Bispecific antibodies that widely target coronavirus

Abstract

Disclosed are bispecific antibodies, including dual variable domain immunoglobulins that specifically bind to a coronavirus spike protein (e.g., SARS-CoV-2). The use of these antibodies for inhibiting coronavirus infection is also disclosed. In addition, methods of detecting coronavirus in a biological sample using the disclosed antibodies are disclosed.

Description

Cross-reference to related applications

[0001] This application 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.

[0002] open field This application relates to bispecific antibodies, such as dual variable domain (DVD) immunoglobulins, which specifically bind to coronavirus spike proteins, and their use in inhibiting or detecting coronavirus infection in subjects.

[0003] sequence list The contents of the electronic sequence list (Sequence.xml; size: 125,213 bytes; and creation date: July 18, 2024) are incorporated herein by reference in their entirety.

[0004] background The coronavirus SARS-CoV-2 emerged in 2019 and has infected 400 million individuals globally, causing over 5 million deaths. While effective vaccines and antibody therapies have been developed, their efficacy is threatened by the emergence of variants that are more resistant to many of these treatments than previous ones. Furthermore, the possibility of other coronaviruses spilling over from animal libraries remains, and preparations must be made for this. Bispecific antibodies that bind to multiple coronavirus antigens are still needed.

[0005] Public Overview This article discloses bispecific antibodies that specifically bind to coronaviruses. In some aspects, DVD immunoglobulin is disclosed, which specifically binds to and neutralizes the coronavirus spike protein.

[0006] In some respects, DVD immunoglobulins comprise heavy and light chains, and among them: a) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 34, 35 and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The structural domain, and the heavy chain constant structural domain; and the light chain contains, in order from N-terminus to C-terminus: V containing SEQ ID NO: 38, 39 and 40. L Structural domain, connector, second V including SEQ ID NO:30, 31 and 32 L The structural domain, as well as the light chain constant structural domain (COV44-79_COV89-22_GS and COV44-79_COV89-22_EL). b) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 6, 7 and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 22, 23 and 24 L The structural domain, as well as the light chain constant structural domains (COV44-62_COV72-37_GS and COV44-62_COV72-37_EL). c) The heavy chain comprises, in the order from the amino (N) terminus to the carboxyl (C) terminus, the first V containing SEQ ID NO: 2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 6, 7 and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 14, 15 and 16 L The structural domain, as well as the heavy chain constant structural domains (COV44-62_COV30-14_GS and COV44-62_COV30-14_EL). d) The heavy chain of the bispecific antibody comprises, in order from N-terminus to C-terminus: the first V containing SEQ ID NO: 2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 6, 7 and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 30, 31 and 32 L The structural domain, as well as the light chain constant structural domain (COV44-62_COV89-22_GS and COV44-62_COV89-22_EL). e) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 14, 15 and 16L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The structural domain, as well as the light chain constant structural domain (COV30-14_COV44-62_GS and COV30-14_COV44-62_EL). f) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 18, 19 and 20. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The structural domain, and the heavy chain constant structural domain; and the light chain contains, in order from N-terminus to C-terminus: V containing SEQ ID NO: 22, 23 and 24. L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The structural domain, as well as the light chain constant structural domains (COV72-37_COV44-62_GS and COV72-37_COV44-62_EL). g) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 26, 27 and 28. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 30, 31 and 32. L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The structural domain, as well as the light chain constant structural domain (COV89-22_COV44-62_GS and COV89-22_COV44-62_EL). h) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 34, 35 and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The structural domain, and the heavy chain constant structural domain; and the light chain contains, in order from N-terminus to C-terminus: V containing SEQ ID NO: 38, 39 and 40. L Structural domain, connector, containing the second V of SEQ ID NO:14, 15 and 16 L The structural domain, as well as the light chain constant structural domain (COV44-79_COV30-14_GS and COV44-79_COV30-14_EL). i) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 14, 15 and 16 L Structural domain, connector, containing the second V of SEQ ID NO:38, 39 and 40 L The structural domain, as well as the light chain constant structural domain (COV30-14_COV44-79_GS and COV30-14_COV44-79_EL). j) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 34, 35 and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The structural domain, and the heavy chain constant structural domain; and the light chain contains, in order from N-terminus to C-terminus: V containing SEQ ID NO: 38, 39 and 40. L Structural domain, connector, second V including SEQ ID NO:22, 23 and 24 L The structural domain, as well as the light chain constant structural domain (COV44-79_COV72-37_GS and COV44-79_COV72-37_EL). k) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 18, 19 and 20. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The structural domain, and the heavy chain constant structural domain; and the light chain contains, in order from N-terminus to C-terminus: V containing SEQ ID NO: 22, 23 and 24. L Structural domain, connector, containing the second V of SEQ ID NO:38, 39 and 40 L The structural domain, as well as the light chain constant structural domain (COV72-37_COV44-79_GS and COV72-37_COV44-79_EL). l) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 26, 27 and 28. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 HThe structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 30, 31 and 32. L Structural domain, connector, containing the second V of SEQ ID NO:38, 39 and 40 L The structural domain, as well as the light chain constant structural domain (COV89-22_COV44-79_GS and COV89-22_COV44-79_EL). m) The heavy chain of the bispecific antibody comprises, in order from N-terminus to C-terminus: the first V containing SEQ ID NO: 42, 43 and 44 H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 46, 47 and 48. L Structural domain, connector, containing the second V of SEQ ID NO: 14, 15 and 16 L The structural domain, as well as the light chain constant structural domain (COV91-27_COV30-14_GS and COV91-27_COV30-14_EL). n) The heavy chain comprises, in order from N-terminus to C-terminus: the first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 14, 15 and 16 L Structural domain, connector, containing the second V of SEQ ID NO:46, 47 and 48 L The structural domain, as well as the light chain constant structural domain (COV30-14_COV91-27_GS and COV30-14_COV91-27_EL). o) The heavy chain comprises, in order from N-terminus to C-terminus: the first V containing SEQ ID NO: 42, 43 and 44 H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 46, 47 and 48. L Structural domain, connector, second V including SEQ ID NO:22, 23 and 24 LThe structural domain, as well as the light chain constant structural domain (COV91-27_COV72-37_GS and COV91-27_COV72-37_EL). p) The heavy chain comprises, in order from N-terminus to C-terminus: the first V containing SEQ ID NO: 18, 19 and 20 H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The structural domain, and the heavy chain constant structural domain; and the light chain contains, in order from N-terminus to C-terminus: V containing SEQ ID NO: 22, 23 and 24. L Structural domain, connector, containing the second V of SEQ ID NO:46, 47 and 48 L The structural domain, as well as the light chain constant structural domain (COV72-37_COV91-27_GS and COV72-37_COV91-27_EL). q) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 42, 43 and 44. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The light chain of the bispecific antibody comprises, in order from N-terminus to C-terminus, a V-terminal domain and a heavy chain constant domain; and the light chain of the bispecific antibody comprises, in order from N-terminus to C-terminus, V-terminus of SEQ ID NO: 46, 47 and 48. L Structural domain, connector, containing the second V of SEQ ID NO: 30, 31 and 32 L The structural domain, and the light chain constant structural domains (COV91-27_COV89-22_GS and COV91-27_COV89-22_EL); or r) The heavy chain comprises, in order from N-terminus to C-terminus: the first V containing SEQ ID NO: 26, 27 and 28 H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The structural domain, and the constant structural domain of the heavy chain; and the light chain contains, in order from the N-terminus to the C-terminus: V containing SEQ ID NO: 30, 31 and 32. L Structural domain, connector, containing the second V of SEQ ID NO:46, 47 and 48 L The structural domain, as well as the light chain constant structural domain (COV89-22_COV91-27_GS and COV89-22_COV91-27_EL).

