WO2025024233A1 - Bispecific antibodies that broadly target coronaviruses - Google Patents

Abstract

Disclosed are bispecific antibodies, including dual variable domain immunoglobulins that specifically bind a coronavirus spike protein, such as SARS-CoV-2. Also disclosed is the use of these antibodies for inhibiting a coronavirus infection. In addition, disclosed are methods for detecting a coronavirus in a biological sample, using the disclosed antibodies.

Description

BISPECIFIC ANTIBODIES THAT BROADLY TARGET CORONAVIRUSES

CROSS REFERENCE TO RELATED APPLICATIONS

This claims the benefit of U.S. Provisional Application No. 63/528,158, filed July 21, 2023, which is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

This relates to bispecific antibodies, such as dual variable domain (DVD) immunoglobulins, that specifically bind a coronavirus spike protein, and their use for inhibiting or detecting a coronavirus infection in a subject.

SEQUENCE LISTING

The contents of the electronic sequence listing (Sequence. xml; Size 125,213 bytes; and Date of Creation: July 18, 2024) is herein incorporated by reference in its entirety.

BACKGROUND

The coronavirus SARS-CoV-2 emerged in 2019 and has infected 400 million individuals and caused more than 5 million deaths worldwide. While effective vaccines and antibody therapeutics have been developed, their efficacy is threatened by the emergence of variants which are more resistant than previous variants to many of these tools. Furthermore, the possibility of further coronavirus spillover from animal reservoirs remains and must be prepared for. A need remains for bispecific antibodies that bind multiple antigens of coronaviruses.

SUMMARY OF THE DISCLOSURE

Bispecific antibodies are disclosed herein that specifically bind a coronavirus. In some aspects, disclosed are DVD immunoglobulin that specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.

In some aspects, a DVD immunoglobulin includes a heavy chain and a light chain, and wherein: a) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, and 40, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain (COV44-79_COV89-22_GS and COV44-79_COV89-22_EL); b) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a Vi. domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second V domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain (COV44-62_COV72-37_GS and COV44-62_COV72-37_EL); c) the heavy chain comprises, in amino (N) to carboxy (C) terminal order: a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 10, 11 and 12, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs: 14, 15 and 16, and a heavy chain constant domain (COV44-62_COV30-14_GS and COV44-62_COV30-14_EL); d) the heavy chain of the bispecific antibody comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, and light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain (COV44-62_COV89-22_GS and COV44-62_COV89-22_EL); e) the heavy chain comprises, in N to C terminal order: a first VH domain comprising SEQ ID NOs: 10, 11 and 12, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order: a VL domain comprising SEQ ID NOs: 14, 15 and 16 a linker, a second VL domain comprising SEQ ID NOs: 6, 7 and 8, and a light chain constant domain (COV30-14_COV44-62_GS and COV30-14_COV44-62_EL); f) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second VL domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain (COV72-37_ COV44-62_GS and COV72-37_ COV44-62_EL); g) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain (COV89-22_ COV44-62_GS and COV89-22_ COV44-62_EL); h) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, and 40, a linker, a second VL domain comprising SEQ ID NOs: 14, 15, andl6, and a light chain constant domain (COV44-79_COV30-14_GS and COV44-79_COV30-14_EL); i) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, and the light chain comprises in N to C terminal order, a VL domain comprising SEQ ID NOs: 14, 15, and 16, a linker, a second VL domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain (COV30-14_COV44-79_GS and COV30-14_COV44-79_EL); j) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, and light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, 40, a linker, a second VL domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain (COV44-79_COV72-37_GS and COV44-79_COV72-37_EL); k) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a V domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second V_L domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain (COV72-37_COV44-79_GS and COV72-37_COV44-79_EL); l) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain (COV89-22_COV44-79_GS and COV89-22_COV44-79_EL); m) the heavy chain of the bispecific antibody comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second VL domain comprising SEQ ID NOs: 14, 15, and 16, and a light chain constant domain (COV91-27_COV30-14_GS and COV91-27_COV30- 14_EL); n) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 14, 15, and 16, a linker, a second VL domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain (COV30-14_COV91-27_GS and COV30-14_COV91-27_EL); o) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second V_L domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain (COV91-27_COV72-37_GS and COV91-27_COV72-37_EL); p) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second V_L domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain (COV72-37_COV91-27_GS and COV72-37_COV91-27_EL); q) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, and the light chain of the bispecific antibody comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain (COV91-27_COV89-22_GS and COV91-27_COV89- 22_EL); or r) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a V domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain (COV89-22_COV91-27_GS and COV89-22_COV91-27_EL).

In some aspects, disclosed are nucleic acid molecules encoding these bispecific antibodies, vectors including these nucleic acid molecules, and host cells including these vectors.

Pharmaceutical compositions including the bispecific antibodies nucleic acid molecules and vectors are also disclosed. In more aspects, disclosed is the use of these pharmaceutical compositions to inhibit a coronavirus infection in a subject.

In yet other aspects, disclosed is the use of the disclosed antibodies and antigen binding fragments for the detection of a coronavirus in a biological sample.

The foregoing and other features of the disclosure will become more apparent from the following detailed description of several aspects which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Individual antibodies show binding specificity to two distinct neutralizing epitopes of coronavirus spike proteins: the fusion peptide and the stem helix. The heat map represents the binding of the bispecific antibodies to peptides that represent fusion peptide and stem helix residues which are highly conserved in spike proteins of SARS-CoV-2 Wuhan Hu-1, SARS-CoV-1, MERS-CoV, HCoV-HKUl, HCoV-OC43 (betacoronavirus genus), HCoV-NL63 (alphacoronavirus genus), and HCoV-229E (alphacoronavirus genus). mAbs specific to the fusion peptide (COV44-62, COV44-79, COV91-27), stem helix (COV89-22, COV30-14 and COV72-37) and the anti-HIV-1 spike protein mAb VRC01 were included as controls for mAb binding experiments. Area under the curve (AUC) values for each antigen are shown after subtracting values for the negative control antigen CD4.

FIGs. 2-10. Tables showing the amino acid sequences of bispecific antibodies that specifically bind both the SARS-CoV-2 fusion peptide and stem helix.

FIGs. 11A-11C. Neutralization curves for SARS COV-2 pseudovirus (USA-WA1 2020 strain). Curves are shown for parental stem helix mAbs (CQV30-14, COV72-37 and COV89-22), parental fusion peptide mAbs (COV44-62, COV44-79 and COV91-27) and representative bispecific antibodies. The dotted line indicates 50% neutralization of infection and error bars show standard deviation of two replicates. FIGs. 12A-12B. Tables evidencing the bispecific antibodies neutralize SARS COV-2 pseudovirus (USA-WA1 2020 strain) infection of ACE2 expressing HeLa cells.

SEQUENCES

The nucleic and amino acid sequences are shown using standard letter abbreviations for nucleotide bases, and three letter code for amino acids, as defined in 37 C.F.R. 1.822. Only one strand of each nucleic acid sequence is shown, but the complementary strand is understood as included by any reference to the displayed strand. The complementarity determining regions (CDRs) are shown in bold in each variable heavy chain domain (VH) and variable light chain domain (VL) shown below.

SEQ ID NO: 1 is the amino acid sequence of the COV44-62 VH. SEQ ID NOs: 2, 3 and 4 are the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively.

QMQLMQSGAEVKKPGASVTVSCKASGDTFSDYRIHWVRQAPGQGLEWMGRMNPKSGDTNFAQ KFQGRVTMTRDMSINTAYMTLSGLTFDDTALYYCASLLIVGGFDPLDDFEVWGQGTMVTISS.

SEQ ID NO: 5 is the amino acid sequence of the COV44-62 VL. SEQ ID NOs: 6, 7, and 8 are the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively.

QSALTQPPSASGSPGQSVTISCSGTSSDVGGYNFVSWYQHHPGKAPKILIYEVTKRPSGVPDRFSGS KSGNTASLTVSGLQAEDEADYYCSSYGGTNNLLFGGGTKLTVL.

SEQ ID NO: 9 is the amino acid sequence of the COV30-14 VH. SEQ ID NOs: 10, 11, and 12 are the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively.

QVQLVQSGAEVKKPGASVKVSCQTSGYTFTSYYMHWVRQAPGQGLEWMGLITPSGDDTYYAQR FQGRVTMTRDTSTSPTYMELSSLTSEDTAVYYCAKMSRAGGFDVWGQGTLVTVSS

SEQ ID NO: 13 is the amino acid sequence of the COV30-14 VL. SEQ ID NOs: 14, 15, and 16 are the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively.

EVVLTQSPGTLSLSPGERATLSCRASQSITGRYLAWYQQKPGQAPRLLMYGESSRVTGIPDRFSGG GSGTDFTLTISRLEPEDFAVYYCQHFASSPPTYTFGQGTKLEIR.

SEQ ID NO: 17 is the amino acid sequence of the COV72-37 VH- SEQ ID NOs: 18, 19, and 20 are the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively.

QVQLVQSGAEVKKPAASVKVSCKASGDTFSSHYMHWVRQAPGQGPEWMGIINPSGSGTAYGQK FQGRLTMTRDTSTSTVYMELSSLTSDDTAVYYCGGGSGGLFAYWGQGTLVTVSS

SEQ ID NO: 21 is the amino acid sequence of the COV72-37 VL. SEQ ID NOs: 22, 23, and 24 are the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively;

EIVLTQSPGTLSLSPGERATLSCRASQIVRSNYLAWYQQKPGQAPRLLIYGASSRATGTPDRFSGGG SGTDFTLT1NRLEPEDFAVYYCLQYDSSPPTYIFGQGTKLE1K.

SEQ ID NO: 25 is the amino acid sequence of the COV89-22 VH. SEQ ID NOs: 26, 27, and 28 are the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively.

QEQLVQSGAEVKKPGASVKVSCKSSGFTFSYFYLHWVRQAPGQGLEWMGIINPRGDGTRYAQKF QGRVTMTRDASTGTLYMELRSLRSEDTAVYYCARGADHGAFDIWGQGTMVTVSS SEQ ID NO: 29 is the amino acid sequence of the COV89-22VL. SEQ ID NOs: 30, 31, and 32 are the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively. EIVLTQSPGTLSLSPGERATLSCRASQSVRRNYFAWYQQKRGQAPRLLIYDASTRATGIPDRFSGSG SGTDFTLTISRLEPEDFAVYYCQQYDSSPPMYIFGQGTKLEIK.

SEQ ID NO: 33 is the amino acid sequence of the COV44-79 VH. SEQ ID NOs: 34, 35, and 36 are the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively. QVQLVESGGGVVQPGRSLRLSCAASGLTFSGYAMHWVRQAPGKGLEWVAVISRDARNKYYADS VKGRFTISRDNSKKTVYLEMNSLRVEDTAVYYCAILIIPGITEPGSPDALDIWGQGTMVSVSS.

SEQ ID NO: 37 is the amino acid sequence of the COV44-79 VL. SEQ ID NOs: 38, 39, and 40 are the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively DIQMTQSPSSMSASVGDRVTITCRASQDISKWLAWYQQRPGKAPKLLIYAASSLQSGVPSRFSGSGS GTDFTLTISSLQPEDFATYYCQQASSFPWSITFGQGTRLEIR.

SEQ ID NO: 41 is the amino acid sequence of the COV91-27 VH- SEQ ID NOs: 42, 43, and 44 are the amino acid sequences of the HCDR1, HCDR2, and HCDR3, respectively. QVQLVESGGGVVHPGGSLRLSCAASGFTFSDFAMNWVRQAPGEGLQWVAIISYDGRNKHFAASV RGRFTISRDNSKNTMFLQMSSLRAEDTAIYYCATLGGWFEEASPTYWGRGTLVTVSS.

SEQ ID NO: 45 is the amino acid sequence of the COV91-27 VL. SEQ ID NOs: 46, 47, and 48 are the amino acid sequences of the LCDR1, LCDR2, and LCDR3, respectively. EVVLTQSPAIVSLSPGERATLSCRASQSVGSRLAWHQQKPGLAPRVLIYDASNRATGIPARFSGSGS GTDFTLTISSIEPEDSAVYYCQQRGDWPLITFGQGTRLEIK.

SEQ ID NO: 49-117 and 125-127 are the amino acid sequences of DVD-immunoglobulin antibody sequences, also shown in FIGs. 2-10.

SEQ ID NO: 118-120 are examples of the amino acid sequences of a stem helix and part of the fusion peptide in the S2 domain of the spike protein.

SEQ ID NO: 121 is the amino acid sequence of the GS linker.

SEQ ID NOs: 122-124 are the amino acid sequences of three different EL linkers where the outer V is a heavy chain, kappa, or lambda, respectively.

DETAILED DESCRIPTION OF SEVERAL ASPECTS

Bispecific antibodies are disclosed herein that include VH and a VL from two different antibodies that specifically bind the spike protein of SARS-CoV-2 and bind the spike protein of at least one additional coronavirus, such as an alphacoronavirus or a betacoronavirus. In some aspects, the monoclonal antibodies bind the S2 domain of the spike protein. The bispecific antibody can be a DVD-Ig. These bispecific antibodies can be used to inhibit a coronavirus infection, such as, but not limited to, a SARS-Cov-2 infection. The monoclonal antibodies and bispecific antibodies also can be used to detect a coronavirus infection. I. Summary of Terms

Unless otherwise noted, technical terms are used according to conventional usage. Definitions of many common terms in molecular biology may be found in Krebs et al. (eds.), Lewin ’s genes XII, published by Jones & Bartlett Learning, 2017. As used herein, the singular forms “a,” “an,” and “the,” refer to both the singular as well as plural, unless the context clearly indicates otherwise. For example, the term “an antigen” includes singular or plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprises” means “includes.” It is further to be understood that any and all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for descriptive purposes, unless otherwise indicated. Although many methods and materials similar or equivalent to those described herein can be used, particular suitable methods and materials are described herein. In case of conflict, the present specification, including explanations of terms, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting. To facilitate review of the various aspects, the following explanations of terms are provided:

About: Unless context indicated otherwise, “about” refers to plus or minus 5% of a reference value. For example, “about” 100 refers to 95 to 105.

Administration: The introduction of an agent, such as a disclosed DVD immunoglobulins, into a subject by a chosen route. Administration can be local or systemic. For example, if the chosen route is intravascular, the agent (such as the DVD-immunoglobulin) is administered by introducing the composition into a blood vessel of the subject. Exemplary routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, transdermal (for example, topical), intranasal, vaginal, and inhalation routes.

Amino acid substitution: The replacement of one amino acid in a polypeptide with a different amino acid.

Antibody and Antigen Binding Fragment: An immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes an analyte (antigen) such as a coronavirus spike protein, such as a spike protein from SARS-CoV-2. The term “antibody” is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen binding fragments, so long as they exhibit the desired antigen-binding activity.

Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Antigen binding fragments include a VH and a VL, and specifically bind the cognate antigen. Examples of antigen binding fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (see, e.g., Kontermann and Diibel (Eds.), Antibody Engineering, Vols. 1-2, 2^nd ed., Spring er- Verlag, 2010).

Antibodies also include genetically engineered forms such as chimeric antibodies (such as humanized murine antibodies) and heteroconjugate antibodies (such as bispecific antibodies).

An antibody may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or may be different. For instance, a naturally occurring immunoglobulin has two identical binding sites, a single-chain antibody or Fab fragment has one binding site, while a bispecific or bifunctional antibody has two different binding sites.

Typically, a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds. Immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable domain genes. There are two types of light chain, lambda (X) and kappa (K). There are five main heavy chain classes (or isotypes) which determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region (or constant domain) and a variable region (or variable domain). In combination, the heavy and the light chain variable regions specifically bind the antigen.

References to “VH” or “VH” refer to the variable region of an antibody heavy chain, including that of an antigen binding fragment, such as Fv, scFv, dsFv or Fab. References to “VL” or “VL” refer to the variable domain of an antibody light chain, including that of an Fv, scFv, dsFv or Fab.

The VH and VL contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs” (see, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 5^th ed., NTH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991). The sequences of the framework regions of different light or heavy chains are relatively conserved within a species. The framework region of an antibody, that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.

