprising an Fc moiety. The recombinant coronavirus antibody, or the antigen binding fragment thereof of any one of claims 1 or 4, comprising a purified antibody IgG antibody, a multivalent antibody, a multispecific antibody, a single chain antibody, an Fab, an Fab', an F(ab')2, an Fv or an scFv. The recombinant coronavirus antibody, or the antigen-binding fragment thereof of any one of claims 1 or 4, for use as a medicament. A nucleic acid molecule, comprising a polynucleotide sequence encoding the recombinant coronavirus antibody, or the antigen-binding fragment thereof of any one of claims 1, 4, 7 or 8. The nucleic acid molecule of claim 16, wherein the polynucleotide sequence comprises, consists essentially of or consists of, any one of the sequences in Figure 8; or a functional sequence variant thereof having at least 70%, at least 75%, at least 80%, at least 85%, at least 88%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity thereto. The nucleic acid molecule of claim 16, comprising a ribonucleic acid (RNA). The nucleic acid molecule of claim 18, comprising an mRNA. The nucleic acid molecule of claim 19, wherein the mRNA is suitable for use and delivery as a therapeutic mRNA. A vector comprising the nucleic acid molecule of claim 16. A cell comprising the vector of claim 21. A cell expressing the recombinant coronavirus antibody, or the antigen binding fragment thereof according to any one of claims 1, 4, 7 or 8. A pharmaceutical composition comprising the recombinant coronavirus antibody, or the antigen binding fragment thereof of any one of claims 1, 4, 7 or 8, and a pharmaceutically acceptable excipient, diluent or carrier. A pharmaceutical composition, comprising the nucleic acid molecule of claim 16, and a pharmaceutically acceptable excipient, diluent or carrier.

74 A pharmaceutical composition comprising the vector of claim 21, and a pharmaceutically acceptable excipient, diluent or carrier. A pharmaceutical composition comprising the cell of claim 23, and a pharmaceutically acceptable excipient, diluent or carrier. The pharmaceutical composition of claim 24, wherein the recombinant coronavirus antibody, or the antigen binding fragment thereof, wherein the Vh and VI domains form a multivalent or multispecific antibody. The pharmaceutical composition of claim 24, wherein the recombinant coronavirus antibody, or the antigen binding fragment thereof binds to a receptor binding domain (RBD) of a coronavirus spike protein. The pharmaceutical composition of claim 24, wherein the recombinant coronavirus antibody, or the antigen binding fragment thereof binds to an NTD domain of a coronavirus spike protein. The pharmaceutical composition of claim 24, wherein the recombinant coronavirus antibody, or the antigen binding fragment thereof binds to a fusion domain of a coronavirus spike protein. A pharmaceutical composition comprising two RBD binding antibodies or antigen binding fragments thereof, wherein the two antibodies or antigen binding fragments thereof have non-overlapping epitopes and a pharmaceutically acceptable excipient, diluent or carrier. A method of treating or preventing coronavirus infection in a subject in need thereof, comprising administering the recombinant coronavirus antibody, or the antigen binding fragment thereof of any one of claims 1, 4, 7 or 8 to the subject. A method of treating or preventing coronavirus infection of a subject in need thereof, comprising administering the nucleic acid molecule of claim 16 to the subject. A method of treating or preventing coronavirus infection in a subject in need thereof, comprising administering the pharmaceutical composition of claim 24 to the subject. The method of claim 33, wherein the recombinant coronavirus antibody, or antigen binding fragment thereof is administered prior to coronavirus exposure or at the same time as coronavirus exposure.