[0007] In some respects, nucleic acid molecules encoding these bispecific antibodies, vectors comprising these nucleic acid molecules, and host cells comprising these vectors have been disclosed.

[0008] Pharmaceutical compositions comprising bispecific antibody nucleic acid molecules and carriers are also disclosed. Furthermore, the use of these pharmaceutical compositions for inhibiting coronavirus infection in subjects is disclosed.

[0009] In other respects, the use of the disclosed antibody and antigen-binding fragments for detecting coronaviruses in biological samples has been disclosed.

[0010] The above and other features of this disclosure will become more apparent from the following detailed description of several aspects with reference to the accompanying drawings.

[0011] Brief description of the attached figures Figure 1 Single antibodies exhibited binding specificity for two distinct neutralizing epitopes (fusion peptide and stem-helix) of the coronavirus spike protein. The heatmap illustrates the binding of bispecific antibodies to peptides representing fusion peptides and stem-helix residues, which are highly conserved in the spike proteins of SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43 (β-coronavirus), HCoV-NL63 (α-coronavirus), and HCoV-229E (α-coronavirus). mAbs specific to the fusion peptides (COV44-62, COV44-79, COV91-27), stem-helices (COV89-22, COV30-14, and COV72-37), and the anti-HIV-1 spike protein mAb VRC01 were included as controls for mAb binding experiments. The area under the curve (AUC) value for each antigen is shown after subtracting the CD4 value from the negative control antigen.

[0012] Figures 2-10 The amino acid sequence of the bispecific antibody that specifically binds to both the SARS-CoV-2 fusion peptide and the stem-spiral coil is shown.

[0013] Figures 11A-11C Neutralization curves for the SARS-CoV-2 pseudovirus (USA-WA1 2020 strain). Curves are shown for parental stem-spiral mAbs (COV30-14, COV72-37, and COV89-22), parental fusion peptide mAbs (COV44-62, COV44-79, and COV91-27), and representative bispecific antibodies. Dashed lines represent 50% neutralization, and error bars show the standard deviation of two replicates.

[0014] Figures 12A-12B The table shows that the bispecific antibody neutralized the infection of SARS-CoV-2 pseudovirus (USA-WA1 2020 strain) in HeLa cells expressing ACE2.

[0015] sequence Nucleic acid and amino acid sequences are displayed using standard letter abbreviations of nucleotide bases and three-letter codes of amino acids as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but any reference to the strand shown should be understood to include the complementary strand. In each variable heavy chain domain (V) shown below H ) and variable light chain structural domain (V L In the diagram, the Complementary Determinant Region (CDR) is shown in bold.

[0016] SEQ ID NO: 1 is COV44-62 V H The amino acid sequences. SEQ ID NO: 2, 3 and 4 are the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively.

[0017] QMQLMQSGAEVKKPGASVTVSCKASGDTFSDYRIHWVRQAPGQGLEWMGRMNPKSGDTNFAQKFQGRVTMTRDMSINTAYMTLSGLTFDDTALYYCASLLIVGGFDPLDDFEVWGQGTMVTISS.

[0018] SEQ ID NO: 5 is COV44-62 V L The amino acid sequences. SEQ ID NO: 6, 7 and 8 are the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively.

[0019] QSALTQPPSASGSPGQSVTISCSGTSSDVGGYNFVSWYQHHPGKAPKILIYEVTKRPSGVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYGGTNNLLFGGGTKLTVL.

[0020] SEQ ID NO: 9 is COV30-14 V H The amino acid sequences. SEQ ID NO: 10, 11 and 12 are the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively.

[0021] QVQLVQSGAEVKKPGASVKVSCQTSGYTFTSYYMHWVRQAPGQGLEWMGLITPSGDDTYYAQRFQGRVTMTRDTSSPTYMELSSLTSEDTAVYYCAKMSRAGGFDVWGQGTLVTVSS.

[0022] SEQ ID NO: 13 is COV30-14 V LThe amino acid sequences. SEQ ID NO: 14, 15 and 16 are the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively.

[0023] EVVLTQSPGTLSLSPGERATLSCRASQSITGRYLAWYQQKPGQAPRLLMYGESSRVTGIPDRFSGGGSGTDFTLTISRLEPEDFAVYYCQHFASSPPTYTFGQGTKLEIR.

[0024] SEQ ID NO: 17 is COV72-37 V H The amino acid sequences. SEQ ID NO: 18, 19 and 20 are the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively.

[0025] QVQLVQSGAEVKKPAASVKVSCKASGDTFSSHYMHWVRQAPGQGPEWMGIINPSGSGTAYGQKFQGRLTMTRDTSTSTVYMELSSLTSDDTAVYYCGGGSGGLFAYWGQGTLVTVSS.