The CDRs are primarily responsible for binding to an epitope of an antigen. The amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (Sequences of Proteins of Immunological Interest, 5^th ed., NIH Publication No. 91-3242, Public Health Service, National Institutes of Health, U.S. Department of Health and Human Services, 1991; “Kabat” numbering scheme), Al-Lazikani et al., (“Standard conformations for the canonical structures of immunoglobulins,” J. Mol. Bio., 273(4):927-948, 1997; “Chothia” numbering scheme), and Lefranc et al. (“IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev. Comp. Immunol., l(\f.55-Tl, 2003; “IMGT” numbering scheme). The CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3 (from the N-terminus to C-terminus), and are also typically identified by the chain in which the particular CDR is located. Thus, a VH CDR3 is the CDR3 from the VH of the antibody in which it is found, whereas a V CDR1 is the CDR1 from the VL of the antibody in which it is found. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

In some aspects, a disclosed monoclonal antibody includes a heterologous constant domain. For example, the antibody includes a constant domain that is different from a native constant domain, such as a constant domain including one or more modifications (such as the “LS” mutation) to increase half-life.

A “monoclonal antibody” is an antibody obtained from a population of substantially homogeneous antibodies, that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, for example, containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier “monoclonal” indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein. In some examples monoclonal antibodies are isolated from a subject. Monoclonal antibodies can have conservative amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. (See, for example, Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014.)

A “humanized” antibody or antigen binding fragment includes a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) antibody or antigen binding fragment. The non-human antibody or antigen binding fragment providing the CDRs is termed a “donor,” and the human antibody or antigen binding fragment providing the framework is termed an “acceptor.” In one aspect, all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they can be substantially identical to human immunoglobulin constant regions, such as at least about 85-90%, such as about 95% or more identical. Hence, all parts of a humanized antibody or antigen binding fragment, except possibly the CDRs, are substantially identical to corresponding parts of natural human antibody sequences.

A “chimeric antibody” is an antibody which includes sequences derived from two different antibodies, which typically are of different species. In some examples, a chimeric antibody includes one or more CDRs and/or framework regions from one human antibody and CDRs and/or framework regions from another human antibody.

A “fully human antibody” or “human antibody” is an antibody which includes sequences from (or derived from) the human genome, and does not include sequence from another species. In some aspects, a human antibody includes CDRs, framework regions, and (if present) an Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using technologies for creating antibodies based on sequences derived from the human genome, for example by phage display or using transgenic animals (see, e.g., Barbas et aL Phage display: A Laboratory Manuel. 1^st Ed. New York: Cold Spring Harbor Laboratory Press, 2004. Print.; Lonberg, Nat. Biotech., 23: 1117-1125, 2005; Lonenberg, Curr. Opin. Immunol., 20:450-459, 2008).

Antibody or antigen binding fragment that neutralizes a coronavirus: An antibody or antigen binding fragment that specifically binds to a coronavirus antigen (such as the spike protein) in such a way as to inhibit a biological function associated with the coronavirus that inhibits infection. The antibody can neutralize the activity of one or more coronaviruses. For example, an antibody or antigen binding fragment that neutralizes the coronavirus, such as SARS-CoV-2 may interfere with the virus by binding it directly and limiting entry into cells. Alternately, an antibody may interfere with one or more post-attachment interactions of the pathogen with a receptor, for example, by interfering with viral entry using the receptor. In some examples, an antibody that is specific for a coronavirus spike protein neutralizes the infectious titer of the coronavirus.

In some aspects, an antibody or antigen binding fragment that specifically binds to a coronavirus, and neutralizes the coronavirus, inhibits infection of cells, for example, by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99% compared to a control antibody or antigen binding fragment.

A “broadly neutralizing antibody” is an antibody that binds to and inhibits the function of related antigens, such as antigens that share at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity antigenic surface of antigen. With regard to an antigen from a pathogen, such as a virus, the antibody can bind to and inhibit the function of an antigen from more than one class and/or subclass of the pathogen. For example, with regard to a coronavirus, the antibody can bind to and inhibit the function of an antigen, such as the spike protein from coronaviruses.

Biological sample: A sample obtained from a subject. Biological samples include all clinical samples useful for detection of disease or infection in subjects, including, but not limited to, cells, tissues, and bodily fluids, such as blood, derivatives and fractions of blood (such as serum), saliva, sputum, nasal swab, cerebrospinal fluid; as well as biopsied or surgically removed tissue, for example tissues that are unfixed, frozen, or fixed in formalin or paraffin. In a particular example, a biological sample is obtained from a subject having or suspected of having a coronavirus infection, such as, but not limited to, a SARS- CoV-2 infection.

Bispecific antibody: A recombinant molecule composed of two different antigen binding domains that consequently binds to two different antigenic epitopes, although both epitopes may be coronavirus epitopes. Bispecific antibodies include chemically or genetically linked molecules of two antigen-binding domains. The antigen binding domains can be linked using a linker. The antigen binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. A bispecific antibody can include one or more constant domains, but does not necessarily include a constant domain. In one example, a bispecific antibody is a DVD-immunoglobulin.

Conditions sufficient to form an immune complex: Conditions which allow an antibody or antigen binding fragment to bind to its cognate epitope to a detectably greater degree than, and/or to the substantial exclusion of, binding to substantially all other epitopes. Conditions sufficient to form an immune complex are dependent upon the format of the binding reaction and typically are those utilized in immunoassay protocols, or those conditions encountered in vivo. See Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, for a description of immunoassay formats and conditions. The conditions employed in the methods are “physiological conditions” which include reference to conditions e.g., temperature, osmolarity, pH) that are typical inside a living mammal or a mammalian cell. While it is recognized that some organs are subject to extreme conditions, the intra-organismal and intracellular environment normally lies around pH 7 (e.g., from pH 6.0 to pH 8.0, such as pH 6.5 to 7.5), contains water as the predominant solvent, and exists at a temperature above 0°C and below 50°C. Osmolarity is within the range that is supportive of cell viability and proliferation.

The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging (MRI), computed tomography (CT) scans, radiography, and affinity chromatography.

Conjugate: A complex of two molecules linked together, for example, linked together by a covalent bond. In one aspect, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to one or more coronaviruses, such as SARS-CoV-2, covalently linked to an effector molecule, such as a detectable label. The linkage can be by chemical or recombinant means. In one aspect, the linkage is chemical, wherein a reaction between the antibody moiety and the effector molecule has produced a covalent bond formed between the two molecules to form one molecule. A peptide linker (short peptide sequence) can optionally be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with separate functionalities, such as an antibody and an effector molecule, they are also sometimes referred to as “chimeric molecules.”

Conservative variants: “Conservative” amino acid substitutions are those substitutions that do not substantially affect or decrease a function of a protein, such as the ability of the protein to interact with a target protein. For example, a coronavirus-specific antibody disclosed herein can include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conservative substitutions compared to a reference antibody sequence and retain specific binding activity for spike protein binding, and/or neutralization activity. The term conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid. Individual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids (for instance less than 5%, in some aspects less than 1%) in an encoded sequence are conservative variations where the alterations result in the substitution of an amino acid with a chemically similar amino acid.

The following six groups are examples of amino acids that are considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Non-conservative substitutions are those that reduce an activity or function of the antibody, such as the ability to specifically bind to a coronavirus spike protein. For instance, if an amino acid residue is essential for a function of the protein, even an otherwise conservative substitution may disrupt that activity. Thus, a conservative substitution does not alter the basic function of a protein of interest.

Contacting: Placement in direct physical association; includes both in solid and liquid form, which can take place either in vivo or in vitro. Contacting includes contact between one molecule and another molecule, for example the amino acid on the surface of one polypeptide, such as an antigen, that contacts another polypeptide, such as a bispecific antibody. Contacting can also include contacting a cell for example by placing an antibody in direct physical association with a cell.

Control: A reference standard. In some aspects, the control is a negative control, such as sample obtained from a healthy patient not infected with a coronavirus. In other aspects, the control is a positive control, such as a tissue sample obtained from a patient diagnosed with a coronavirus infection. In still other aspects, the control is a historical control or standard reference value or range of values (such as a previously tested control sample, such as a group of patients with known prognosis or outcome, or group of samples that represent baseline or normal values).

A difference between a test sample and a control can be an increase or conversely a decrease. The difference can be a qualitative difference or a quantitative difference, for example a statistically significant difference. In some examples, a difference is an increase or decrease, relative to a control, of at least about 5%, such as at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, or at least about 500%.

Coronavirus: A family of positive-sense, single-stranded RNA viruses that are known to cause severe respiratory illness. Viruses currently known to infect human from the coronavirus family are from the alphacoronavirus and betacoronavirus genera. Additionally, it is believed that the gammacoronavirus and deltacoronavirus genera may infect humans in the future.

Non-limiting examples of betacoronaviruses include SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome coronavirus (SARS-CoV), Human coronavirus HKU1 (HKUl-CoV), Human coronavirus OC43 (OC43-CoV), Murine Hepatitis Virus (MHV-CoV), Bat SARS-like coronavirus WIV1 (WIVl-CoV), and Human coronavirus HKU9 (HKU9- CoV). Non-limiting examples of alphacoronaviruses include human coronavirus 229E (229E-CoV), human coronavirus NL63 (NL63-CoV), porcine epidemic diarrhea virus (PEDV), and Transmissible gastroenteritis coronavirus (TGEV). A non-limiting example of a deltacoronaviruses is the Swine Delta Coronavirus (SDCV).

The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. The coronavirus virion includes a viral envelope containing type I fusion glycoproteins referred to as the spike (S) protein. Most coronaviruses have a common genome organization with the replicase gene.

Degenerate variant: In the context of the present disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide (such as an antibody heavy or light chain) that includes a sequence that is degenerate as a result of the genetic code. There are 20 natural amino acids, most of which are specified by more than one codon. Therefore, all degenerate nucleotide sequences encoding a peptide are included as long as the amino acid sequence of the peptide encoded by the nucleotide sequence is unchanged.

Detectable marker: A detectable molecule (also known as a label) that is conjugated directly or indirectly to a second molecule, such as a bispecific antibody, to facilitate detection of the second molecule. For example, the detectable marker can be capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT scans, MRIs, ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). Methods for using detectable markers and guidance in the choice of detectable markers appropriate for various purposes are discussed for example in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4^th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements, 2017).

Detecting: To identify the existence, presence, or fact of something. Effective amount: A quantity of a specific substance sufficient to achieve a desired effect in a subject to whom the substance is administered. For instance, this can be the amount necessary to inhibit a coronavirus infection, such as a SARS-CoV-2 infection, or to measurably alter outward symptoms of such an infection.

In one example, a desired response is to inhibit or reduce or prevent SARS-CoV-2 infection. The SARS-CoV-2 infection does not need to be completely eliminated or reduced or prevented for the method to be effective. For example, administration of an effective amount of the immunogen can induce an immune response that decreases the SARS-CoV-2 infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the SARS-CoV-2) by a desired amount, for example by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable SARS-CoV-2 infection), as compared to a suitable control. Additional coronavirus infections can also be inhibited as a result of using the presently disclosed antibodies.

In some aspects, administration of an effective amount of a disclosed DVD-immunoglobulin that binds to a coronavirus spike protein can reduce or inhibit infection (for example, as measured by infection of cells, or by number or percentage of subjects infected by the coronavirus or by an increase in the survival time of infected subjects, or reduction in symptoms associated with the infection) by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable infection), as compared to a suitable control.

The effective amount of a DVD-immunoglobulin that specifically binds the coronavirus spike protein that is administered to a subject to inhibit infection may vary depending upon a number of factors associated with that subject, for example the overall health and/or weight of the subject. An effective amount can be determined by varying the dosage and measuring the resulting response, such as, for example, a reduction in pathogen titer. Effective amounts also can be determined through various in vitro, in vivo or in situ immunoassays.

An effective amount encompasses a fractional dose that contributes in combination with previous or subsequent administrations to attaining an effective response. For example, an effective amount of an agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment lasting several days or weeks. However, the effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration. A unit dosage form of the agent can be packaged in an amount, or in multiples of the effective amount, for example, in a vial (e.g., with a pierceable lid) or syringe having sterile components.

Effector molecule: A molecule intended to have or produce a desired effect; for example, a desired effect on a cell to which the effector molecule is targeted, or a detectable marker. Effector molecules can include, for example, polypeptides and small molecules. Some effector molecules may have or produce more than one desired effect.

Epitope: An antigenic determinant. These are particular chemical groups or peptide sequences on a molecule that are antigenic, such that they elicit a specific immune response, for example, an epitope is the region of an antigen to which B and/or T cells respond. An antibody can bind to a particular antigenic epitope, such as an epitope on a coronavirus spike protein.

Expression: Transcription or translation of a nucleic acid sequence. For example, an encoding nucleic acid sequence (such as a gene) can be expressed when its DNA is transcribed into RNA or an RNA fragment, which in some examples is processed to become mRNA. An encoding nucleic acid sequence (such as a gene) may also be expressed when its mRNA is translated into an amino acid sequence, such as a protein or a protein fragment. In a particular example, a heterologous gene is expressed when it is transcribed into an RNA. In another example, a heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include controls on transcription, translation, RNA transport and processing, degradation of intermediary molecules such as mRNA, or through activation, inactivation, compartmentalization, or degradation of specific protein molecules after they are produced.

Dual variable domain immunoglobulin: A bi-specific antibody that includes two heavy chain variable domains and two light chain variable domains. Unlike IgG, however, both heavy and light chains of a DVD-immunoglobulin molecule contain an additional variable domain (VD) connected via a linker sequence at the N-termini of the VH and VL of an existing monoclonal antibody (mAb). Thus, when the heavy and the light chains combine, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites, see Jakob et al., Mabs 5: 358-363, 2013, incorporated herein by reference, see Fig. 1 of Jakob et al. for schematic and space-filling diagrams. A DVD-immunoglobulin molecule functions to bind two different antigens on each DFab simultaneously.

Expression Control Sequences: Nucleic acid sequences that regulate the expression of a heterologous nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcriptional terminators, a start codon (ATG) in front of a protein-encoding gene, splice signals for introns, maintenance of the correct reading frame of that gene to permit proper translation of mRNA and stop codons. The term “control sequences” is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

Expression vector: A vector including a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed. An expression vector includes sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system. Non-limiting examples of expression vectors include cosmids, plasmids (e.g., naked or contained in liposomes) and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) that incorporate the recombinant polynucleotide.

A polynucleotide can be inserted into an expression vector that contains a promoter sequence which facilitates the efficient transcription of the inserted genetic sequence of the host. The expression vector typically contains an origin of replication, a promoter, as well as specific nucleic acid sequences that allow phenotypic selection of the transformed cells.

Fc region: The constant region of an antibody excluding the first heavy chain constant domain. Fc region generally refers to the last two heavy chain constant domains of IgA, IgD, and IgG, and the last three heavy chain constant domains of IgE and IgM. An Fc region may also include part or all of the flexible hinge N-terminal to these domains. For IgA and IgM, an Fc region may or may not include the tailpiece, and may or may not be bound by the J chain. For IgG, the Fc region is typically understood to include immunoglobulin domains C 2 and Cy3 and optionally the lower part of the hinge between Cyl and Cy2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is usually defined to include residues following C226 or P230 to the Fc carboxyl-terminus, wherein the numbering is according to EU numbering. For IgA, the Fc region includes immunoglobulin domains Ca2 and Ca3 and optionally the lower part of the hinge between Cal and Ca2.

Heterologous: Originating from a different genetic source. A nucleic acid molecule that is heterologous to a cell originated from a genetic source other than the cell in which it is expressed. In one specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein, such as a DVD- immunoglobulin , is expressed in a cell, such as a mammalian cell. Methods for introducing a heterologous nucleic acid molecule in a cell or organism are well known, for example transformation with a nucleic acid, including electroporation, lipofection, particle gun acceleration, and homologous recombination.

Host cell: Cells in which a vector can be propagated, and its DNA expressed. The cell may be prokaryotic or eukaryotic, such as bacterial cells and mammalian cells, such as E. coli or human cells. The term also includes any progeny of the subject host cell. It is understood that all progeny may not be identical to the parental cell since there may be mutations that occur during replication. However, such progeny are included when the term “host cell” is used.

IgA: A polypeptide belonging to the class of antibodies that are substantially encoded by a recognized immunoglobulin alpha gene. In humans, this class or isotype includes IgAi and IgA,. IgA antibodies can exist as monomers, polymers (referred to as plgA) of predominantly dimeric form, and secretory IgA. The constant chain of wild-type IgA contains an 18-amino-acid extension at its C-terminus called the tail piece (tp). Polymeric IgA is secreted by plasma cells with a 15-kDa peptide called the J chain linking two monomers of IgA through the conserved cysteine residue in the tail piece.