75 A method of treating or preventing coronavirus infection comprising administering a therapeutic or prophylactic amount of a composition comprising an antibody or antigen binding fragment thereof comprising a Vh and VI sequence of any one of claims 1, 4, 7 or 8, wherein the Vh and VI sequences are comprised in a bi- or tri-specific antibody format or in a multivalent antibody format. An isolated monoclonal antibody or antigen-binding fragment thereof that binds to a coronavirus spike protein or fragment thereof, comprising a heavy chain, light chain, or both a heavy chain and a light chain, wherein the heavy chain comprises CDRs DY AMY, AISSSGGGTYHVESVKG and AFYDPGSYYNPRPYGMDV; and wherein the light chain comprises CDRs RASQDINNYLA, AASTLQS and QQLHTYPPIT. The antibody of claim 38, wherein DY AMY comprises CDR1, AISSSGGGTYHVESVKG comprises CDR2 and AFYDPGSYYNPRPYGMDV comprises CDR3; and wherein RASQDINNYLA comprises CDR1 , AASTLQS comprises CDR2 and QQLHTYPPIT comprises CDR3. The antibody or fragment of claim 38, wherein the coronavirus spike protein comprises a human coronavirus spike protein. The antibody or fragment of claim 38, wherein the antibody comprises a fully human or humanized antibody. The antibody or fragment of claim 38, wherein the antibody comprises a monospecific, bispecific, or multispecific antibody. The antibody or fragment of claim 38, wherein the antibody comprises IgG. The antibody or fragment of claim 38, wherein the antibody comprises a single chain antibody. The antibody or fragment of claim 38, wherein the antibody or fragment has a binding affinity of at least 1 x 1 O'⁹ M. The antibody or fragment of claim 38, further comprising a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

76 The antibody or fragment of claim 38, comprising DH1294. The antibody or fragment of claim 38, wherein the antibody or fragment competes with binding of DH1294. The antibody or fragment of claim 38, wherein the antibody or fragment is linked to a therapeutic agent. The antibody or fragment of claim 38, wherein the antibody comprises a single chain fragment. An isolated antibody or fragment thereof that binds to a human coronavirus spike protein, comprising a heavy chain variable region amino acid sequence SEQ ID NO: 19, or a sequence at least 90% identical thereto. An isolated antibody or fragment thereof that binds to a human coronavirus spike protein, comprising a light chain variable region amino acid sequence SEQ ID NO: 20, or a sequence at least 90% identical thereto. An isolated antibody or fragment thereof that binds to a human coronavirus spike protein, comprising a heavy chain variable region amino acid sequence SEQ ID NO: 19, or a sequence at least 90% identical thereto; and comprising a light chain variable region amino acid sequence SEQ ID NO: 20, or a sequence at least 90% identical thereto. An isolated scFV that binds to a human coronavirus spike protein, comprising a heavy chain variable region amino acid sequence SEQ ID NO: 19, or a sequence at least 90% identical thereto. An isolated scFV that binds to a human coronavirus spike protein, comprising a light chain variable region amino acid sequence SEQ ID NO: 20, or a sequence at least 90% identical thereto. An isolated scFV that binds to a human coronavirus spike protein, comprising a heavy chain variable region amino acid sequence SEQ ID NO: 19, or a sequence at least 90% identical thereto; and a light chain variable region amino acid sequence SEQ ID NO: 20, or a sequence at least 90% identical thereto. An isolated bispecific antibody comprising the fragment of any one of claims 38-56 and a second antigen-binding fragment having specificity to a molecule on an immune cell.

77 The bispecific antibody of claim 57, wherein the fragment and the second fragment each is independently selected from an Fab fragment, a single-chain variable fragment (scFv), or a single-domain antibody. The bispecific antibody of claim 57, further comprising an Fc fragment. A nucleic acid encoding the antibody, fragment or scFV of any one of claims 38-56. A nucleic acid encoding the bispecific antibody of claim 57. A pharmaceutical composition comprising the antibody or fragment of any one of claims 38-56, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 62, further comprising at least one additional therapeutic agent. A pharmaceutical composition comprising the bispecific antibody of claim 57, and a pharmaceutically acceptable carrier or excipient. The pharmaceutical composition of claim 64, further comprising at least one additional therapeutic agent.