[0026] SEQ ID NO: 21 is COV72-37 V L The amino acid sequences. SEQ ID NO: 22, 23 and 24 are the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively; EIVLTQSPGTLSLSPGERATLSCRASQIVRSNYLAWYQQKPGQAPRLLIYGASSRATGTPDRFSGGGSGTDFTLTINRLEPEDFAVYYCLQYDSSPPTYIFGQGTKLEIK.

[0027] SEQ ID NO: 25 is COV89-22 V H The amino acid sequences. SEQ ID NO: 26, 27 and 28 are the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively.

[0028] QEQLVQSGAEVKKPGASVKVSCKSSGFTFSYFYLHWVRQAPGQGLEWMGIINPRGDGTRYAQKFQGRVTMTRDASTGTLYMELRSLRSEDTAVYYCARGADHGAFDIWGQGTMVTVSS.

[0029] SEQ ID NO: 29 is COV89-22VL The amino acid sequences. SEQ ID NO: 30, 31 and 32 are the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively.

[0030] EIVLTQSPGTLSLSPGERATLSCRASQSVRRNYFAWYQQKRGQAPRLLIYDASTRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYDSSPPMYIFGQGTKLEIK.

[0031] SEQ ID NO: 33 is COV44-79 V H The amino acid sequences. SEQ ID NO: 34, 35 and 36 are the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively.

[0032] QVQLVESGGGVVQPGRSLRLSCAASGLTFSGYAMHWVRQAPGKGLEWVAVISRDARNKYYADSVKGRFTISRDNSKKTVYLEMNSLRVEDTAVYYCAILIIPGITEPGSPDALDIWGQGTMVSVSS.

[0033] SEQ ID NO: 37 is COV44-79 V L The amino acid sequences. SEQ ID NO: 38, 39 and 40 are the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively. DIQMTQSPSSMSASVGDRVTITCRASQDISKWLAWYQQRPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQASSFPWSITFGQGTRLEIR.

[0034] SEQ ID NO: 41 is COV91-27 V H The amino acid sequences. SEQ ID NO: 42, 43 and 44 are the amino acid sequences of HCDR1, HCDR2 and HCDR3, respectively.

[0035] QVQLVESGGGVVHPGGSLRLSCAASGFTFSDFAMNWVRQAPGEGLQWVAIISYDGRNKHFAASVRGRFTISRDNSKNTMFLQMSSLRAEDTAIYYCATLGGWFEEASPTYWGRGTLVTVSS.

[0036] SEQ ID NO: 45 is COV91-27 V L The amino acid sequences. SEQ ID NO: 46, 47 and 48 are the amino acid sequences of LCDR1, LCDR2 and LCDR3, respectively.

[0037] EVVLTQSPAIVSLSPGERATLSCRASQSVGSRLAWHQQKPGLAPRVLIYDASNRATGIPARFSGSGSGTDFTLTISSIEPEDSAVYYCQQRGDWPLITFGQGTRLEIK.

[0038] SEQ ID NO: 49-117 and 125-127 are the amino acid sequences of the DVD-immunoglobulin antibody sequence, and also... Figures 2-10 It is displayed in the middle.

[0039] SEQ ID NO: 118-120 is an example of the amino acid sequence of the stem helix and part of the fusion peptide in the S2 domain of the spike protein.

[0040] SEQ ID NO: 121 is the amino acid sequence of the GS linker.

[0041] SEQ ID NO: 122-124 are amino acid sequences of three different EL linkers, where the outer V is the heavy chain, κ, or λ, respectively.

[0042] Detailed description of many aspects This article discloses bispecific antibodies, which include V from two different antibodies. H and V L The monoclonal antibody binds to the S2 domain of the spike protein, specifically binding to the spike protein of SARS-CoV-2 and also to the spike protein of at least one other coronavirus (e.g., alpha or beta coronavirus). In some respects, these monoclonal antibodies bind to the S2 domain of the spike protein. The bispecific antibody may be DVD-Ig. These bispecific antibodies can be used to inhibit coronavirus infection, for example, but not limited to SARS-CoV-2 infection. Monoclonal and bispecific antibodies can also be used to detect coronavirus infection.

[0043] I. Terminology Summary Unless otherwise stated, technical terms are used according to convention. Definitions of many commonly used molecular biology terms can be found in Krebs. et al. (eds.), Lewin's genes XIIJones & Bartlett Learning, 2017. Unless the context clearly indicates otherwise, as used herein, the singular forms “a,” “an,” and “the” refer to both the singular and the plural. For example, the term “antigen” includes both singular and plural antigens and can be considered equivalent to the phrase “at least one antigen.” As used herein, the term “comprising” means “including.” It should be further understood that, unless otherwise stated, any and all base sizes or amino acid sizes given with respect to nucleic acids or polypeptides, as well as all molecular weight or molecular mass values, are approximate and provided for descriptive purposes. Although many methods and materials similar to or equivalent to those described and used herein may be used, particularly suitable methods and materials are described herein. In case of conflict, this specification (including the explanations of terms) shall prevail. Furthermore, the materials, methods, and examples are illustrative only and are not intended to be limiting. For ease of review of the various aspects, the following explanations of terms are provided: Approximately: Unless the context otherwise indicates, “approximately” means plus or minus 5% of a reference value. For example, “approximately” 100 means 95 to 105.

[0044] Administration: The reagent (e.g., publicly disclosed DVD immunoglobulin) is introduced into the subject via a selected route. Administration may be local or systemic. For example, if the selected route is intravascular, the reagent (e.g., DVD-immunoglobulin) is administered by introducing the composition into the subject's blood vessels. Exemplary routes of administration include, but are not limited to, oral, injectable (e.g., subcutaneous, intramuscular, intradermal, intraperitoneal, and intravenous), sublingual, rectal, percutaneous (e.g., topical), intranasal, vaginal, and inhalation routes.

[0045] Amino acid substitution: One amino acid in a polypeptide is replaced by a different amino acid.