IgG: A polypeptide belonging to the class or isotype of antibodies that are substantially encoded by a recognized immunoglobulin gamma gene. In humans, this class includes IgGi, IgGz, IgG ?, and IgG4.

Immune complex: The binding of antibody, antigen binding fragment (such as a scFv), or a bispecific antibody (such as a DVD-immunoglobulin) to a soluble antigen forms an immune complex. The formation of an immune complex can be detected through conventional methods, for instance immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (for example, Western blot), magnetic resonance imaging, CT scans, radiography, and affinity chromatography.

Inhibiting or treating a disease: Inhibiting the full development of a disease or condition, for example, in a subject who is at risk for a disease such as a coronavirus infection. “Treatment” refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition after it has begun to develop. The term “ameliorating,” with reference to a disease or pathological condition, refers to any observable beneficial effect of the treatment. Inhibiting a disease can include preventing or reducing the risk of the disease, such as preventing or reducing the risk of viral infection. The beneficial effect can be evidenced, for example, by a delayed onset of clinical symptoms of the disease in a susceptible subject, a reduction in severity of some or all clinical symptoms of the disease, a slower progression of the disease, a reduction in the viral load, an improvement in the overall health or well-being of the subject, or by other parameters that are specific to the particular disease. A “prophylactic” treatment is a treatment administered to a subject who does not exhibit signs of a disease or exhibits only early signs for the purpose of decreasing the risk of developing pathology.

The term “reduces” is a relative term, such that an agent reduces a disease or condition if the disease or condition is quantitatively diminished following administration of the agent, or if it is diminished following administration of the agent, as compared to a reference agent. Similarly, the term “prevents” does not necessarily mean that an agent completely eliminates the disease or condition, so long as at least one characteristic of the disease or condition is eliminated. Thus, a composition that reduces or prevents an infection, can, but does not necessarily completely, eliminate such an infection, so long as the infection is measurably diminished, for example, by at least about 50%, such as by at least about 70%, or about 80%, or even by about 90% the infection in the absence of the agent, or in comparison to a reference agent.

Isolated: A biological component (such as a nucleic acid, peptide, protein or protein complex, for example an antibody) that has been substantially separated, produced apart from, or purified away from other biological components in the cell of the organism in which the component naturally occurs, that is, other chromosomal and extra-chromosomal DNA and RNA, and proteins. Thus, isolated nucleic acids, peptides and proteins include nucleic acids and proteins purified by standard purification methods. The term also embraces nucleic acids, peptides and proteins prepared by recombinant expression in a host cell, as well as, chemically synthesized nucleic acids. An isolated nucleic acid, peptide or protein, for example an antibody, can be at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% pure.

Kabat position: A position of a residue in an amino acid sequence that follows the numbering convention delineated by Kabat et al. (Sequences of Proteins of Immunological Interest, 5^th Edition, Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIH Publication No. 91-3242, 1991).

Linker: A bi-functional molecule that can be used to link two molecules into one contiguous molecule, for example, to link a detectable marker to an antibody or link two antibodies to form a bispecific antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

The terms “conjugating,” “joining,” “bonding,” or “linking” can refer to making two molecules into one contiguous molecule; for example, linking two polypeptides into one contiguous polypeptide, or covalently attaching an effector molecule or detectable marker radionuclide or other molecule to a polypeptide, such as a DVD-immunoglobulin . The linkage can be either by chemical or recombinant means. “Chemical means” refers to a reaction between the antibody moiety and the effector molecule such that there is a covalent bond formed between the two molecules to form one molecule.

Nucleic acid (molecule or sequence): A deoxyribonucleotide or ribonucleotide polymer or combination thereof including without limitation, cDNA, mRNA, genomic DNA, and synthetic (such as chemically synthesized) DNA or RNA. The nucleic acid can be double stranded (ds) or single stranded (ss). Where single stranded, the nucleic acid can be the sense strand or the antisense strand. Nucleic acids can include natural nucleotides (such as A, T/U, C, and G), and can include analogs of natural nucleotides, such as labeled nucleotides.

“cDNA” refers to a DNA that is complementary or identical to an mRNA, in either single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences of nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, to serve as templates for synthesis of other polymers and macromolecules in biological processes having either a defined sequence of nucleotides (i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of mRNA produced by that gene produces the protein in a cell or other biological system. Both the coding strand, the nucleotide sequence of which is identical to the mRNA sequence and is usually provided in sequence listings, and non-coding strand, used as the template for transcription, of a gene or cDNA can be referred to as encoding the protein or other product of that gene or cDNA. Unless otherwise specified, a “nucleotide sequence encoding an amino acid sequence” includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. Nucleotide sequences that encode proteins and RNA may include introns.

Operably linked: A first nucleic acid sequence is operably linked with a second nucleic acid sequence when the first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence. For instance, a promoter, such as the CMV promoter, is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence. Generally, operably linked DNA sequences are contiguous and, where necessary to join two protein-coding regions, in the same reading frame.

Pharmaceutically acceptable carriers: The pharmaceutically acceptable carriers of use are conventional. Remington: The Science and Practice of Pharmacy, 22^nd ed., London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for pharmaceutical delivery of the disclosed agents.

The nature of the carrier can depend on the particular mode of administration being employed. For instance, parenteral formulations usually include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle. For solid compositions (e.g., powder, pill, tablet, or capsule forms), conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, added preservatives (such as non-natural preservatives), and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate. In particular examples, the pharmaceutically acceptable carrier is sterile and suitable for parenteral administration to a subject for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in a unit dosage form such as a pill or in a selected quantity in a vial. Unit dosage forms can include one dosage or multiple dosages (for example, in a vial from which metered dosages of the agents can selectively be dispensed).

Polypeptide: A polymer in which the monomers are amino acid residues that are joined together through amide bonds. When the amino acids are alpha-amino acids, either the L-optical isomer or the D- optical isomer can be used, the L-isomers being preferred. The terms “polypeptide” or “protein” as used herein are intended to encompass any amino acid sequence and include modified sequences such as glycoproteins. A polypeptide includes both naturally occurring proteins, as well as those that are recombinantly or synthetically produced. A polypeptide has an amino terminal (N-terminal) end and a carboxy-terminal end. In some aspects, the polypeptide is a disclosed antibody or a fragment thereof.

Purified: The term purified does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide preparation is one in which the peptide or protein (such as an antibody) is more enriched than the peptide or protein is in its natural environment within a cell. In one aspect, a preparation is purified such that the protein or peptide represents at least 50% of the total peptide or protein content of the preparation.

Recombinant: A recombinant nucleic acid is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. This artificial combination can be accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, for example, by genetic engineering techniques. A recombinant protein is one that has a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two otherwise separated segments of sequence. In several aspects, a recombinant protein is encoded by a heterologous (for example, recombinant) nucleic acid that has been introduced into a host cell, such as a bacterial or eukaryotic cell. The nucleic acid can be introduced, for example, on an expression vector having signals capable of expressing the protein encoded by the introduced nucleic acid or the nucleic acid can be integrated into the host cell chromosome.

SARS-CoV-2: Also known as Wuhan coronavirus or 2019 novel coronavirus, SARS-CoV-2 is a positive-sense, single stranded RNA virus of the genus betacoronavirus that has emerged as a highly fatal cause of severe acute respiratory infection. The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. The SARS-CoV-2 virion includes a viral envelope with large spike glycoproteins. The SARS-CoV-2 genome, like most coronaviruses, has a common genome organization with the replicase gene included in the 5'-two thirds of the genome, and structural genes included in the 3'-third of the genome. The SARS-CoV-2 genome encodes the canonical set of structural protein genes in the order 5' - spike (S) - envelope (E) - membrane (M) and nucleocapsid (N) - 3'. Symptoms of SARS-CoV-2 infection include fever and respiratory illness, such as dry cough and shortness of breath. Cases of severe infection can progress to severe pneumonia, multi-organ failure, and death. The time from exposure to onset of symptoms is approximately 2 to 14 days.

Standard methods for detecting viral infection may be used to detect SARS-CoV-2 infection, including but not limited to, assessment of patient symptoms and background and genetic tests such as reverse transcription-polymerase chain reaction (rRT-PCR). The test can be done on patient samples such as respiratory or blood samples.

Spike (S) protein (Coronavirus): A class I fusion glycoprotein initially synthesized as a precursor protein of approximately 1256 amino acids in size for SARS-CoV, and 1273 for SARS-CoV-2. Individual precursor S polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease between approximately position 679/680 for SARS-CoV, and 685/686 for SARS-CoV-2, to generate separate SI and S2 polypeptide chains, which remain associated as S1/S2 protomers within the homotrimer and is therefore a trimer of heterodimers. The SI subunit is distal to the virus membrane and contains the receptor-binding domain (RBD) that is believed to mediate virus attachment to its host receptor. The S2 subunit contains the fusion protein machinery, such as the fusion peptide, two heptad-repeat sequences (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and the cytosolic tail domain.

An exemplary sequence of SARS-CoV S protein is provided in GENBANK® GI: 30795145, as available on February 1, 2022. An exemplary sequence of the HKUl-CoV S protein is provided in GENBANK® GI: 123867264, as available on February 1, 2022. An exemplary sequence of OC43-CoV S protein is provided in GENBANK® GI: 744516696, as available on February 1, 2022. An exemplary sequence of NL63-CoV S protein is provided in GENBANK® GI: 71153773, as available on February 1, 2022. An exemplary sequence of 229E-CoV S protein is provided in GENBANK® GI: 1060650120, as available on February 1, 2022. The S protein includes both the SI and S2 domains.

The numbering used in the disclosed SARS-CoV-2 S proteins and fragments thereof is relative to the S protein of SARS-CoV-2, the sequence of which was deposited as NCBI Ref. No. YP 009724390.1, as available on February 1, 2022, which is incorporated by reference herein in its entirety.

Sequence identity: The identity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the percentage identity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences. Homologs and variants of a VL or a VH of an antibody that specifically binds a target antigen are typically characterized by possession of at least about 75% sequence identity, for example at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over the full-length alignment with the amino acid sequence of interest.

Any suitable method may be used to align sequences for comparison. Non-limiting examples of programs and alignment algorithms are described in: Smith and Waterman, Adv. Appl. Math. 2(4):482-489, 1981; Needleman and Wunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85(8):2444-2448, 1988; Higgins and Sharp, Gene, 73(l):237-244, 1988; Higgins and Sharp, Bioinformatics. 5(2): 151-3, 1989; Corpet, Nucleic Acids Res. 16(22): 10881-10890, 1988; Huang et al. Bioinformatics. 8(2): 155-165, 1992; and Pearson, Methods Mol. Biol. 24:307-331, 1994., Altschul et al., J. Mol. Biol. 215(3):403-410, 1990, presents a detailed consideration of sequence alignment methods and homology calculations. The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215(3):403-410, 1990) is available from several sources, including the National Center for Biological Information and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Blastn is used to compare nucleic acid sequences, while blastp is used to compare amino acid sequences. Additional information can be found at the NCBI web site.

Generally, once two sequences are aligned, the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is present in both sequences. The percent sequence identity between the two sequences is determined by dividing the number of matches either by the length of the sequence set forth in the identified sequence, or by an articulated length (such as 100 consecutive nucleotides or amino acid residues from a sequence set forth in an identified sequence), followed by multiplying the resulting value by 100.

Specifically bind: When referring to an antibody, antigen binding fragment, or bispecific antibody, refers to a binding reaction which determines the presence of a target protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated conditions, an antibody binds preferentially to a particular target protein, peptide or polysaccharide (such as an antigen present on the surface of a pathogen, for example a coronavirus spike protein and does not bind in a significant amount to other proteins present in the sample or subject. With regard to a spike protein, the epitope may be present on the spike protein of more than one type of coronavirus, such that the antibody binds to the spike protein on more than one types of virus, but does not bind to other proteins, such as proteins from other viruses or other proteins (non-spike) of coronavirus. Specific binding can be determined by standard methods. See Harlow & Lane, Antibodies, A Laboratory Manual, 2^nd ed., Cold Spring Harbor Publications, New York (2013), for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity .

With reference to an antibody-antigen complex, specific binding of the antigen and antibody has a KD of less than about 10’⁷ Molar, such as less than about 10'⁸ Molar, 10'⁹, or even less than about IO¹⁰ Molar. KD refers to the dissociation constant for a given interaction, such as a polypeptide ligand interaction or an antibody antigen interaction. For example, for the bimolecular interaction of an antibody or antigen binding fragment and an antigen it is the concentration of the individual components of the bimolecular interaction divided by the concentration of the complex.

An antibody that specifically binds to an epitope on a coronavirus spike protein, such as the S domain, RBD domain, or NTD domain, can bind molecules/agents including this domain, including viruses, substrate to which the spike protein is attached, or the protein in a biological specimen. It is, of course, recognized that a certain degree of non-specific interaction may occur between an antibody and a non-target. Typically, specific binding results in a much stronger association between the antibody and a spike protein than between the antibody other different coronavirus proteins (such as the E, M or N protein) or from noncoronavirus proteins. Specific binding typically results in greater than 2-fold, such as greater than 5-fold, greater than 10-fold, or greater than 100-fold increase in amount of bound antibody (per unit time) to a protein including the epitope or cell or tissue expressing the target epitope as compared to a protein or cell or tissue lacking this epitope. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats are appropriate for selecting antibodies or other ligands specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein.

Subject: Living multi-cellular vertebrate organisms, a category that includes human and nonhuman mammals, such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, and the like. In an example, a subject is a human. In a particular example, the subject is a human. In an additional example, a subject is selected that is in need of inhibiting a SARS-CoV-2 infection. For example, the subject is either uninfected and at risk of the SARS-CoV-2 infection or is infected and in need of treatment.

Transformed: A transformed cell is a cell into which a nucleic acid molecule has been introduced by molecular biology techniques. As used herein, the term transformed and the like (e.g., transformation, transfection, transduction, etc.) encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transduction with viral vectors, transformation with plasmid vectors, and introduction of DNA by electroporation, lipofection, and particle gun acceleration.

Vector: An entity containing a nucleic acid molecule (such as a DNA or RNA molecule) bearing a promoter(s) that is operationally linked to the coding sequence of a protein of interest and can express the coding sequence. Non-limiting examples include a naked or packaged (lipid and/or protein) DNA, a naked or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may be replicationincompetent, or a virus or bacterium or other microorganism that may be replication-competent. A vector is sometimes referred to as a construct. Recombinant DNA vectors are vectors having recombinant DNA. A vector can include nucleic acid sequences that permit it to replicate in a host cell, such as an origin of replication. A vector can also include one or more selectable marker genes and other genetic elements.

Viral vectors are recombinant nucleic acid vectors having at least some nucleic acid sequences derived from one or more viruses. In some aspects, a viral vector includes a nucleic acid molecule encoding a disclosed DVD-immunoglobulin or antigen binding fragment that specifically binds to a coronavirus spike protein and neutralizes the coronavirus. In some aspects, the viral vector is an adeno-associated virus (AAV) vector.

Under conditions sufficient for: A phrase that is used to describe any environment that permits a desired activity.

II. Description of Several Aspects

Bispecific antibodies, including DVD-immunoglobulins, are provided herein that include two isolated monoclonal antibodies or antigen binding fragments that specifically bind a coronavirus spike protein. The monoclonal antibodies and antigen binding fragments included in the bispecific antibody specifically bind to a coronavirus spike protein and neutralize SARS-CoV-2 and at least one additional betacoronavirus or alphacoronavirus. The antibodies and antigen binding fragments can be fully human. The disclosed DVD-immunoglobulins can neutralize a coronavirus, such as, but not limited to, SARS-CoV- 2. In some aspects, the disclosed DVD-immunoglobulins can inhibit a coronavirus infection in vivo, and can be administered prior to, or after, an infection with a coronavirus, such as, but not limited to, SARS-CoV-2. In addition, disclosed herein are compositions comprising the DVD-immunoglobulins and a pharmaceutically acceptable carrier. Nucleic acids encoding the DVD-immunoglobulins, and expression vectors (such as adeno-associated virus (AAV) viral vectors) comprising these nucleic acids are also provided. The DVD-immunoglobulins, nucleic acid molecules, host cells, and compositions can be used for research, diagnostic, treatment and prophylactic purposes. For example, the disclosed DVD- immunoglobulins can be used to diagnose a subject with a coronavirus infection or can be administered to inhibit a coronavirus infection in a subject.

Monoclonal Antibodies that Specifically Bind a Coronavirus Spike protein and Antigen Binding Fragments Thereof included in a Bispecific Antibody

The discussion of bispecific antibodies herein refers to molecules that include two isolated monoclonal antibodies or antigen binding fragments thereof, that specifically bind a coronavirus spike protein. These monoclonal antibodies and antigen binding fragments include heavy and/or light chain variable domains (or antigen binding fragments thereof) comprising a CDR1, CDR2, and/or CDR3 with reference to the IMGT numbering scheme (unless the context indicates otherwise). Various CDR numbering schemes (such as the Kabat, Chothia or IMGT numbering schemes) can be used to determine CDR positions in a monoclonal antibody, or antigen binding fragment thereof, that is included in the disclosed bispecific antibodies. The amino acid sequence and the CDRs of the heavy and light chain of the monoclonal antibody according to the IMGT numbering scheme these are exemplary only. In some aspects, the CDRs are determined using Kabat positioning.

In some aspects, the bispccific antibody includes at last one monoclonal antibody (or antigen binding fragment thereof), that includes the heavy and light chain CDRs of COV44-62, COV30-14, COV72- 37, COV89-22, COV44-79, and COV91-27 antibodies described below. In other aspects, the bispecific antibody includes two monoclonal antibodies (or antigen binding fragment thereof) that includes the heavy and light chain CDRs of one of COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27 antibodies described below. The bispecific antibody can be a DVD-immunoglobulin.

In some aspects, the bispecific antibody includes the heavy and light chain variable regions of one or two of the COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27 antibodies described below. In more aspects, the bispecific antibody includes the heavy and light chain CDRs of one or two of the COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27 antibodies described below. In more aspects, the bispecific antibody includes the heavy and the light chain variable regions of two of the COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27 antibodies described below. In more aspects, the bispecific antibody includes the heavy and light chain CDRs of two of the COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27 antibodies described below. The bispecific antibody can be a DVD-immunoglobulin. In some aspects, at least one of the monoclonal antibodies included in the bispecific antibody binds an S domain of the coronavirus spike protein. In other aspects, at least one of the monoclonal antibodies included in the bispecific antibody binds a stem helix in the S2 domain of the spike protein, such as LQPELDSFKEELDKYFKNHTS (SEQ ID NO: 118), such as LDSFKEELDKYF (SEQ ID NO: 119). In further aspects, at least one of the monoclonal antibodies included in the bispecific antibody binds an epitope of the fusion peptide in the S2 domain of the spike protein, such as SFIEDLLFNKVTLA (SEQ ID NO: 120).

In some aspects, at least one of the monoclonal antibodies included in the bispecific antibody binds specifically binds a spike protein from at least three betacoronaviruses selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, and OC43. In other aspects, at least one of the monoclonal antibodies included in the bispecific antibody binds a betacoronavirus and an alphacoronavirus. In more aspects, at least one of the monoclonal antibodies included in the bispecific antibody binds a spike protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63 and 229E.

The following monoclonal antibodies, or antigen biding fragment thereof, can be included in a disclosed bi-specific antibody, such as a DVD-immunoglobulin. a. Monoclonal antibody COV44-62

In some aspects, the antibody or antigen binding fragment is based on or derived from the COV44- 62 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In some examples, the antibody or antigen binding fragment comprises a Anand a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the COV44-62 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen binding fragment neutralizes a betacoronavirus and an alphacoronavirus. In some aspects, the antibody or antigen binding fragment specifically binds a spike protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63 and 229E.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 1, and specifically binds to a coronavirus spike, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In additional aspects, the antibody or antigen binding fragment comprises a VH and a V independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3 and 4, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7 and 8, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 2, 3 and 4, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 6, 7 and 8, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 1, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 1, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 5, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 5, and the antibody or antigen binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In this aspect, variations due to sequence identify fall outside the CDRs. The coronavirus can be SARS-CoV- 2

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 1, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 5, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In some aspects, the antibody or antigen binding fragment comprises a Vn and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 1 and 5, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the disclosed antibodies inhibit viral entry and/or replication. These antibodies and antigen binding fragments can be included in the disclosed bispccific antibody, such as a DVD- immunoglobulin. b. Monoclonal antibody COV30-14

In some aspects, the antibody or antigen binding fragment is based on or derived from the COV30- 14 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.

In some examples, the antibody or antigen binding fragment comprises a Vnand a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the COV30-14 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen binding fragment specifically binds a spike protein from at least three betacoronaviruses selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, and OC43. In some aspects, the antibody or antigen binding fragment neutralizes a betacoronavirus and an alphacoronavirus. In some aspects, the antibody or antigen binding fragment specifically binds a stem-helix in the S2 domain of the spike protein. In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to a coronavirus spike, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11 and 12, respectively, and/or a V comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15 and 16, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 10, 11 and 12, respectively, a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 14, 15 and 16, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 9, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 9, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 13, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 13, and the antibody or antigen binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In this aspect, variations due to sequence identify fall outside the CDRs. The coronavirus can be SARS-CoV- 2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 9, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 13, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 9 and 13, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the disclosed antibodies inhibit viral entry and/or replication. These antibodies and antigen binding fragments can be included in the disclosed bispecific antibody, such as a DVD- immunoglobulin. c. Monoclonal antibody COV72-37

In some aspects, the antibody or antigen binding fragment is based on or derived from the COV72- 37 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.

In some examples, the antibody or antigen binding fragment comprises a Vu nd a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the COV72-37 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen binding fragment specifically binds a spike protein from at least three betacoronaviruses selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, and OC43. In some aspects, the antibody or antigen binding fragment neutralizes a betacoronavirus and an alphacoronavirus. In some aspects, the antibody or antigen binding fragment specifically binds a stem-helix in the S2 domain of the spike protein.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 17, and specifically binds to a coronavirus spike, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 21, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In additional aspects, the antibody or antigen binding fragment comprises a VH and a V independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 18, 19 and 20, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 22, 23 and 24, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 18, 19 and 20, respectively, a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 22, 23 and 24, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 17, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 17, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 21, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 21, and the antibody or antigen binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In this aspect, variations due to sequence identify fall outside the CDRs. The coronavirus can be SARS-CoV- T In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 17, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 21, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 17 and 21, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the disclosed antibodies inhibit viral entry and/or replication. These antibodies and antigen binding fragments can be included in the disclosed bispecific antibody, such as a DVD- immunoglobulin. d. Monoclonal antibody COV89-22

In some aspects, the antibody or antigen binding fragment is based on or derived from the COV89- 22 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.

In some examples, the antibody or antigen binding fragment comprises a Vnand a V comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the COV89-22 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen binding fragment specifically binds a spike protein from at least three betacoronaviruses selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, and OC43. In some aspects, the antibody or antigen binding fragment neutralizes a betacoronavirus and an alphacoronavirus. In some aspects, the antibody or antigen binding fragment specifically binds a stem-helix in the S2 domain of the spike protein.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 25, and specifically binds to a coronavirus spike, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27 and 28, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31 and 32, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 26, 27 and 28, respectively, a VLComprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 30, 31 and 32 respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 25, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 25, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 29, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 29, and the antibody or antigen binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In this aspect, variations due to sequence identify fall outside the CDRs. The coronavirus can be SARS-CoV- 2

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 25, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 29, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 25 and 29, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the disclosed antibodies inhibit viral entry and/or replication. These antibodies and antigen binding fragments can be included in the disclosed bispecific antibody, such as a DVD- immunoglobulin. e. Monoclonal antibody COV44-79

In some aspects, the antibody or antigen binding fragment is based on or derived from the COV44- 79 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.

In some examples, the antibody or antigen binding fragment comprises a Vnand a VL comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the COV44-79 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen binding fragment neutralizes a betacoronavirus and an alphacoronavirus. In some aspects, the antibody or antigen binding fragment specifically binds a spike protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63 and 229E.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a coronavirus spike, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35 and 36, respectively, and/or a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 38, 39 and 40, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 34, 35 and 36, respectively, a VL comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 38, 39 and 40, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 33, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 33, and wherein the VL comprises an amino acid sequence at least 90% identical to SEQ ID NO: 37, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 37, and the antibody or antigen binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In this aspect, variations due to sequence identify fall outside the CDRs. The coronavirus can be SARS-CoV- 2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 33, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 37, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 33 and 37, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the disclosed antibodies inhibit viral entry and/or replication. These antibodies and antigen binding fragments can be included in the disclosed bispecific antibody, such as a DVD- immunoglobulin. f. Monoclonal antibody COV91-27

In some aspects, the antibody or antigen binding fragment is based on or derived from the COV91- 27 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus.

In some examples, the antibody or antigen binding fragment comprises a Vuand a Vi. comprising the HCDR1, the HCDR2, the HCDR3, the LCDR1, the LCDR2, and the LCDR3, respectively (for example, according to IMGT, Kabat or Chothia), of the COV91-27 antibody, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen binding fragment specifically binds a spike protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63 and 229E. In some aspects, the antibody or antigen binding fragment neutralizes a betacoronavirus and an alphacoronavirus.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 41, and specifically binds to a coronavirus spike, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising an amino acid sequence at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequence set forth as SEQ ID NO: 45, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In additional aspects, the antibody or antigen binding fragment comprises a VH and a VL independently comprising amino acid sequences at least 90% (such as at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) identical to the amino acid sequences set forth as SEQ ID NOs: 41 and 45, respectively, and specifically binds to a coronavirus spike protein and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 42, 43, and 44, respectively, and/or a V Comprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 46, 47 and 48, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising a HCDR1, a HCDR2, and a HCDR3 as set forth as SEQ ID NOs: 42, 43 and 44, respectively, a V_Lcomprising a LCDR1, a LCDR2, and a LCDR3 as set forth as SEQ ID NOs: 46, 47 and 48, respectively, wherein the VH comprises an amino acid sequence at least 90% identical to SEQ ID NO: 41, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 41, and wherein the V comprises an amino acid sequence at least 90% identical to SEQ ID NO: 45, such as 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 45, and the antibody or antigen binding fragment specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In this aspect, variations due to sequence identify fall outside the CDRs. The coronavirus can be SARS-CoV- 2.

In some aspects, the antibody or antigen binding fragment comprises a VH comprising the amino acid sequence set forth as SEQ ID NO: 41, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In more aspects, the antibody or antigen binding fragment comprises a VL comprising the amino acid sequence set forth as SEQ ID NO: 45, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. In some aspects, the antibody or antigen binding fragment comprises a VH and a VL comprising the amino acid sequences set forth as SEQ ID NOs: 41 and 45, respectively, and specifically binds to a coronavirus spike protein, and neutralizes a coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the disclosed antibodies inhibit viral entry and/or replication. These antibodies and antigen binding fragments can be included in the disclosed bispecific antibody, such as a DVD- immunoglobulin.

Additional Description of Antibodies, Antigen Binding Fragments, DVD-Immunoglobulins

Any suitable method can be used to design and produce a bispecific antibody, such as crosslinking two or more antibodies, antigen binding fragments (such as scFvs) of the same type or of different types. Exemplary methods of making multispecific antibodies, such as bispecific antibodies, include those described in PCT Pub. No. WO2013/163427, which is incorporated by reference herein in its entirety. Nonlimiting examples of suitable crosslinkers include those that are heterobifunctional, having two distinctly reactive groups separated by an appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional (such as disuccinimidyl suberate).

A multi-specific antibody, such as a bispecific antibody, may have any suitable format that allows for binding to the coronavirus spike protein by the antibody or antigen binding fragment as provided herein. Bispecific single chain antibodies can be encoded by a single nucleic acid molecule. Non-limiting examples of bispecific single chain antibodies, as well as methods of constructing such antibodies are provided in U.S. Pat. Nos. 8,076,459, 8,017,748, 8,007,796, 7,919,089, 7,820,166, 7,635,472, 7,575,923, 7,435,549, 7,332,168, 7,323,440, 7,235,641, 7,229,760, 7,112,324, 6,723,538. Additional examples of bispecific single chain antibodies can be found in PCT application No. WO 99/54440; Mack et al., J. Immunol., 158(8):3965- 3970, 1997; Mack ct a/., Proc. Natl. Acad. Sci. U.S.A., 92(15):7021-7025, 1995; Kufer et a/., Cancer Immunol. Immunother., 45(3-4): 193-197, 1997; Loffler et al., Blood, 95(6):2098-2103, 2000; and Briihl et al., J. Immunol., 166(4):2420-2426, 2001. Production of bispecific Fab-scFv (“bibody”) molecules are described, for example, in Schoonjans et al. (J. Immunol., 165(12):7050-7057, 2000) and Willems et al. (J. Chromatogr. B Analyt. Technol. Biomed Life Sci. 786(1-2): 161-176, 2003). For bibodies, a scFv molecule can be fused to one of the VL-CL (L) or VH-CH1 chains, e.g., to produce a bibody one scFv is fused to the C-term of a Fab chain.

Antigen binding fragments can be included in the bispecilic antibodies of the present disclosure, such as Fab, F( b')2, and Fv which include a heavy chain and light chain and specifically bind a coronavirus spike protein. These antibody fragments retain the ability to selectively bind with the antigen and are “antigen-binding” fragments, and thus can be included in a bispecific antibody. Non-limiting examples of such fragments include:

(1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain;

(3) (Fab'h, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, a genetically engineered fragment containing the VL and VL expressed as two chains; and

(5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the VH and the VL linked by a suitable polypeptide linker as a genetically fused single chain molecule (see, e.g., Ahmad et al., Clin. Dev. Immunol., 2012, doi: 10.1155/2012/980250; Marbry and Snavely, IDrugs, 13(8) :543-549, 2010). The intramolecular orientation of the Vu-domain and the VL- domain in a scFv, is not decisive for the provided antibodies (e.g., for the provided multispecific antibodies). Thus, scFvs with both possible arrangements (Vu-domain-linker domain-V -domain; V -domain-linker domain-Vn-domain) may be used.

(6) A dimer of a single chain antibody tscFVi). defined as a dimer of a scFV. This has also been termed a “mini antibody.”

Any suitable method of producing the above-discussed antigen binding fragments may be used. Non-limiting examples are provided in Harlow and Lane, Antibodies: A Laboratory Manual, 2^nd, Cold Spring Harbor Laboratory, New York, 2013.

Antigen binding fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in a host cell (such as an E. coli cell) of DNA encoding the fragment. Antigen binding fragments can also be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antigen binding fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent lightheavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

An antibody or antigen binding fragment included in the bispecific antibodies disclosed herein can be a human antibody or fragment thereof; these are of use in the presently disclosed bispecific antibodies. Chimeric antibodies are also provided and are of use in the bispecific antibodies. The antibody or antigen binding fragment can include any suitable framework region, such as (but not limited to) a human framework region from another source, or an optimized framework region. Alternatively, a heterologous framework region, such as, but not limited to a mouse or monkey framework region, can be included in the heavy or light chain of the antibodies. These molecules can also be included the disclosed bispecific antibodies.

The bispecific antibody, such as a DVD-immunogloubulin, can be of any isotype. The antibody bispecific antibody, or DVD-immunogloubulin, can be, for example, an IgA, IgM or an IgG antibody, such as IgGi, IgG₂, IgGs, or IgG4. The class of an antibody, bispecific antibody, or DVD-immunogloubulin, that specifically binds to a coronavirus spike protein can be switched with another. In one aspect, a nucleic acid molecule encoding VL or VH is isolated such that it does not include any nucleic acid sequences encoding the constant region of the light or heavy chain, respectively. A nucleic acid molecule encoding VL or VH is then operatively linked to a nucleic acid sequence encoding a CL or CH from a different class of immunoglobulin molecule. This can be achieved, for example, using a vector or nucleic acid molecule that comprises a CL or CH chain. For example, an antibody that specifically binds the spike protein, that was originally IgG may be class switched to an IgA. Class switching can be used to convert one IgG subclass to another, such as from IgGi to IgG₂, IgG ;. or IgG₄.