[0046] Antibody and antigen-binding fragment: Immunoglobulin, antigen-binding fragment, or derivative thereof, which specifically bind to and recognize an analyte (antigen), such as the spike protein of a coronavirus, for example, the spike protein from SARS-CoV-2. The term "antibody" is used in the broadest sense herein and encompasses a wide range of antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antigen-binding fragments, provided they exhibit the desired antigen-binding activity.

[0047] Non-limiting examples of antibodies include, for example, intact immunoglobulins and their variants and fragments that retain binding affinity for antigens. Antigen-binding fragments include V... H and V LAntigen-binding fragments specifically bind to homologous antigens. Examples of antigen-binding fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabody; linear antibody; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen-binding fragments that are either generated by modifying intact antibodies or synthesized de novo using recombinant DNA methods (see, e.g., Kontermann and Dübel (Eds.)). Antibody Engineering Vols. 1-2, 2 nd ed., Springer-Verlag, 2010).

[0048] Antibodies also include genetically engineered forms, such as chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugated antibodies (e.g., bispecific antibodies).

[0049] Antibodies can have one or more binding sites. If there are more than one binding site, these binding sites can be the same as each other or they can be different from each other. For example, naturally occurring immunoglobulins have two identical binding sites, single-chain antibodies or Fab fragments have one binding site, while bispecific antibodies or bifunctional antibodies have two different binding sites.

[0050] Naturally occurring immunoglobulins typically consist of heavy (H) chains and light (L) chains linked together by disulfide bonds. Immunoglobulin genes include κ, λ, α, γ, δ, ε, and μ constant region genes, as well as numerous immunoglobulin variable domain genes. Two types of light chains exist: lambda (λ) and kappa (κ). Five major heavy chain classes (or isotypes) exist, which determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE.

[0051] Each heavy and light chain contains constant regions (or constant domains) and variable regions (or variable domains). The variable regions of the heavy and light chains combine to specifically bind antigens.

[0052] Mentioning "V" H "VH" or "VH" refers to the variable region of the antibody heavy chain, including the variable region of antigen-binding fragments (such as Fv, scFv, dsFv, or Fab). Mentioning "V"... L "VL" or "VL" refers to the variable domain of the antibody light chain, including the variable domains of Fv, scFv, dsFv, or Fab.

[0053] V H and V L A "frame" region containing three high-variable regions, also known as a "complementary determination region" or "CDR" (see, for example, Kabat). et al. , Sequences of Proteins of Immunological Interest , 5 th (ed., NIHP Publication No. 91-3242, Public Health Service, National Institutes of Health, US Department of Health and Human Services, 1991). The framework region sequences of different light or heavy chains are relatively conserved within species. The antibody's framework region, i.e., the combined framework region that makes up the light and heavy chains, is used for localization and alignment of CDRs in three-dimensional space.

[0054] CDRs are primarily responsible for binding to epitopes of antigens. The amino acid sequence boundaries of a given CDR can be readily determined using any of many well-known methods, including Kabat. et al. ( Sequences of Proteins of Immunological Interest , 5 th ed., NIH Publication No. 91-3242, Public HealthService, National Institutes of Health, US Department of Health and HumanServices, 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-likedomains,” Dev. Comp. Immunol. , 27(1):55-77, 2003; those described in the “IMGT” numbering scheme). The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3 (from the N end to the C end), and are usually also identified by the chain in which the specific CDR is located. Therefore, V H CDR3 is the V of the antibody it belongs to. H The CDR3 sequence, while V LCDR1 is the V of the antibody it belongs to. L CDR1. Light chain CDRs are sometimes referred to as LCDR1, LCDR2, and LCDR3. Heavy chain CDRs are sometimes referred to as HCDR1, HCDR2, and HCDR3.

[0055] In some respects, disclosed monoclonal antibodies include heterologous constant domains. For example, the antibody may include a constant domain that differs from the natural constant domain, such as a constant domain containing one or more modifications (e.g., an "LS" mutation) to extend its half-life.

[0056] “Monoclonal antibody” is an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies comprising that group are identical and / or bind to the same epitopes, except for possible variant antibodies, such as those containing naturally occurring mutations or variants generated during the preparation of the monoclonal antibody, which are typically present in very small amounts. In contrast to polyclonal antibody formulations, which typically comprise different antibodies targeting different determinants (epitopes), each monoclonal antibody in a monoclonal antibody formulation targets a single determinant on the antigen. Therefore, the modifier “monoclonal” indicates that the antibody is characterized by being obtained from a substantially homogeneous group of antibodies and should not be construed as requiring the antibody to be produced by any particular method. For example, monoclonal antibodies can be prepared using a variety of techniques, including but not limited to hybridoma methods, recombinant DNA protocols, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, which are described herein, along with other exemplary methods for preparing monoclonal antibodies. In some instances, monoclonal antibodies are isolated from a subject. Monoclonal antibodies may have conserved amino acid substitutions that have substantially no effect on antigen binding or other immunoglobulin function. (See, for example, Greenfield (Ed.)) Antibodies: A Laboratory Manual , 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014.) "Humanized" antibody or antigen-binding fragments consist of a human framework region and one or more core-receiving regions (CDRs) derived from non-human (e.g., mouse, rat, or synthetic) antibody or antigen-binding fragments. The non-human antibody or antigen-binding fragment providing the CDR is called the "donor," and the human antibody or antigen-binding fragment providing the framework is called the "acceptor." In one respect, in humanized immunoglobulins, all CDRs are derived from the donor immunoglobulin. Constant regions are not necessarily present, but if present, they can be substantially identical to the constant regions of human immunoglobulins, for example, at least about 85-90% identical, for example, about 95% or higher. Therefore, apart from the possible CDRs, all portions of the humanized antibody or antigen-binding fragment are substantially identical to the corresponding portions of the natural human antibody sequence.

[0057] A "chimeric antibody" is an antibody that comprises sequences derived from two different antibodies, typically from different species. In some instances, a chimeric antibody includes one or more CDRs and / or frame regions from one human antibody and CDRs and / or frame regions from another human antibody.

[0058] A "fully human antibody" or "human antibody" is an antibody that includes sequences from (or derived from) the human genome but excludes sequences from other species. In some respects, human antibodies include the CDR, frame region, and (if present) Fc region from (or derived from) the human genome. Human antibodies can be identified and isolated using techniques for constructing antibodies based on sequences derived from the human genome (e.g., via phage display or using transgenic animals) (see, for example, 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).