A DVD-immunogloubulin can be derivatized or linked to another molecule (such as another peptide or protein). In general, an antibody or antigen binding fragment in the bispecific antibody is derivatized such that the binding to the spike protein is not affected adversely by the derivatization or labeling. For example, the antibody or antigen binding fragment can be functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (for example, another bi-specific antibody or a diabody to create a multispecific antibody), a detectable marker, an effector molecule, or a protein or peptide that can mediate association of the antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag).

In several aspects, each antibody or antigen binding fragment included in the DVD- immunogloubulin specifically binds the coronavirus spike protein with an affinity (e.g., measured by KD) of no more than 1.0 x 10'⁸M, no more than 5.0 x 10'⁸ M, no more than 1.0 x 10'⁹M, no more than 5.0 x 10'⁹M, no more than 1.0 x 10¹⁰M, no more than 5.0 x 10¹⁰M, or no more than 1.0 x 10¹¹ M. KD can be measured, for example, by a radiolabeled antigen binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen. In one assay, solution binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of (¹²⁵I)-labclcd antigen in the presence of a titration scries of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol. 293(4):865-881, 1999). To establish conditions for the assay, MICROTITER® multi-well plates (Thermo Scientific) are coated overnight with 5 pg/ml of a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23° C.). In a non-adsorbent plate (NUNC™ Catalog #269620), 100 LIM or 26 pM [¹²⁵I]-antigen are mixed with serial dilutions of a Fab of interest (e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta et al., Cancer Res. 57(20):4593- 4599, 1997). The Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g., for one hour). The solution is then removed, and the plate is washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. When the plates have dried, 150 pl/well of scintillant (MICROSCINT™-20; PerkinElmer) is added, and the plates are counted on a TOPCOUNT™ gamma counter (PerkinElmer) for ten minutes. Concentrations of each Fab that give less than or equal to 20% of maximal binding are chosen for use in competitive binding assays. In another assay, KD can be measured using surface plasmon resonance assays using a BIACORE®- 2000 or a BIACORE®-3000 (BIAcore, Inc., Piscataway, N.J.) at 25° C with immobilized antigen CM5 chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor chips (CM5, BIACORE®, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N- hydroxysuccinimide (NHS) according to the supplier's instructions. Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/ml (~0.2 pM) before injection at a flow rate of 5 1/minute to achieve approximately 10 response units (RU) of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) at 25° C at a flow rate of approximately 25 1/min. Association rates (k_on) and dissociation rates (k_Off) are calculated using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams. The equilibrium dissociation constant (KD) is calculated as the ratio k_Off/k_On- See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds 10⁶ M^-1 s^-1 by the surface plasmon resonance assay above, then the on-rate can be determined by using a fluorescent quenching technique that measures the increase or decrease in fluorescence emission intensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25° C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in the presence of increasing concentrations of antigen as measured in a spectrometer, such as a stop-flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCO™ spectrophotometer (ThermoSpectronic) with a stirred cuvette. Affinity can also be measured by high throughput SPR using the Carterra LSA. In some aspects, a multi-specific antibody, such as a bi-specific antibody, for example, a DVD-immunoglobulin, is provided that comprises an antibody or antigen binding fragment that specifically binds a coronavirus spike protein with a particular affinity.

The bispccific tctravalcnt immunoglobulin known as the DVD-immunoglobulin or DVD-IG™ is disclosed in Wu et al., MAbs. 2009;1:339-47, doi: 10.4161/mabs.1.4.8755, incorporated herein by reference. See also Nat Biotechnol. 2007 Nov;25(l l): 1290-7. doi: 10.1038/nbtl345. Epub 2007 Oct 14., also incorporated herein by reference. A DVD-immunoglobulin molecule includes two heavy chains and two light chains. Unlike IgG, however, both heavy and light chains of a DVD-immunoglobulin molecule contain an additional variable domain (VD) connected via a linker sequence at the N-termini of the VH and VL of an existing monoclonal antibody (mAb). Thus, when the heavy and the light chains combine, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites, see Jakob et aL, Mabs 5'. 358-363, 2013, incorporated herein by reference, see Fig. 1 of Jakob et al. for schematic and space-filling diagrams. A DVD-immunoglobulin molecule functions to bind two different antigens on each DFab simultaneously.

The outermost or N-terminal variable domain is termed VD1 and the innermost variable domain is termed VD2; the VD2 is proximal to the C-terminal CHI or CL. As disclosed in Jakob et al., supra. DVD- immunoglobulin molecules can be manufactured and purified to homogeneity in large quantities, have pharmacological properties similar to those of a conventional IgGi, and show in vivo efficacy. In some aspects, the disclosed bispecific antibody is in a DVD-immunoglobulin format. Exemplary amino acid sequences of DVD-immunoglobulin are listed in FIGs. 2-9. In this nomenclature, the outer variable domain (VD1) is listed first, the inner domain (VD2) is listed second, and the linker is listed third. The constant domain, such as an IgG or IgM constant domain, is linked to the inner domain using a linker. The following linkers can be utilized:

GS linker.

GGGGSGGGGSGGGG (SEQ ID NO: 121)

There are three additional EL linkers, one for heavy chain, one where the outer is kappa and one where the outer is lambda.

EL linker (when outer V is heavy chain) ASTKGPSVFPLAP (SEQ ID NO: 122)

EL linker (when outer V is kappa)

TVAAPSVFIFPP (SEQ ID NO: 123)

EL linker (when outer V is lambda)

QPKAAPSVTLFPP (SEQ ID NO: 124)

1. Exemplary DVD-lmmunoglobulins

Exemplary amino acid sequences for the VH and VL of bispecific antibodies, specifically DVD- immunoglobulins, are provided as SEQ ID NOs: 49-1 17 and 125-127, and are shown in FIGs. 2-9. Additional exemplary bispecific antibodies are disclosed below:

A. COV44-62_COV30-14_GS and COV44-62_COV30-14_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV44-62 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV30-14. Aspects of the VH and V domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispccific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs: 14, 15, and 16, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker. In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 1, a linker, a second VH domain comprising SEQ ID NO: 9, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 5 a linker, and a second VL domain comprising SEQ ID NO: 13, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 125. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 126. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 125 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 126.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 127. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 49. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 127 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 49.

B. COV30-14_ COV44-62_GS and COV30-14_ COV44-62_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV30-14 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the V of COV44-62. Aspects of the Vn and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 14, 15, and 16, a linker, a second VL domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 9, a linker, a second VH domain comprising SEQ ID NO: 1, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 13, a linker, and a second VL domain comprising SEQ ID NO: 5, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 50. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 51. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 50 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 51. In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 52. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 53. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 52 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 53.

C: COV44-62 COV72-37 GS and COV44-62_COV72-37_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV44-62 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV72-37. Aspects of the VH and V domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 2,3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first Vn domain comprising SEQ ID NO: 1, a linker, a second Vu domain comprising SEQ ID NO: 17, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 5, a linker, and a second VL domain comprising SEQ ID NO: 21, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 54. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 55. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 54 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 55.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 56. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 57. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 56 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 57.

D: COV72-37 COV44-62_GS and COV72-37 COV44-62_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV72-37 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV44-62. Aspects of the VH and Vi. domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody. In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second VL domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 17, a linker, a second VH domain comprising SEQ ID NO: 1, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 21, a linker, and a second VL domain comprising SEQ ID NO: 5, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 58. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 59. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 58 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 59.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 60. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 61. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 60 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 61.

E: COV44-62_COV89-22_GS and COV44-62_COV89-22_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV44-62 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV89-22. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 1, a linker, a second VH domain comprising SEQ ID NO: 25, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 5, a linker, and a second VL domain comprising SEQ ID NO: 29, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 62. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 63. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:62 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 63.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 64. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 65. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 64 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 65.

F: COV89-22_ COV44-62J3S and COV89-22_ COV44-62_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the Vu and the VL of COV89-22 as the VD1, and the VD2 of the DVD-immunoglobulin includes Vu and the VL of COV44-62. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bi specific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first Vn domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second Vn domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 25, a linker, a second VH domain comprising SEQ ID NO: 1, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 29, a linker, and a second VL domain comprising SEQ ID NO: 5, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 66. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 67. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:66 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 67.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 68. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 69. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 68 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 69.

G: COV44-79_COV30-14_GS and COV44-79_COV30-14_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV44-79 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV30-14. Aspects of the VH and V domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, and 40, a linker, a second VL domain comprising SEQ ID NOs: 14, 15, and!6, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 33, a linker, a second VH domain comprising SEQ ID NO: 9, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bi specific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 37, a linker, and a second VL domain comprising SEQ ID NO: 13, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 70. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 71. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:70 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 71.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 72. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 73. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 72 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 73.

H: COV30-14_COV44-79_GS and COV30-14_COV44-79_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV30-14 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV44-79. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 14, 15, and 16, a linker, a second VL domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 9, a linker, a second VH domain comprising SEQ ID NO: 33, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a V domain comprising SEQ ID NO: 13, a linker, and a second VL domain comprising SEQ ID NO: 37, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 74. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 75. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:74 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 75.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 76. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 77. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 76 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 77.

I: COV44-79_COV72-37_GS and COV44-79_COV72-37_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV44-79 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV72-37. Aspects of the VH and VL domains of these antibodies arc disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, 40, a linker, a second VL domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 33, a linker, a second VH domain comprising SEQ ID NO: 17, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 37, a linker, and a second Vi. domain comprising SEQ ID NO: 21, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker. In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 78. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 79. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:78 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 79.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 80. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 81. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 80 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 81.

J: COV72-37_COV44-79_GS and COV72-37_COV44-79_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV72-37 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV44-79. Aspects of the VH and V domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second VL domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 17, a linker, a second VH domain comprising SEQ ID NO: 33, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 21, a linker, and a second VL domain comprising SEQ ID NO: 37, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 82. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 83. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:82 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 83.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 84. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 85. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 84 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 85. K: COV44-79_COV89-22_GS and COV44-79_COV89-22_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV44-79as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV89-22. Aspects of the VH and V domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, and 40, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 33, a linker, a second VH domain comprising SEQ ID 25, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 37, a linker, and a second VL domain comprising SEQ ID NO: 29, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 86. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 87. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:86 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 87.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 88. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 89. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 88 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 89.

L: COV89-22_COV44-79_GS and COV89-22_COV44-79_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV89-22 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV44-79. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a Vi. domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO 25, a linker, a second VH domain comprising SEQ ID NO: 33, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 29 a linker, and a second VL domain comprising SEQ ID NO: 37, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 90. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 91. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NQ:90 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 91.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 92. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 93. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 92 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 93.

M: COV91-27_COV30-14_GS and COV91-27_COV30-14_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the Vn and the VL of COV91-27 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the V of COV30-14. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispccific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second VL domain comprising SEQ ID NOs: 14, 15, and 16, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 41, a linker, a second VH domain comprising SEQ ID NO: 9, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 45, a linker, and a second VL domain comprising SEQ ID NO: 13, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 94. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ I DNO: 95. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:94 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 95.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 96. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 97. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 96 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 97.

N: COV30-14_COV91-27_GS and COV30-14_COV91-27_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV30-14 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV91-27. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 14, 15, and 16, a linker, a second VL domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 9, a linker, a second VH domain comprising SEQ ID NO: 41, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 13, a linker, and a second VL domain comprising SEQ ID NO: 45, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 98. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 99. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:98 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 99.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 100. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 101. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 100 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 101.

O: COV91-27_COV72-37_GS and COV91-27_COV72-37_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the Vi. of COV91-27 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV72-37. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second V domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO 41, a linker, a second VH domain comprising SEQ ID NO: 17, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 45, a linker, and a second VL domain comprising SEQ ID NO: 21, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 102. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 103. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 102 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 103.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 104. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 105. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 104 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 105.

P: COV72-37 COV91-27 GS and COV72-37_COV91-27_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV72-37 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV91-27. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second VL domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 17, a linker, a second VH domain comprising SEQ ID NO: 41, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 21, a linker, and a second VL domain comprising SEQ ID NO: 45, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 106. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 107. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 106 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 107.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 108. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 109. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 108 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 109.

Q: COV91-27_COV89-22_GS and COV91-27_COV89-22_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV91-27 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the V of COV89-22. Aspects of the VH and VL domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bi specific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispccific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 41, a linker, a second VH domain comprising SEQ ID NO: 25, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 45, a linker, and a second VL domain comprising SEQ ID NO: 29, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 110. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 111. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:110 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 111.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 112. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 113. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 112 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 113.

R: COV89-22_COV91-27_GS and COV89-22_COV91-27_EL

In some aspects, the VD1 of the DVD-immunoglobulin includes the VH and the VL of COV89-22 as the VD1, and the VD2 of the DVD-immunoglobulin includes VH and the VL of COV91-27. Aspects of the VH and V domains of these antibodies are disclosed above; any of these aspects can be used in the DVD- immunoglobulin bispecific antibody.

In more aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In other aspects, the heavy chain of the bispecific antibody includes, in N-to C terminal order, a first VH domain comprising SEQ ID NO: 25, a linker, a second VH domain comprising SEQ ID NO: 41, and a heavy chain constant domain, such as an IgG or IgA constant domain. The light chain of the bispecific antibody includes, in N to C terminal order, a VL domain comprising SEQ ID NO: 29, a linker, and a second VL domain comprising SEQ ID NO: 45, and a light chain constant domain, such as an IgG or IgA constant domain. In some aspects, the linker can be a GS linker. In other aspects, the linker can be an EL linker.

In one aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 114. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 115. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO:114 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 115.

In another aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 116. In another aspect, the light chain of the DVD-immunoglobulin includes SEQ ID NO: 117. In a further aspect, the heavy chain of the DVD-immunoglobulin includes SEQ ID NO: 116 and the light chain of the DVD- immunoglobulin includes SEQ ID NO: 117.

2. Variants

In some aspects, amino acid sequence variants of the DVD-immunoglobulins are provided. For example, it may be desirable to improve the binding affinity and/or other biological properties of the DVD- immunoglobulin. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody VH domain and/or Vi. domain, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g., antigen-binding.

In some aspects, variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the CDRs and the framework regions of the DVD-immunoglobulin. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC. These can be included in the DVD-immunoglobulin.

The variants typically retain amino acid residues necessary for correct folding and stabilizing between the VH and the VL regions, and will retain the charge characteristics of the residues in order to preserve the low pl and low toxicity of the molecules. Amino acid substitutions can be made in the VH and the VL regions to increase yield. In some aspects, the bispecific antibody is a DVD-immunoglobulin and includes up to 10 (such as up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, or up to 9) amino acid substitutions in SEQ ID NOs: 49-120. In other aspects, the DVD-immunoglobulin includes a heavy chain domain and/or light chain domain at least 95%, such as 96%, 97%, 98% or 99% identical to one of SEQ ID NOs: 49-120, wherein any changes are in the framework regions (not the CDRs).

In some aspects, substitutions, insertions, or deletions may occur within one or more CDRs so long as such alterations do not substantially reduce the ability of the DVD-immunoglobulin to bind antigen when included in the bispecific antibody. For example, conservative alterations (e.g., conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in CDRs. In some aspects of the variant VH and V sequences provided above, each CDR either is unaltered, or contains no more than one, two or three amino acid substitutions. In some aspects of the variant VH and VL sequences provided above, only the framework residues arc modified so the CDRs arc unchanged.

To increase binding affinity of the DVD-immunoglobulin, the VL and VH segments can be randomly mutated, such as within HCDR3 region or the LCDR3 region, in a process analogous to the in vivo somatic mutation process responsible for affinity maturation of antibodies during a natural immune response. Thus in vitro affinity maturation can be accomplished by amplifying VH and VL regions using PCR primers complementary to the HCDR3 or LCDR3, respectively. In this process, the primers have been “spiked” with a random mixture of the four nucleotide bases at certain positions such that the resultant PCR products encode VH and VL segments into which random mutations have been introduced into the VH and/or VL CDR3 regions. These randomly mutated VH and VL segments can be tested to determine the binding affinity for the spike protein.

In some aspects, a DVD-immunoglobulin is altered to increase or decrease the extent to which the antibody or antigen binding fragment is glycosylated. Addition or deletion of glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where a bispecific antibody, such as a DVD-immunogloblin, includes an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. See, e.g., Wright et al. Trends Biotechnol. 15(l):26-32, 1997. The oligosaccharide may include various carbohydrates, e.g., mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the “stem” of the biantennary oligosaccharide structure. In some aspects, modifications of the oligosaccharide in a DVD-immunoglobulin may be made in order to create variants with certain improved properties.

In one aspect, variants of the disclosed DVD-immunoglobulins are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (e.g., complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region; however, Asn297 may also be located about ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., US Patent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to “defucosylated” or “fucose-deficient” antibody variants include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; W02005/053742; WO 2002/031140; Okazaki et al., J. Mol. Biol., 336(5): 1239-1249, 2004; Yamane-Ohnuki et al., Biotechnol. Bioeng. 87(5):614-622, 2004. Examples of cell lines capable of producing defucosylated antibodies include Lee 13 CHO cells deficient in protein fucosylation (Ripka et al., Arch. Biochem. Biophys. 249(2):533-545, 1986; US Pat. Appl. No. US 2003/0157108 and WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki etal., Biotechnol. Bioeng., 87(5): 614-622, 2004; Kanda et al. , Biotechnol. Bioeng., 94(4):680-688, 2006; and W02003/085107).

DVD-immunoglobulin variants are further provided with bisected oligosaccharides, e.g., in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet et al.y, U.S. Pat. No. 6,602,684 (Umana et al.),- and US 2005/0123546 (Umana et al.). Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, e.g., in WO 1997/30087; WO 1998/58964; and WO 1999/22764. In several aspects, the constant region of the DVD-immunoglobulin includes one or more amino acid substitutions to optimize in vivo half-life of the antibody. The serum half-life of IgG Abs is regulated by the neonatal Fc receptor (FcRn). Thus, in several aspects, the bispecific antibody comprises an amino acid substitution that increases binding to the FcRn. Non-limiting examples of such substitutions include substitutions at IgG constant regions T250Q and M428L (see, e.g., Hinton et al., J Immunol., 176( 1) :346- 356, 2006); M428L and N434S (the “LS” mutation, see, e.g., Zalevsky, et al., Nature Biotechnol.. 28(2): 157-159, 2010); N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759- 1769, 2006); T307A, E380A, and N434A (see, e.g., Petkova et al., Int. Immunol., 18(12): 1759-1769, 2006); and M252Y, S254T, and T256E (see, e.g., Dall’Acqua et al., J. Biol. Chem., 281 (33) :23514-23524, 2006). The disclosed DVD- immunoglobulin can be linked to or include an Fc polypeptide including any of the substitutions listed above, for example, the Fc polypeptide can include the M428L and N434S substitutions.

In some aspects, a DVD-immunoglobulin provided herein may be further modified to contain additional nonproteinaceous moieties. The moieties suitable for derivatization of the bispecific antibody include but are not limited to water soluble polymers. Non-limiting examples of water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-l,3-dioxolane, poly- 1,3,6- trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in an application under defined conditions, etc.

Conjugates

The DVD-immunoglobulin that specifically bind to a coronavirus spike protein, as disclosed herein, can be conjugated to an agent, such as an effector molecule or detectable marker. Both covalent and noncovalent attachment means may be used. Various effector molecules and detectable markers can be used, including (but not limited to) toxins and radioactive agents such as¹²⁵1,³²P,¹⁴C,³H and³⁵S and other labels, target moieties, enzymes and ligands, etc. The choice of a particular effector molecule or detectable marker depends on the particular target molecule or cell, and the desired biological effect.

The procedure for attaching a detectable marker to an antibody varies according to the chemical structure of the effector. Polypeptides typically contain a variety of functional groups, such as carboxyl (- COOH), free amine (-NH2) or sulfhydryl (-SH) groups, which are available for reaction with a suitable functional group on a polypeptide to result in the binding of the effector molecule or detectable marker. Alternatively, the DVD-immunoglobulins is derivatized to expose or attach additional reactive functional groups. The derivatization may involve attachment of any suitable linker molecule. The linker is capable of forming covalent bonds to both the antibody or antigen binding fragment and to the effector molecule or detectable marker. Suitable linkers include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers. Where theDVD-immunoglobulins and the effector molecule or detectable marker are polypeptides, the linkers may be joined to the constituent amino acids through their side chains (such as through a disulfide linkage to cysteine) or the alpha carbon, or through the amino, and/or carboxyl groups of the terminal amino acids.

In view of the large number of methods that have been reported for attaching a variety of radiodiagnostic compounds, radiotherapeutic compounds, labels (such as enzymes or fluorescent molecules), toxins, and other agents to antibodies, a suitable method for attaching a given agent to a DVD- immunoglobulins can be determined.

The DVD-immunoglobulins can be conjugated with a detectable marker; for example, a detectable marker capable of detection by ELISA, spectrophotometry, flow cytometry, microscopy or diagnostic imaging techniques (such as CT, computed axial tomography (CAT), MRI, magnetic resonance tomography (MTR), ultrasound, fiberoptic examination, and laparoscopic examination). Specific, non-limiting examples of detectable markers include fluorophores, chemiluminescent agents, enzymatic linkages, radioactive isotopes and heavy metals or compounds (for example super paramagnetic iron oxide nanocrystals for detection by MRI). For example, useful detectable markers include fluorescent compounds, including fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylamine-l-napthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors and the like. Bioluminescent markers are also of use, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). An antibody, antigen binding fragment, such as a DVD-immunoglobulin, can also be conjugated with enzymes that are useful for detection, such as horseradish peroxidase, 0- galactosidase, luciferase, alkaline phosphatase, glucose oxidase and the like. When a DVD-immunoglobulin is conjugated with a detectable enzyme, it can be detected by adding additional reagents that the enzyme uses to produce a reaction product that can be discerned. For example, when the agent horseradish peroxidase is present, the addition of hydrogen peroxide and diaminobenzidine leads to a colored reaction product, which is visually detectable. A DVD- immunoglobulin may also be conjugated with biotin, and detected through indirect measurement of avidin or streptavidin binding. It should be noted that the avidin itself can be conjugated with an enzyme or a fluorescent label.

The DVD-immunoglobulin can be conjugated with a paramagnetic agent, such as gadolinium. Paramagnetic agents such as superparamagnetic iron oxide are also of use as labels. DVD-immunoglobulins can also be conjugated with lanthanides (such as europium and dysprosium), and manganese. ADVD- immunoglobulins may also be labeled with a predetermined polypeptide epitope recognized by a secondary reporter (such as leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags).

The DVD-immunoglobulins can also be conjugated with a radiolabeled amino acid, for example, for diagnostic purposes. For instance, the radiolabel may be used to detect a coronavirus by radiography, emission spectra, or other diagnostic techniques. Examples of labels for polypeptides include, but are not limited to, the following radioisotopes:³H,¹⁴C,³’S,⁹⁰Y,^99mTc,^1HIn,¹²⁵I,¹³¹I. The radiolabels may be detected, for example, using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted illumination. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

The average number of detectable marker moieties per DVD-immunoglobulin in a conjugate can range, for example, from 1 to 20 moieties per antibody or antigen binding fragment, or per bispecific antibody. In some aspects, the average number of effector molecules or detectable marker moieties per antibody, antigen binding fragment, such as a DVD-immunoglobulin n the conjugate range from about 1 to about 2, from about 1 to about 3, about 1 to about 8; from about 2 to about 6; from about 3 to about 5; or from about 3 to about 4. The loading (for example, effector molecule per antibody ratio) of a conjugate may be controlled in different ways, for example, by: (i) limiting the molar excess of effector molecule-linker intermediate or linker reagent relative to antibody, (ii) limiting the conjugation reaction time or temperature, (iii) partial or limiting reducing conditions for cysteine thiol modification, (iv) engineering by recombinant techniques the amino acid sequence of the antibody such that the number and position of cysteine residues is modified for control of the number or position of linker-effector molecule attachments.

Polynucleotides and Expression

Nucleic acid molecules (for example, cDNA or RNA molecules) encoding the amino acid sequences of DVD-immunoglobulins and conjugates that specifically bind to a coronavirus spike protein, as disclosed herein, are provided. Nucleic acids encoding these molecules can readily be produced using the amino acid sequences provided herein (such as the CDR sequences, VH and VL sequences, and DVD-immunoglobulin sequences shown in FIGs. 2-9), sequences available in the art (such as framework or constant region sequences), and the genetic code. In several aspects, nucleic acid molecules can encode the VH, the VL, or both the VH and V (for example in a bicistronic expression vector) of the two antibodies of the bispecific antibody. In several aspects, the nucleic acid molecules can be expressed in a host cell (such as a mammalian cell) to produce a disclosed antibody or antigen binding fragment.

The genetic code can be used to construct a variety of functionally equivalent nucleic acid sequences, such as nucleic acids which differ in their sequence, but which encode the same antibody sequence, or encode a conjugate or fusion protein including the DVD-immunoglobulins.

Nucleic acid molecules encoding the DVD-immunoglobulins, and conjugates that specifically bind to a coronavirus spike protein, can be prepared by any suitable method including, for example, cloning of appropriate sequences or by direct chemical synthesis by standard methods. Chemical synthesis produces a single stranded oligonucleotide. This can be converted into double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.

Exemplary nucleic acids can be prepared by cloning techniques. Examples of appropriate cloning and sequencing techniques can be found, for example, in Green and Sambrook (Molecular Cloning: A Laboratory Manual, 4^th ed., New York: Cold Spring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) (Current Protocols in Molecular Biology, New York: John Wiley and Sons, including supplements).

Nucleic acids can also be prepared by amplification methods. Amplification methods include the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), and the self-sustained sequence replication system (3SR).

The nucleic acid molecules can be expressed in a recombinantly engineered cell such as bacteria, plant, yeast, insect and mammalian cells. The antibodies, antigen binding fragments, and conjugates can be expressed as individual proteins including the VH and/or VL (linked to an effector molecule or detectable marker as needed), or can be expressed as a fusion protein. Any suitable method of expressing and purifying antibodies and antigen binding fragments may be used; non-limiting examples are provided in Al-Rubeai (Ed.), Antibody Expression and Production, Dordrecht; New York: Springer, 2011). The nucleic acid sequences can optionally encode a leader sequence.

To create a scFv the VH- and Vi.-encoding DNA fragments can be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly4-Ser)j, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the V and VH domains joined by the flexible linker (see, e.g., Bird et al., Science, 242(4877) :423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85(16):5879-5883, 1988; McCafferty et al., Nature, 348:552-554, 1990; Kontermann and Diibcl (Eds.), Antibody Engineering, Vols. 1-2, 2^nd cd., Spring cr-Vcrlag, 2010; Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, a cleavage site can be included in a linker, such as a furin cleavage site.

The single chain antibody may be monovalent, if only a single VH and VL are used, bivalent, if two VH and VL are used, or polyvalent, if more than two VH and VL are used. Bispecific or polyvalent antibodies may be generated that bind specifically to a coronavirus spike protein and another antigen. The encoded VH and VL optionally can include a furin cleavage site between the VH and VL domains. Linkers can also be encoded, such as when the nucleic acid molecule encodes a bi-specific antibody in DVD-immunoglobulin format.

One or more DNA sequences encoding the DVD-immunoglobulins, or conjugates, can be expressed in vitro by DNA transfer into a suitable host cell. The cell may be prokaryotic or eukaryotic. Numerous expression systems available for expression of proteins including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO, HeLa and myeloma cell lines, can be used to express the disclosed antibodies and antigen binding fragments. Methods of stable transfer, meaning that the foreign DNA is continuously maintained in the host may be used. Hybridomas expressing the antibodies of interest are also encompassed by this disclosure.

The expression of nucleic acids encoding the bispecific antibodies (such as DVD-immunoglobulin antibodies) described herein can be achieved by operably linking the DNA or cDNA to a promoter (which is either constitutive or inducible), followed by incorporation into an expression cassette. The promoter can be any promoter of interest, including a cytomegalovirus promoter. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassettes can be suitable for replication and integration in either prokaryotes or eukaryotes. Typical expression cassettes contain specific sequences useful for regulation of the expression of the DNA encoding the protein. For example, the expression cassettes can include appropriate promoters, enhancers, transcription and translation terminators, initiation sequences, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, sequences for the maintenance of the correct reading frame of that gene to permit proper translation of mRNA, and stop codons. The vector can encode a selectable marker, such as a marker encoding drug resistance (for example, ampicillin or tetracycline resistance).

To obtain high level expression of a cloned gene, it is desirable to construct expression cassettes which contain, for example, a strong promoter to direct transcription, a ribosome binding site for translational initiation (e.g., internal ribosomal binding sequences), and a transcription/translation terminator. For E. coli, this can include a promoter such as the T7, trp, lac, or lamda promoters, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences can include a promoter and/or an enhancer derived from, for example, an immunoglobulin gene, HTLV, SV40 or cytomegalovirus, and a polyadenylation sequence, and can further include splice donor and/or acceptor sequences (for example, CMV and/or HTLV splice acceptor and donor sequences). The cassettes can be transferred into the chosen host cell by any suitable method such as transformation or electroporation for E. coli and calcium phosphate treatment, electroporation or lipofcction for mammalian cells. Cells transformed by the cassettes can be selected by resistance to antibiotics conferred by genes contained in the cassettes, such as the amp, gpt, neo and hyg genes.

Modifications can be made to a nucleic acid encoding a polypeptide described herein without diminishing its biological activity. Some modifications can be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications include, for example, termination codons, sequences to create conveniently located restriction sites, and sequences to add a methionine at the amino terminus to provide an initiation site, or additional amino acids (such as poly His) to aid in purification steps.

Once expressed, the DVD-immunoglobulins and conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, and the like (see, generally, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009). The DVD-immunoglobulins and conjugates need not be 100% pure. Once purified, partially or to homogeneity as desired, if to be used prophylatically, the polypeptides should be substantially free of endotoxin. Methods for expression of DVD-immunoglobulins and conjugates from mammalian cells, and bacteria such as E. coli have been described and are applicable to the antibodies disclosed herein. See, e.g., Greenfield (Ed.), Antibodies: A Laboratory Manual, 2^nd ed. New York: Cold Spring Harbor Laboratory Press, 2014, Simpson et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, 2009, and Ward et al., Nature 341(6242):544- 546, 1989.

Methods and Compositions

1. Inhibiting a coronavirus infection

Methods are disclosed herein for the inhibition of a coronavirus infection in a subject. These methods include the administration on of an effective amount (that is, an amount effective to inhibit the infection in the subject) of a disclosed DVD-immunoglobulin, or a nucleic acid encoding such a DVD- immunoglobulins, to the subject. Combinations of these DVD-immunoglobulins are also of use.

The disclosed methods can include administering to the subject an effective amount (that is, an amount effective to inhibit the infection in the subject) of a disclosed DVD-immunoglobulins, or a nucleic acid encoding such a DVD-immunoglobulins, to a subject at risk of a coronavirus infection or having the coronavirus infection. The methods can be used pre -exposure or post-exposure.

The infection does not need to be completely eliminated or inhibited for the method to be effective. For example, the method can decrease the infection by a desired amount, for example by at least 10%, at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination or prevention of detectable coronavirus infection) as compared to the coronavirus infection in the absence of the treatment. In some aspects, the subject can also be treated with an effective amount of an additional agent, such as an anti-viral agent.

In some aspects, administration of an effective amount of a DVD-immunoglobulin, or nucleic acid molecule encoding the DVD-immunoglobulin, inhibits the establishment of an infection and/or subsequent disease progression in a subject, which can encompass any statistically significant reduction in activity (for example, growth or invasion) or symptoms of the coronavirus infection in the subject.

Methods are disclosed herein for the inhibition of a coronavirus replication in a subject. The methods include administering to the subject an effective amount (that is, an amount effective to inhibit replication in the subject) of a disclosed DVD-immunoglobulin, or a nucleic acid encoding such a DVD- immunoglobulin, to a subject at risk of a coronavirus infection or having a coronavirus infection. The methods can be used pre-exposure or post-exposure.

Methods are disclosed for treating a coronavirus infection in a subject. Methods are also disclosed for preventing a coronavirus infection in a subject. These methods include administering one or more of the disclosed DVD-immunoglobulins, or nucleic acid molecule encoding the one or more DVD- immunoglobulins, or a composition including such molecules, to the subject. DVD-immunoglobulins can be administered by intravenous infusion. Doses can vary, but generally range between about 0.5 mg/kg to about 50 mg/kg, such as a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg. In some aspects, the dose of the DVD-immunoglobulin can be from about 0.5 mg/kg to about 5 mg/kg, such as a dose of about 1 mg/kg, about 2 mg/kg, about 3 mg/kg, about 4 mg/kg or about 5 mg/kg. The DVD-immunoglobulin is administered according to a dosing schedule determined by a medical practitioner. In some examples, the DVD-immunoglobulin is administered weekly, every two weeks, every three weeks or every four weeks.