[0059] Neutralizing antibodies or antigen-binding fragments of coronaviruses: These are antibodies or antigen-binding fragments that specifically bind to coronavirus antigens (such as the spike protein), inhibiting coronavirus-related biological functions to suppress infection. The antibody can neutralize the activity of one or more coronaviruses. For example, antibodies or antigen-binding fragments that neutralize coronaviruses (such as SARS-CoV-2) can interfere with the virus by directly binding to it and restricting its entry into cells. Alternatively, the antibody can interfere with one or more post-attachment interactions between the pathogen and a receptor, for example, by interfering with viral entry using the receptor. In some instances, antibodies specific to the coronavirus spike protein neutralize the infectious titer of the coronavirus.

[0060] In some respects, antibodies or antigen-binding fragments that specifically bind to and neutralize coronaviruses inhibit cell infection by, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%, compared to control antibodies or antigen-binding fragments.

[0061] "Broad-spectrum neutralizing antibodies" are antibodies that bind to and inhibit the function of relevant antigens, such as antigens with at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the surface of the antigen. For antigens from pathogens (e.g., viruses), antibodies can bind to and inhibit the function of antigens from more than one class and / or subclass of that pathogen. For example, for coronaviruses, antibodies can bind to antigens (e.g., the spike protein from the coronavirus) and inhibit their function.

[0062] Biological samples: Samples obtained from a subject. Biological samples include all clinical samples that can be used to detect disease or infection in a subject, including but not limited to cells, tissues, and body fluids such as blood, blood derivatives and fractions (e.g., serum), saliva, sputum, nasal swabs, cerebrospinal fluid; and tissues obtained through biopsy or surgical removal, such as tissues that have not been fixed, frozen, or fixed in formalin or paraffin. In specific instances, biological samples are obtained from subjects who have or are suspected of having a coronavirus infection (e.g., but not limited to SARS-CoV-2 infection).

[0063] Bispecific antibodies are recombinant molecules consisting of two distinct antigen-binding domains that bind to two distinct antigenic epitopes, although both epitopes can be coronavirus epitopes. Bispecific antibodies comprise molecules with two antigen-binding domains chemically or genetically linked. The antigen-binding domains can be linked using adapters. The antigen-binding domains can be monoclonal antibodies, antigen-binding fragments (e.g., Fab, scFv), or combinations thereof. Bispecific antibodies may include one or more constant domains, but do not necessarily include constant domains. In one example, a bispecific antibody is DVD-immunoglobulin.

[0064] Conditions sufficient for immune complex formation: conditions that allow an antibody or antigen-binding fragment to bind to its homologous epitope to a detectable degree, and / or substantially exclude its binding to substantially all other epitopes. The conditions sufficient for immune complex formation depend on the form of the binding reaction and are generally those utilized in the immunoassay protocol or encountered in vivo. For a description of immunoassay forms and conditions, see Greenfield (Ed.). Antibodies: A Laboratory Manual , 2 nd ed. New York: Cold Spring Harbor Laboratory Press, 2014. The conditions used in these methods are “physiological conditions,” which include references to typical conditions within living mammals or mammalian cells (e.g., temperature, osmotic pressure, pH). Although it is recognized that some organs are subjected to extreme conditions, the in vivo and intracellular environment is generally at around pH 7 (e.g., pH 6.0 to pH 8.0, or pH 6.5 to 7.5), contains water as the primary solvent, and exists at temperatures above 0°C and below 50°C. Osmotic pressure is within the range supporting cell viability and proliferation.

[0065] The formation of immune complexes can be detected by conventional methods, such as immunohistochemistry (IHC), immunoprecipitation (IP), flow cytometry, immunofluorescence microscopy, ELISA, immunoblotting (e.g., Western blotting), magnetic resonance imaging (MRI), computed tomography (CT), radiography, and affinity chromatography.

[0066] Conjugates: Complexes of two molecules linked together (e.g., covalently). On one hand, an antibody is linked to an effector molecule; for example, an antibody that specifically binds to one or more coronaviruses (e.g., SARS-CoV-2) is covalently linked to an effector molecule (e.g., a detectable marker). This linking can be achieved through chemical or recombinant means. On the other hand, this linking is chemical, where a reaction between the antibody portion and the effector molecule creates a covalent bond between the two molecules, forming a single molecule. Optionally, a peptide linker (a short peptide sequence) may be included between the antibody and the effector molecule. Because conjugates can be prepared from two molecules with independent functions (e.g., an antibody and an effector molecule), they are sometimes also referred to as "chimeric molecules."

[0067] Conservative variants: "Conservative" amino acid substitutions are substitutions that substantially do not affect or reduce protein function (e.g., the protein's ability to interact with target proteins). For example, the coronavirus-specific antibodies disclosed herein may include up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 conserved amino acid substitutions compared to a reference antibody sequence, while maintaining specific binding activity and / or neutralizing activity against the spike protein. The term "conservative variant" also includes the use of substituted amino acids in place of unsubstituted parental amino acids.

[0068] The individual substitutions, deletions, or additions of a single amino acid or a small subset of amino acids (e.g., less than 5%, less than 1% in some respects) in the coding sequence are conserved variations in which these changes result in the substitution of the amino acid with a chemically similar amino acid.

[0069] The following six groups are examples of amino acids that are considered to be conserved substitutes for each other: 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).

[0070] Non-conserved substitutions are substitutions that reduce antibody activity or function (such as the ability to bind specifically to the coronavirus spike protein). For example, if an amino acid residue is essential for the function of a protein, even a seemingly conserved substitution may disrupt that activity. Therefore, conserved substitutions do not alter the fundamental function of the protein of interest.

[0071] Contact: This refers to direct physical association; it includes both solid and liquid forms, and can occur in vivo or in vitro. Contact includes the contact between one molecule and another, for example, the contact of an amino acid on the surface of one polypeptide (e.g., an antigen) with another polypeptide (e.g., a bispecific antibody). Contact can also include contact with cells, for example, by placing an antibody in direct physical association with a cell.

[0072] Control: A reference standard. In some respects, a control is a negative control, such as a sample obtained from a healthy patient who has never been infected with coronavirus. In other respects, a control is a positive control, such as a tissue sample obtained from a patient diagnosed with coronavirus. In still other respects, a control is a historical control or a standard reference value or numerical range (e.g., a control sample previously tested, such as a patient population with a known prognosis or outcome, or a sample group representing baseline or normal values).

[0073] The difference between the test sample and the control can be an increase or conversely a decrease. The difference can be qualitative or quantitative, such as a statistically significant difference. In some instances, the difference is an increase or decrease of at least about 5% relative to the control, for example, 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%.

[0074] Coronaviruses: A family of positive-sense single-stranded RNA viruses known to cause severe respiratory illness. Currently, viruses infecting humans are known to belong to the genera *Alphacoronavirus* and *Betacoronavirus*. Furthermore, the genera *Gammacoronavirus* and *Deltacoronavirus* are considered potential future infectors.

[0075] Non-limiting examples of β-coronaviruses include SARS-CoV-2, Middle East Respiratory Syndrome Coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV), human coronavirus HKU1 (HKU1-CoV), human coronavirus OC43 (OC43-CoV), mouse hepatitis virus (MHV-CoV), bat SARS-like coronavirus WIV1 (WIV1-CoV), and human coronavirus HKU9 (HKU9-CoV). Non-limiting examples of α-coronaviruses 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 δ-coronavirus is porcine δ-coronavirus (SDCV).

[0076] The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. Coronavirus particles include a viral envelope containing a type I fusion glycoprotein called the spike (S) protein. Most coronaviruses share a genome organization with the replicase gene.

[0077] Degenerate variants: In the context of this disclosure, a “degenerate variant” refers to a polynucleotide encoding a polypeptide (e.g., an antibody heavy or light chain) that includes sequences degenerate due to the genetic code. There are 20 naturally occurring amino acids, most of which are designated by more than one codon. Therefore, all degenerate nucleotide sequences encoding peptides are included, provided that the amino acid sequence of the peptide encoded by the nucleotide sequence remains unchanged.

[0078] Detectable biomarkers: Detectable molecules (also called markers) are directly or indirectly conjugated to a second molecule (e.g., a bispecific antibody) to facilitate the detection of the second molecule. For example, detectable biomarkers can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques (e.g., CT scans, MRI, ultrasound, fiber optic examination, and laparoscopy). Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked conjugates, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). Methods of using detectable biomarkers and guidelines for selecting suitable detectable biomarkers for various purposes are provided, for example, in Green and Sambrook (…). Molecular Cloning: A Laboratory Manual , 4 th ed., New York: ColdSpring Harbor Laboratory Press, 2012) and Ausubel et al. (Eds.) ( Current Protocols in Molecular Biology This is discussed in *New York: John Wiley and Sons, including the supplement, 2017*.

[0079] Detection: Identifying the presence, occurrence, or fact of something. Effective amount: The amount of a substance sufficient to achieve the desired effect in a subject administered the substance. For example, this could be the amount necessary to suppress coronavirus infection (such as SARS-CoV-2 infection) or to measurably alter the external symptoms of such infection.

[0080] In one instance, the desired response is to suppress, reduce, or prevent SARS-CoV-2 infection. Effectiveness of the method does not necessarily require complete elimination, reduction, or prevention of SARS-CoV-2 infection. For example, administration of an effective amount of immunogen can induce an immune response to reduce SARS-CoV-2 infection (e.g., by cell infection or by the number or percentage of subjects infected with SARS-CoV-2) by a desired amount, such as 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) compared to a suitable control. The antibodies disclosed herein can also suppress other coronavirus infections.

[0081] In some respects, administration of an effective amount of publicly available DVD-immunoglobulin that binds to the coronavirus spike protein can reduce or inhibit the desired amount of infection (e.g., by cellular infection, or by the number or percentage of subjects infected with the coronavirus, or by an increase in the survival time of infected subjects, or a reduction in infection-related symptoms), for example, by a reduction or inhibition of 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) compared to a suitable control.

[0082] The effective amount of DVD-immunoglobulin, which specifically binds to the coronavirus spike protein, administered to subjects to suppress infection can vary depending on a number of factors relevant to the subject, such as the subject's overall health and / or weight. The effective amount can be determined by altering the dose and measuring the resulting response, for example, a decrease in pathogen titer. The effective amount can also be determined by a variety of in vitro, in vivo, or in situ immunoassays.

[0083] An effective dose encompasses a fraction of the dose that, in combination with previous or subsequent administrations, contributes to achieving an effective response. For example, an effective dose of the reagent may be administered as a single dose or multiple doses (e.g., daily) over a course of treatment lasting several days or weeks. However, the effective dose can vary depending on the subject being treated, the severity and type of the condition being treated, and the method of administration. The reagent may be packaged in unit dose form in the amount of the effective dose or multiples thereof, for example, in vials with sterile components (e.g., with a puncture cap) or syringes.

[0084] Effector molecules: Molecules designed to have or produce a desired effect (e.g., a desired effect on cells targeted by the effector molecule); or detectable markers. Effector molecules can include, for example, peptides and small molecules. Some effector molecules may have or produce more than one desired effect.

[0085] Epitopes: Antigenic determinants. These are specific chemical groups or peptide sequences on a molecule that are antigenic, causing them to elicit a specific immune response. For example, an epitope is an antigenic region that triggers a B cell and / or T cell response. Antibodies can bind to specific antigenic epitopes (such as epitopes on the coronavirus spike protein).

[0086] Expression: The transcription or translation of a nucleic acid sequence. For example, when DNA encoding a nucleic acid sequence (e.g., a gene) is transcribed into RNA or RNA fragments, that sequence can be expressed; in some instances, these RNAs or RNA fragments are processed into mRNA. The nucleic acid sequence encoding a nucleic acid sequence (e.g., a gene) can also be expressed when mRNA is translated into an amino acid sequence (e.g., a protein or protein fragment). In a specific instance, a heterologous gene is expressed when it is transcribed into RNA. In another instance, the heterologous gene is expressed when its RNA is translated into an amino acid sequence. Regulation of expression can include control over transcription, translation, RNA transport and processing, degradation of intermediate molecules (e.g., mRNA), or activation, inactivation, compartmentalization, or degradation of specific protein molecules after their production.