In some aspects, the method of inhibiting the infection in a subject further comprises administration of one or more additional agents to the subject. Additional agents of interest include, but are not limited to, anti-viral agents such as hydroxychloroquine, arbidol, remdesivir, favipiravir, baricitinib, lopinavir/ritonavir, Zinc ions, and interferon beta- lb, or their combinations.

In some aspects, the method comprises administration of a first DVD-immunoglobulin as disclosed herein and a second DVD-immunoglobulin. In some aspects, 2, 3, 4, 5, 6, 7, 8, 9 or 10 of the disclosed DVD-immunoglobulins can be administered to the subject. Nucleic acid molecules are also of use in these aspects. Combinations of these nucleic acid molecules encoding a disclosed DVD-immunoglobulins, such as one, two, three, four, or five of these nucleic acid molecules, can be administered to the subject.

In some aspects, a subject is administered DNA or RNA encoding a disclosed DVD- immunoglobulin to provide in vivo antibody production, for example using the cellular machinery of the subject. Any suitable method of nucleic acid administration may be used; non-limiting examples are provided in U.S. Patent No. 5,643,578, U.S. Patent No. 5,593,972 and U.S. Patent No. 5,817,637. U.S. Patent No. 5,880,103 describes several methods of delivery of nucleic acids encoding proteins to an organism. One approach to administration of nucleic acids is direct administration with plasmid DNA, such as with a mammalian expression plasmid. The nucleotide sequence encoding the disclosed DVD- immunoglobulin can be placed under the control of a promoter to increase expression. The methods include liposomal delivery of the nucleic acids. Such methods can be applied to the production of a bispecific antibody. In some aspects, a disclosed DVD-immunoglobulin is expressed in a subject using the pVRC8400 vector (described in Barouch et al., J. Virol., 79(14), 8828-8834, 2005, which is incorporated by reference herein).

In several aspects, a subject (such as a human subject at risk of a coronavirus infection or having a coronavirus infection) can be administered an effective amount of an AAV viral vector that comprises one or more nucleic acid molecules encoding a disclosed DVD-immunoglobulin. The AAV viral vector is designed for expression of the nucleic acid molecules encoding a disclosed bispecific antibody, and administration of the effective amount of the AAV viral vector to the subject leads to expression of an effective amount of a DVD-immunoglobulin in the subject. Non-limiting examples of AAV viral vectors that can be used to express a disclosed DVD-immunoglobulin in a subject include those provided in Johnson et al., Nat. Med., 15(8):901-906, 2009 and Gardner et al., Nature, 519(7541):87-91, 2015, each of which is incorporated by reference herein in its entirety. In one aspect, a nucleic acid encoding a disclosed DVD-immunoglobulin is introduced directly into tissue. For example, the nucleic acid can be loaded onto gold microspheres by standard methods and introduced into the skin by a device such as Bio-Rad’s HELIOS™ Gene Gun. The nucleic acids can be “naked,” consisting of plasmids under control of a strong promoter.

Typically, the DNA is injected into muscle, although it can also be injected directly into other sites. Dosages for injection are usually around 0.5 ,ug/kg to about 50 mg/kg, and typically are about 0.005 mg/kg to about 5 mg/kg (see, e.g., U.S. Patent No. 5,589,466).

Single or multiple administrations of a composition including a disclosed DVD-immunoglobulin, or nucleic acid molecule encoding such a DVD-immunoglobulin, can he administered depending on the dosage and frequency as required and tolerated by the patient. The dosage can be administered once but may be applied periodically until either a desired result is achieved or until side effects warrant discontinuation of therapy. Generally, the dose is sufficient to inhibit a coronavirus infection without producing unacceptable toxicity to the patient.

Data obtained from cell culture assays and animal studies can be used to formulate a range of dosage for use in humans. The dosage normally lies within a range of circulating concentrations that include the ED50, with little or minimal toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. The effective dose can be determined from cell culture assays and animal studies.

The coronavirus spike protein- specific DVD-immunoglobulin or nucleic acid molecule encoding such molecule, or a composition including such molecule, can be administered to subjects in various ways, including local and systemic administration, such as, e.g., by injection subcutaneously, intravenously, intraarterially, intraperitoneally, intramuscularly, intradermally, or intrathecally. In an aspect, the DVD- immunoglobulin or nucleic acid molecule encoding the DVD-immunoglobulin, or a composition including such molecules, is administered by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal or intrathecal injection once a day. The DVD-immunoglobulin or nucleic acid molecule encoding the DVD-immunoglobulin, or a composition including such molecules, can also be administered by direct injection at or near the site of disease. A further method of administration is by osmotic pump (e.g., an Alzet pump) or mini-pump (e.g., an Alzet mini-osmotic pump), which allows for controlled, continuous and/or slow-release delivery of the DVD-immunoglobulin or nucleic acid molecule encoding the DVD-immunoglobulin, or a composition including such molecules, over a pre-determined period. The osmotic pump or mini-pump can be implanted subcutaneously, or near a target site.

2. Compositions

Compositions are provided that include one or more of the DVD-immunoglobulins, or nucleic acid molecules encoding such DVD-immunoglobulins, that are disclosed herein in a pharmaceutically acceptable carrier. In some aspects, the composition comprises two, three, four or more DVD-immunoglobulins. The compositions are useful, for example, for example, for the inhibition or detection of a coronavirus infection, such as, but not limited to, a SARS-CoV-2 infection. In some aspects, the compositions are of use for inhibiting an alphacoronavirus or a betacoronavirus infection. In other aspects, the compositions are of use for inhibiting a SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, or a OC43 infection. In further aspects, the compositions are of use for inhibiting a SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63 or a 229E infection.

The compositions can be prepared in unit dosage forms, such as in a kit, for administration to a subject. The amount and timing of administration are at the discretion of the administering physician to achieve the desired purposes. The DVD-immunoglobulin or nucleic acid molecule encoding the DVD- immunoglobulin can be formulated for systemic or local administration. In one example, the DVD- immunoglobulin or nucleic acid molecule encoding the DVD-immunoglobulin is formulated for parenteral administration, such as intravenous administration.

In some aspects, the DVD-immunoglobulinor nucleic acid molecule included in the composition is at least 70% (such as at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99%) pure. In some aspects, the composition contains less than 10% (such as less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even less) of macromolecular contaminants, such as other mammalian (e.g., human) proteins.

The compositions for administration can include a solution of the DVD-immunoglobulin, conjugate, or nucleic acid molecule encoding the DVD-immunoglobulin, dissolved in a pharmaceutically acceptable carrier, such as an aqueous carrier. A variety of aqueous carriers can be used, for example, buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by any suitable technique. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of DVD-immunoglobulin or nucleic acid molecules in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the subject’s needs.

A typical composition for intravenous administration comprises about 0.01 to about 30 mg/kg of DVD-immunoglobulin per subject per day (or the corresponding dose of a conjugate including the antibody or antigen binding fragment). Any suitable method may be used for preparing administrable compositions; non-limiting examples are provided in such publications as Remington: The Science and Practice of Pharmacy, 22^nd ed., London, UK: Pharmaceutical Press, 2013. In some aspects, the composition can be a liquid formulation including one or more DVD- immunoglobulins in a concentration range from about 0.1 mg/ml to about 20 mg/ml, or from about 0.5 mg/ml to about 20 mg/ml, or from about 1 mg/ml to about 20 mg/ml, or from about 0.1 mg/ml to about 10 mg/ml, or from about 0.5 mg/ml to about 10 mg/ml, or from about 1 mg/ml to about 10 mg/ml. A DVD-immunoglobulin, or a nucleic acid encoding a DVD-immunoglobulin, can be provided in lyophilized form, and rehydrated with sterile water before administration, although they are also provided in sterile solutions of known concentration. A solution including the DVD-immunoglobulin, or a nucleic acid encoding the DVD-immunoglobulin, can then be added to an infusion bag containing 0.9% sodium chloride, USP, and typically administered at a dosage of from 0.5 to 15 mg/kg of body weight. Considerable experience is available in the art for the administration of antibody drugs, which have been marketed in the U.S. since the approval of Rituximab in 1997. DVD-immunoglobulins, or a nucleic acid encoding a DVD- immunoglobulin, can be administered by slow infusion, rather than in an intravenous push or bolus. In one example, a higher loading dose is administered, with subsequent, maintenance doses being administered at a lower level. For example, an initial loading dose of 4 mg/kg may be infused over a period of some 90 minutes, followed by weekly maintenance doses for 4-8 weeks of 2 mg/kg infused over a 30-minute period if the previous dose was well tolerated.

Controlled-release parenteral formulations can be made as implants, oily injections, or as particulate systems. For a broad overview of protein delivery systems see, Banga, Therapeutic Peptides and Proteins: Formulation, Processing, and Delivery Systems, Lancaster, PA: Technomic Publishing Company, Inc., 1995. Particulate systems include microspheres, microparticles, microcapsules, nanocapsules, nanospheres, and nanoparticles. Microcapsules contain the active protein agent, such as a cytotoxin or a drug, as a central core. In microspheres, the active protein agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 Jim are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of approximately 5 pm so that only nanoparticles are administered intravenously. Microparticles are typically around 100 urn in diameter and are administered subcutaneously or intramuscularly. See, for example, Kreuter, Colloidal Drug Delivery Systems, J. Kreuter (Ed.), New York, NY : Marcel Dekker, Inc., pp. 219-342, 1994; and Tice and Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization. Applications, A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.

Polymers can be used for ion-controlled release of the compositions disclosed herein. Any suitable polymer may be used, such as a degradable or nondegradable polymeric matrix designed for use in controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for controlled release of proteins. In yet another aspect, liposomes are used for controlled release as well as drug targeting of the lipid-capsulated drug.

2. Methods of detection and diagnosis

Methods are also provided for the detection of the presence of a coronavirus spike protein in vitro or in vivo. In one example, the presence of a coronavirus spike protein is detected in a biological sample from a subject and can be used to identify a subject with an infection.

In aspects, the method detects the presence of at least one coronavirus in the biological same. In some aspects, the method detects the presence of an alphacoronavirus or a betacoronavirus in a biological sample. In other aspects, the method detects the presence of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, or a OC43 in a biological sample. In further aspects, the method detects the presence of SARS- CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63 or a 229E in a biological sample. In further aspects, the methods detects the presence of at least SARS-CoV-2 in the biological sample.

The sample can be any sample, including, but not limited to, tissue from biopsies, autopsies and pathology specimens. Biological samples also include sections of tissues, for example, frozen sections taken for histological purposes. Biological samples further include body fluids, such as blood, serum, plasma, sputum, spinal fluid or urine. The method of detection can include contacting a cell or sample, with an antibody, antigen binding fragment, such as a DVD-immunoglobulin, that specifically binds to a coronavirus spike protein, or conjugate thereof (e.g., a conjugate including a detectable marker) under conditions sufficient to form an immune complex, and detecting the immune complex e.g., by detecting a detectable marker conjugated to the antibody or antigen binding fragment).

In one aspect, the DVD-immunoglobulin is directly labeled with a detectable marker. In another aspect, the DVD-immunoglobulin is unlabeled and a secondary antibody or other molecule that can bind the primary DVD-immunoglobulin is utilized for detection. The secondary antibody is chosen that can specifically bind the specific species and class of the first antibody. For example, if the first bispecific includes a human IgG, then the secondary antibody may be an anti-human-IgG. Other molecules that can bind to DVD-immunoglobulins include, without limitation, Protein A and Protein G, both of which are available commercially. Suitable labels for the DVD-immunoglobulin or secondary antibody are known and described above, and include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.

In some aspects, the disclosed DVD-immunoglobulins are used to test vaccines. For example, to test if a vaccine composition including a coronavirus spike protein or fragment thereof assumes a conformation including the epitope of a disclosed DVD-immunoglobulin. Thus, provided herein is a method for testing a vaccine, wherein the method comprises contacting a sample containing the vaccine, such as a coronavirus spike protein immunogen, with a disclosed DVD-immunoglobulin, under conditions sufficient for formation of an immune complex, and detecting the immune complex, to detect the vaccine including the epitope of interest in the sample. In one example, the detection of the immune complex in the sample indicates that vaccine component, such as the immunogen assumes a conformation capable of binding the DVD-immunoglobulin.

EXAMPLES

Bispecific antibody VH and V/./VK sequences were constructed using the sequences of mAbs specific to the fusion peptide (COV44-62, COV44-79, COV91-27) and stem helix (COV89-22, COV30-14 and COV72-37). Matched pairs of bispecific antibody VH and V /VK sequences were then commercially cloned into plasmids in a DVD-Ig format and expressed as recombinant antibody by transient transfection of Expi293 cells. Supernatants containing the expressed recombinant antibodies were mixed with multiplexed beads conjugated to SARS-CoV-2 fusion peptide, SARS-CoV-2 stem helix and CD4 (negative control). The beads were incubated with supernatants at room temperature then washed with buffer, and binding of the expressed bispecific antibody to both the fusion peptide and stem helix antigen was detected using an anti-human IgG Alexa Fluor 647 secondary antibody. Samples were acquired on the iQue Screener Plus flow cytometer and data were analyzed with FlowJo. AUC analyses were performed with GraphPad Prism and reported after correction using the AUC of the negative control CD4 population.

A heat map was generated, representing the binding of DVD-immunoglobulin to peptides that represent fusion peptide and stem helix residues which are highly conserved in spike proteins of SARS- CoV-2 Wuhan Hu-1, SARS-CoV-1, MERS-CoV, HCoV-HKUl, HCoV-OC43 (betacoronavirus genus), as well as HCoV-NL63 and HCoV-229E (alphacoronavirus genus). mAbs specific to the fusion peptide (COV44-62, COV44-79, COV91-27), stem helix (COV89-22, COV30-14 and COV72-37) and the anti- HIV-1 spike protein mAb VRC01 were included as controls for mAb binding experiments. FIG. 1 shows the area under the curve (AUC) values for each antigen after subtracting values for the negative control antigen CD4. Of the expressed antibodies, 30 were confirmed as bispecific due to their ability to simultaneously bind the fusion peptide and stem helix (compared to the control mAbs which bind only either the stem helix or fusion peptide). The breadth and potency of neutralization of the bispecific antibodies is assessed against SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-OC43, HCoV-NL63.

To evaluate the functionality of the bispecific antibodies, titrations of recombinantly expressed antibodies were co-incubated with pseudovirus which expressed the SARS COV-2 spike (USA-WA1 2020 strain) and the firefly luciferase reporter gene. HeLa cells expressing the SARS-COV-2 receptor ACE2 (HeLa-ACE2) were inoculated with the antibody -pseudovirus mixture and luciferase activity was measured 48h post-infection (p.i.) to assess the resulting level of HcLa-ACE2 infection. 50% neutralizing titer (NT50) was calculated using 5-parameter non-linear regression. All parental stem helix mAbs (SH; COV30-14, COV72-37 and COV89-22) and parental fusion peptide mAbs (FP; COV44-62, COV44-79 and COV91-27) achieved at least 50% neutralization of the SARS COV-2 pseudovirus over the concentration range tested, with COV89-22 being the most potent SH mAb (NT50 = 1.15pg/mL) and COV44-62 being the most potent FP mAb (NT50 = 0.82pg/mL). Of the 27 bispecific antibodies tested, n=15 achieved at least 50% neutralization of the SARS COV-2 pseudovirus over the concentration range tested with NT50 potencies ranging from 0.28 - 30.25 pg/mL. NT50 was not determined for 7 bispecific antibodies due to low recovery from transfection supernatants (FIGs. 11 A-l 1C).

FIGS. 12A-12B are Tables that show that bispecific antibodies neutralize SARS COV-2 pseudovirus (USA-WA1 2020 strain) infection of ACE2 expressing HeLa cells. Where tested, 50% neutralizing titer (NT50) denotes the concentration of antibody at which infection is neutralized by 50% and was calculated using 5-parameter non-linear regression. The highest concentration tested is indicated for antibodies that did not achieve 50% neutralization. In view of the many possible aspects to which the principles of our invention may be applied, it should be recognized that illustrated aspects are only examples of the invention and should not be considered a limitation on the scope of the invention. Rather, the scope of the invention is defined by the following claims. We therefore claim as our invention all that comes within the scope and spirit of these claims.