[0087] Bivariate domain immunoglobulins: Bispecific antibodies comprising two heavy chain variable domains and two light chain variable domains. However, unlike IgG, both the heavy and light chains of DVD-immunoglobulin molecules contain additional variable domains (VDs), which are linked to the V domains of existing monoclonal antibodies (mAbs) via adapter sequences. H and V L The N-terminus. Therefore, when the heavy and light chains combine, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites, see Jakob et al., Mabs 5: 358-363, 2013, which is incorporated herein by reference; schematic diagrams and space-filling diagrams can be found in Jakob et al. Figure 1 DVD-immunoglobulin molecules have the ability to bind to two different antigens on each DFab simultaneously.

[0088] Expression control sequence: A nucleic acid sequence that regulates the expression of a heterologous nucleic acid sequence to which it is operatively linked. The expression control sequence is operatively linked to the nucleic acid sequence in controlling and regulating transcription and (if applicable) translation of the nucleic acid sequence. Therefore, the expression control sequence may include a suitable promoter, enhancer, transcription terminator, start codon (ATG) preceding a protein-coding gene, splicing signals for introns, and maintenance of the correct reading frame of the gene to allow correct translation of the mRNA and stop codon. The term "control sequence" is intended to include at least the components whose presence can affect expression, and may also include additional components whose presence is advantageous, such as leader sequences and fusion coupler sequences. The expression control sequence may include a promoter.

[0089] Expression vectors: Vectors comprising recombinant polynucleotides containing an expression control sequence operatively linked to a nucleotide sequence to be expressed. Expression vectors include sufficient cis-acting elements for expression; other elements for expression may be provided by a host cell or an in vitro expression system. Non-limiting examples of expression vectors include clomiphenes, plasmids (e.g., naked plasmids or plasmids contained in liposomes), and viruses (e.g., lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses) incorporating recombinant polynucleotides.

[0090] Polynucleotides can be inserted into expression vectors containing promoter sequences that facilitate efficient transcription of the inserted gene sequence by the host. Expression vectors typically contain an origin of replication, a promoter, and specific nucleic acid sequences that allow for phenotypic selection of transformed cells.

[0091] Fc region: The antibody constant region excluding the first heavy chain constant domain. The Fc region typically 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. The Fc region may also include part or all of the flexible hinge located at the N-terminus of these domains. For IgA and IgM, the Fc region may or may not include a tailpiece and may or may not bind to the J chain. For IgG, the Fc region is generally understood to include immunoglobulin domains Cγ2 and Cγ3, and optionally the lower portion of the hinge between Cγ1 and Cγ2. Although the boundaries of the Fc region may vary, the human IgG heavy chain Fc region is generally defined as including residues from C226 or P230 to the C-terminus of the Fc group, numbered according to EU designations. For IgA, the Fc region includes immunoglobulin domains Cα2 and Cα3, and optionally the lower portion of the hinge between Cα1 and Cα2.

[0092] Heterologous: Derived from a different genetic source. A nucleic acid molecule heterologous to a cell originates from a genetic source other than the cell in which it is expressed. In a specific, non-limiting example, a heterologous nucleic acid molecule encoding a protein (e.g., DVD-immunoglobulin) is expressed in a cell (e.g., a mammalian cell). Methods for introducing heterologous nucleic acid molecules into cells or organisms are well-known, such as nucleic acid transformation, including electroporation, liposome transfection, gene gun acceleration, and homologous recombination.

[0093] Host cell: The cell in which the vector can reproduce and express its DNA. The cell can be a prokaryotic or eukaryotic cell, such as a bacterial cell or a mammalian cell, such as *E. coli* or a human cell. The term also includes any progeny of the host cell. It should be understood that all progeny may not be identical to the parent cell due to mutations that can occur during replication. However, when using the term "host cell," these progeny are included.

[0094] IgA: A polypeptide belonging to a class of antibodies essentially encoded by the well-known immunoglobulin α gene. In humans, this class, or isotype, includes IgA1 and IgA2. IgA antibodies can exist as monomers, polymers primarily in the form of dimers (called pIgA), and secreted IgA. The constant chain of wild-type IgA contains an 18-amino acid extension at its C-terminus, called the tail fragment (tp). Polymeric IgA is secreted by plasma cells and has a 15-kDa peptide (called the J chain) linking two IgA monomers via conserved cysteine ​​residues in the tail fragment.

[0095] IgG: A class of antibodies or isotypes of polypeptides that are essentially encoded by the well-known immunoglobulin γ gene. In humans, this class includes IgG1, IgG2, IgG3, and IgG4.

[0096] Immune complexes are formed when antibodies, antigen-binding fragments (e.g., scFv), or bispecific antibodies (e.g., DVD-immunoglobulin) bind to soluble antigens. The formation of immune complexes can be detected by conventional methods such as immunohistochemistry, immunoprecipitation, flow cytometry, immunofluorescence microscopy, ELISA, Western blotting (e.g., Western blotting), magnetic resonance imaging, CT scans, radiographic imaging, and affinity chromatography.

[0097] Suppressing or treating disease: Suppressing the full development of a disease or condition in subjects at risk of illness, such as coronavirus infection. "Treatment" refers to a therapeutic intervention that reduces the signs or symptoms of a disease or pathological condition after it has begun to develop. The term "reduction," in relation to a disease or condition, refers to any observable beneficial effect of treatment. Suppressing disease can include preventing or reducing the risk of disease, such as preventing or reducing the risk of viral infection. Beneficial effects can be demonstrated, for example, by delayed onset of clinical symptoms of the disease in susceptible subjects, reduction in the severity of some or all of the clinical symptoms of the disease, slowed disease progression, reduced viral load, improvement in the overall health or well-being of the subject, or by other parameters specific to the disease. "Preventative" treatment is a treatment administered to subjects who do not exhibit signs of disease or only exhibit early signs, with the aim of reducing the risk of illness.