Claims

We claim:

1. A dual variable domain immunoglobulin (DVD-Ig) that specifically binds to a coronavirus spike protein, and neutralizes a coronavirus, wherein the (DVD) immunoglobulin comprises a heavy chain and a light chain, and wherein: a) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, and 40, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain (COV44-79_COV89-22_GS and COV44-79_COV89-22_EL); b) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a V domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain (COV44-62_COV72-37_GS and COV44-62_COV72-37_ELg c) the heavy chain comprises, in amino (N) to carboxy (C) terminal order: a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 10, 11 and 12, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs:14, 15 and 16, and a heavy chain constant domain (COV44-62_COV30-14_GS and COV44-62_COV30-14_EL); d) the heavy chain of the bispecific antibody comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 2, 3, and 4, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, and light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 6, 7, and 8, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain (COV44-62_COV89-22_GS and COV44-62_COV89-22_EL); e) the heavy chain comprises, in N to C terminal order: a first VH domain comprising SEQ ID NOs: 10, 11 and 12, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order: a VL domain comprising SEQ ID NOs: 14, 15 and 16 a linker, a second VL domain comprising SEQ ID NOs: 6, 7 and 8, and a light chain constant domain (COV30-14_COV44-62_GS and COV30-14_COV44-62_EL); f) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second VL domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain (COV72-37_ COV44-62_GS and COV72-37_ COV44-62_EL); g) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second VH domain comprising SEQ ID NOs: 2, 3, and 4, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 6, 7, and 8, and a light chain constant domain (COV89-22_ COV44-62_GS and COV89-22_ COV44-62_EL); h) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, and 40, a linker, a second VL domain comprising SEQ ID NOs: 14, 15, andl6, and a light chain constant domain (COV44-79_COV30-14_GS and COV44-79_COV30-14_EL); i) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, and the light chain comprises in N to C terminal order, a VL domain comprising SEQ ID NOs: 14, 15, and 16, a linker, a second VL domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain (COV30-14_COV44-79_GS and COV30-I4 COV44-79 EL): j) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 34, 35, and 36, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, and light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 38, 39, 40, a linker, a second VL domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain (COV44-79_COV72-37_GS and COV44-79_COV72-37_EL); k) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second Vn domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second V_L domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain (COV72-37_COV44-79_GS and COV72-37_COV44-79_EL); l) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second VH domain comprising SEQ ID NOs: 34, 35, and 36, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second VL domain comprising SEQ ID NOs: 38, 39, and 40, and a light chain constant domain (COV89-22_COV44-79_GS and COV89-22_COV44-79_EL); m) the heavy chain of the bispecific antibody comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 10, 11, and 12, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second VL domain comprising SEQ ID NOs: 14, 15, and 16, and a light chain constant domain (COV91-27_COV30-14_GS and COV91-27_COV30- 14_EL); n) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 10, 11, and 12, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a Vi. domain comprising SEQ ID NOs: 14, 15, and 16, a linker, a second VL domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain (COV30-14_COV91-27_GS and COV30-14_COV91-27_EL); o) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 18, 19, and 20, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second VL domain comprising SEQ ID NOs: 22, 23, and 24, and a light chain constant domain (COV91-27_COV72-37_GS and COV91-27_COV72-37_EL); p) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 18, 19, and 20, a linker, a second VH domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a V domain comprising SEQ ID NOs: 22, 23, and 24, a linker, a second V_L domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain (COV72-37_COV91-27_GS and COV72-37_COV91-27_EL); q) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 42, 43, and 44, a linker, a second VH domain comprising SEQ ID NOs: 26, 27, and 28, and a heavy chain constant domain, and the light chain of the bispecific antibody comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 46, 47, and 48, a linker, a second VL domain comprising SEQ ID NOs: 30, 31, and 32, and a light chain constant domain (COV91-27_COV89-22_GS and COV91-27_COV89- 22_EL); or r) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NOs: 26, 27, and 28, a linker, a second Vn domain comprising SEQ ID NOs: 42, 43, and 44, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NOs: 30, 31, and 32, a linker, a second V_L domain comprising SEQ ID NOs: 46, 47, and 48, and a light chain constant domain (COV89-22_COV91-27_GS and COV89-22_COV91-27_EL).

2. The DVD-immunoglobulin of claim 1, wherein: a) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 33, a linker, a second VH domain comprising SEQ ID 25, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 37, a linker, and a second VL domain comprising SEQ ID NO: 29, and a light chain constant domain; b) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 1, a linker, a second VH domain comprising SEQ ID NO: 17, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 5, a linker, and a second VL domain comprising SEQ ID NO: 21, and a light chain constant domain; c) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO:

1, a linker, a second VH domain comprising SEQ ID NO: 9, and a heavy chain constant domain; and the light chain comprises, , in N to C terminal order, a VL domain comprising SEQ ID NO: 5 a linker, and a second Vi. domain comprising SEQ ID NO: 13, and a light chain constant domain; d) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 1, a linker, a second VH domain comprising SEQ ID NO: 25, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 5, a linker, and a second VL domain comprising SEQ ID NO: 29, and a light chain constant domain; e) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 9, a linker, a second VH domain comprising SEQ ID NO: 1, and a heavy chain constant domain, such as an IgG or IgA constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 13, a linker, and a second VL domain comprising SEQ ID NO: 5, and a light chain constant domain; f) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 17, a linker, a second VH domain comprising SEQ ID NO: 1, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 21, a linker, and a second VL domain comprising SEQ ID NO: 5, and a light chain constant domain; g) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 25, a linker, a second VH domain comprising SEQ ID NO: 1, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 29, a linker, and a second VL domain comprising SEQ ID NO: 5, and a light chain constant domain; h) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 33, a linker, a second VH domain comprising SEQ ID NO: 9, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 37, a linker, and a second VL domain comprising SEQ ID NO: 13, and a light chain constant domain; i) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 9, a linker, a second VH domain comprising SEQ ID NO: 33, and a heavy chain constant domain, and the light chain of the bispecific antibody comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 13, a linker, and a second VL domain comprising SEQ ID NO: 37, and a light chain constant domain j) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 33, a linker, a second VH domain comprising SEQ ID NO: 17, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 37, a linker, and a second VL domain comprising SEQ ID NO: 21, and a light chain constant domain; k) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 17, a linker, a second VH domain comprising SEQ ID NO: 33, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 21, a linker, and a second VL domain comprising SEQ ID NO: 37, and a light chain constant domain; l) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO 25, a linker, a second VH domain comprising SEQ ID NO: 33, and a heavy chain constant domain, and the light chain of comprises, in N to C terminal order, a Vi. domain comprising SEQ ID NO: 29 a linker, and a second VL domain comprising SEQ ID NO: 37, and a light chain constant domain; m) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 41, a linker, a second VH domain comprising SEQ ID NO: 9, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 45, a linker, and a second VL domain comprising SEQ ID NO: 13, and a light chain constant domain; n) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 9, a linker, a second VH domain comprising SEQ ID NO: 41, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 13, a linker, and a second VL domain comprising SEQ ID NO: 45, and a light chain constant domain; o) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 41, a linker, a second VH domain comprising SEQ ID NO: 17, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 45, a linker, and a second VL domain comprising SEQ ID NO: 21, and a light chain constant domain; p) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 17, a linker, a second VH domain comprising SEQ ID NO: 41, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 21, a linker, and a second VL domain comprising SEQ ID NO: 45, and a light chain constant domain; q) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 41, a linker, a second VH domain comprising SEQ ID NO: 25, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 45, a linker, and a second VL domain comprising SEQ ID NO: 29, and a light chain constant domain; or r) the heavy chain comprises, in N to C terminal order, a first VH domain comprising SEQ ID NO: 25, a linker, a second VH domain comprising SEQ ID NO: 41, and a heavy chain constant domain, and the light chain comprises, in N to C terminal order, a VL domain comprising SEQ ID NO: 29, a linker, and a second VL domain comprising SEQ ID NO: 45, and a light chain constant domain.

3. The DVD-immunoglobulin of claim 1 of claim 2, wherein the linker comprises SEQ ID NO: 121(GS).

4. The DVD-immunoglobulin of claim 1 or claim 2, wherein the linker comprises one of SEQ ID NO: 122-124 (EL).

5. The DVD-immunoglobulin of any one of claims 1-4, wherein: a) the heavy chain comprises SEQ ID NO: 88 and the light chain comprises SEQ ID NO: 89 (COV44-79_ COV89-22_EL); b) the heavy chain comprises SEQ ID NO: 54 and the light chain comprises SEQ ID NO: 55 (COV44-62_COV72-37_GS); c) the heavy chain comprises SEQ ID NO: 127and the light chain comprises SEQ ID NO: 49 (COV44-62_COV30- 14_EL) ; d) the heavy chain comprises SEQ ID NO: 56 and the light chain comprises SEQ ID NO: 57 (COV44-62_COV72-37_EL); e) the heavy chain comprises SEQ ID NO: 64 and the light chain comprises SEQ ID NO: 65 (COV44-62 COV89-22 EL) ; f) the heavy chain comprises SEQ ID NO: 125and the light chain comprises SEQ ID NO: 126 (COV44-62_COV30- 14_GS); g) the heavy chain comprises SEQ ID NO: 50 and the light chain comprises SEQ ID NO: 51 (CO V30- 14_CO V44-62_GS ) ; h) the heavy chain comprises SEQ ID NO: 52 and the light chain comprises SEQ ID NO: 53 (CO V30- 14_CO V44-62_EL) ; i) the heavy chain comprises SEQ ID NO: 58 and the light chain comprises SEQ ID NO: 59 (COV72-37_COV44-62_GS); j) the heavy chain comprises SEQ ID NO: 60 and the light chain comprises SEQ ID NO: 61 (COV72-37_COV44-62_EL); k) the heavy chain comprises SEQ ID NO: 62 and the light chain comprises SEQ ID NO: 63 (COV44-62_COV89-22_GS); l) the heavy chain comprises SEQ ID NO: 66 and the light chain comprises SEQ ID NO: 67 (COV89-22_COV44-62_GS); m) the heavy chain comprises SEQ ID NO: 68 and the light chain comprises SEQ ID NO: 69 (COV89-22 COV44-62 EL) ; n) the heavy chain comprises SEQ ID NO: 70 and the light chain comprises SEQ ID NO: 71 (COV44-79_COV30-14_GS); o) the heavy chain comprises SEQ ID NO: 72 and the light chain comprises SEQ ID NO: 73 (COV44-79_COV30- 14_EL) ; p) the heavy chain comprises SEQ ID NO: 74 and the light chain comprises SEQ ID NO: 75 (COV30-14_COV44-79_GS); q) the heavy chain comprises SEQ ID NO: 76 and the light chain comprises SEQ ID NO: 77 (COV30- 14_COV44-79_EL) ; r) the heavy chain comprises SEQ ID NO: 78 and the light chain comprises SEQ ID NO: 79 (COV44-79_COV72-37_GS); s) the heavy chain comprises SEQ ID NO: 80 and the light chain comprises SEQ ID NO: 81 (COV44-79_COV72-37_EL); t) the heavy chain comprises SEQ ID NO: 82 and the light chain comprises SEQ ID NO: 83 (COV72-37_ COV44-79_GS); u) the heavy chain comprises SEQ ID NO: 84 and the light chain comprises SEQ ID NO: 85 (COV72-37_ COV44-79_EL); v) the heavy chain comprises SEQ ID NO: 86 and the light chain comprises SEQ ID NO: 87 (COV44-79_ COV89-22_GS); x) the heavy chain comprises SEQ ID NO: 90 and the light chain comprises SEQ ID NO: 91 (COV89-22_ COV44-79_GS); y) the heavy chain comprises SEQ ID NO: 92 and the light chain comprises SEQ ID NO: 93 (COV89-22_ COV44-79_EL); z) the heavy chain comprises SEQ ID NO: 94 and the light chain comprises SEQ ID NO: 95 (COV91-27_ COV30-14_GS); aa) the heavy chain comprises SEQ ID NO: 96 and the light chain comprises SEQ ID NO: 97 (COV91-27_ COV30-14_EL); ab) the heavy chain comprises SEQ ID NO: 98 and the light chain comprises SEQ ID NO: 99 (COV30-14_ COV91-27_GS); ac) the heavy chain comprises SEQ ID NO: 100 and the light chain comprises SEQ ID NO: 101 (COV30- 14_ COV91 -27_EL) ; ad) the heavy chain comprises SEQ ID NO: 102 and the light chain comprises SEQ ID NO: 103 (COV91-27_ COV72-37_GS); ae) the heavy chain comprises SEQ ID NO: 104 and the light chain comprises SEQ ID NO: 105 (COV91-27_ COV72-37 EL); af) the heavy chain comprises SEQ ID NO: 106 and the light chain comprises SEQ ID NO: 107 (COV72-37_ COV91-27_GS); ag) the heavy chain comprises SEQ ID NO: 108 and the light chain comprises SEQ ID NO: 109 (COV72-37_ COV91-27 EL); ah) the heavy chain comprises SEQ ID NO: 110 and the light chain comprises SEQ ID NO: 111 (COV91-27_ COV89-22_GS); ai) the heavy chain comprises SEQ ID NO: 112 and the light chain comprises SEQ ID NO: 113 (COV91-27_ COV89-22 EL); aj) the heavy chain comprises SEQ ID NO: 114 and the light chain comprises SEQ ID NO: 115 (COV89-22_ COV91-27_GS); or ak) the heavy chain comprises SEQ ID NO: 116 and the light chain comprises SEQ ID NO: 117 (COV89-22_ COV91-27 EL).

6. The DVD-immunoglobulin of any one of claims 1-5, wherein the heavy chain constant domain and the light chain constant domain are an IgG heavy chain constant domain and an IgG light chain constant domain.

7. The DVD-immunoglobulin of any one of claims 1-5, wherein the heavy chain constant domain and the light chain constant domain are an IgA heavy chain constant domain and an IgA light chain constant domain.

8. The DVD- immunoglobulin of claim any one of claims 1-7, linked to a detectable marker.

9. An isolated nucleic acid molecule encoding the DVD-immunoglobulin of any one of claims 1-7.

10. The nucleic acid molecule of claim 9, operably linked to a promoter.

11. A vector comprising the nucleic acid molecule of claim 9 or claim 10.

12. The vector of claim 11, wherein the vector is a viral vector.

13. A host cell comprising the nucleic acid molecule of claim 9 or claim 10, or the vector of claim 11 or claim 12.

14. A pharmaceutical composition comprising an effective amount of the DVD- immunoglobulin of any one of claims 1-7, the nucleic acid molecule of claim 9 or claim 10, or the vector of claim 11 or 12; and a pharmaceutically acceptable carrier.

15. A method of producing an antibody or antigen binding fragment that specifically binds to a SARS-CoV protein, comprising: expressing one or more nucleic acid molecules encoding the DVD-immunoglobulin of any one of claims 1-7 in a host cell; and purifying the antibody or antigen binding fragment.

16. A method of detecting the presence of SARS-CoV or SARS-CoV-2 in a biological sample from a subject, comprising: contacting the biological sample with an effective amount of the DVD- immunoglobulin of any one of claims 1-7 under conditions sufficient to form an immune complex; and detecting the presence of the immune complex in the biological sample, wherein the presence of the immune complex in the biological sample indicates the presence of the SARS-CoV or SARS-CoV-2 in the sample.

17. The method of claim 16, wherein detecting the detecting the presence of the immune complex in the biological sample indicates that the subject has a SARS-CoV or SARS-CoV-2 infection.

18. A method of inhibiting a coronavirus infection in a subject, comprising administering an effective amount of the DVD-immunoglobulin of any one of claims 1-7, the nucleic acid molecule of claim 9 or claim 10, the vector of claim 11 or claim 12, or the pharmaceutical composition of claim 14 to the subject, wherein the subject has or is at risk of a coronavirus infection.

19. The method of claim 18, wherein the coronavirus is SARS-CoV-2.

20. Use of the DVD-immunoglobulin of any one of claims 1-7, the nucleic acid molecule of claim 9 or claim 10, the vector of claim 11 or claim 12, or the pharmaceutical composition of claim 14, to inhibit a coronavirus infection in a subject or to detect the presence of a coronavirus in a biological sample.