[0098] The term "reduction" is a relative term, meaning that the agent reduces a disease or condition if the disease or condition is quantitatively reduced after application, or if the disease or condition is reduced after application of the agent compared to a reference agent. Similarly, the term "prevention" does not necessarily mean that the agent completely eliminates a disease or condition, but only eliminates at least one characteristic of the disease or condition. Therefore, a composition that reduces or prevents infection can (but does not necessarily completely) eliminate such infection, provided that the infection is measurably reduced, for example, by at least about 50% in the absence of the agent or compared to a reference agent, such as by at least about 70%, or about 80%, or even about 90%.

[0099] Isolated: A biological component (e.g., nucleic acid, peptide, protein, or protein complex, such as an antibody) that has been substantially isolated, produced, or purified from other biological components (i.e., other chromosomal and extrachromosomal DNA and RNA, and proteins) naturally present in the somatic cells of an organism. Therefore, isolated nucleic acids, peptides, and proteins include nucleic acids and proteins purified by standard purification methods. The term also includes nucleic acids, peptides, and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acids. Isolated nucleic acids, peptides, or proteins (e.g., antibodies) may 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.

[0100] Kabat position: The position of a residue in the amino acid sequence, following the Kabat sequence. et al. ( Sequences of Proteins of Immunological Interest , 5 thThe numbering convention established by the Department of Health and Human Services, Public Health Service, National Institutes of Health, Bethesda, NIHP Publication No. 91-3242, 1991.

[0101] Linkers: Bifunctional molecules that can be used to link two molecules into a single continuous molecule, for example, linking a detectable marker to an antibody, or linking two antibodies together to form a bispecific antibody. Non-limiting examples of peptide linkers include glycine-serine linkers.

[0102] The terms "conjugation," "linking," "bonding," or "connection" can refer to the formation of a single, continuous molecule from two molecules; for example, linking two polypeptides into a single continuous polypeptide, or covalently attaching an effector molecule, a detectable biomarker, a radionuclide, or other molecule to a polypeptide, such as DVD-immunoglobulin. This connection can be achieved through chemical or recombinant methods. "Chemical methods" refer to the reaction between an antibody moiety and an effector molecule, resulting in a covalent bond between the two molecules to form a single molecule.

[0103] Nucleic acid (molecule or sequence): deoxyribonucleotides or ribonucleotide polymers or combinations thereof, including but not limited to cDNA, mRNA, genomic DNA, and synthetic (e.g., chemically synthesized) DNA or RNA. Nucleic acids can be double-stranded (ds) or single-stranded (ss). In the case of single-stranded nucleic acids, the nucleic acid can be a sense strand or an antisense strand. Nucleic acids can include natural nucleotides (e.g., A, T / U, C, and G) and can include analogues of natural nucleotides, such as labeled nucleotides.

[0104] "cDNA" refers to single-stranded or double-stranded DNA that is complementary to or identical to mRNA.

[0105] "Encoding" refers to the inherent property of a specific nucleotide sequence in a polynucleotide (such as a gene, cDNA, or mRNA) as a template for the synthesis of other polymers and macromolecules in biological processes. It has a defined nucleotide sequence (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the resulting biological characteristics. Therefore, if the transcription and translation of mRNA produced by a gene produces a protein in a cell or other biological system, then that gene encodes a protein. Both the coding strand of a gene or cDNA (whose nucleotide sequence is identical to the mRNA sequence and is usually provided in the sequence listing) and the non-coding strand (used as a transcription template) can be considered to encode a protein or other product of that gene or cDNA. Unless otherwise stated, "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and encode the same amino acid sequence. Nucleotide sequences encoding proteins and RNA may include introns.

[0106] Operable ligation: The first and second nucleic acid sequences are operably ligated when they are functionally related. For example, if a promoter (e.g., the CMV promoter) affects the transcription or expression of a coding sequence, then the promoter and coding sequence are operably ligated. Typically, operably ligated DNA sequences are contiguous and located within the same reading frame when it is necessary to link two protein-coding regions.

[0107] Pharmaceutically acceptable carrier: The pharmaceutically acceptable carrier used is conventional. Remington: The Science and Practice of Pharmacy, 22 nd ed. , London, UK: Pharmaceutical Press, 2013, describes compositions and formulations suitable for drug delivery of the disclosed reagents.

[0108] The nature of the carrier can depend on the specific method of administration employed. For example, parenteral preparations typically include injections containing pharmaceutically and physiologically acceptable liquids such as water, physiological saline, balanced salt solutions, glucose solutions, glycerol, etc., as solvents. For solid compositions (e.g., in powder, pill, tablet, or capsule form), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to biologically neutral carriers, the pharmaceutical composition to be administered may also contain small amounts of non-toxic excipients, such as wetting agents or emulsifiers, added preservatives (e.g., non-natural preservatives), and pH buffers, such as sodium acetate or sorbitan monolaurate. In certain instances, pharmaceutically acceptable carriers are sterile and suitable for parenteral administration to subjects, for example, by injection. In some aspects, the active agent and pharmaceutically acceptable carrier are provided in unit dosage forms (e.g., pills) or in selected amounts in vials. Unit dosage forms may include one dose or multiple doses (e.g., in a vial from which a dose of reagent can be selectively dispensed).

[0109] Polypeptide: A polymer in which monomers are linked together by an amide bond of amino acid residues. When the amino acid is an α-amino acid, either an L-optical isomer or a D-optical isomer may be used, with the L-isomer being preferred. As used herein, the terms “polypeptide” or “protein” are intended to cover any amino acid sequence and include modified sequences, such as glycoproteins. Polypeptides include naturally occurring proteins as well as proteins produced through recombinant or synthetic processes. Polypeptides have an amino terminus (N-terminus) and a carboxyl terminus. In some respects, a polypeptide is a disclosed antibody or a fragment thereof.

[0110] Purified: The term "purified" does not require absolute purity; rather, it is intended as a relative term. Thus, for example, a purified peptide formulation is one in which peptides or proteins (e.g., antibodies) are more enriched than in their native intracellular environment. In one aspect, the formulation is purified such that proteins or peptides constitute at least 50% of the total peptide or protein content in the formulation.

[0111] Recombination: Recombinant nucleic acids are nucleic acids having a non-naturally occurring sequence or having a sequence artificially combined from two originally separate sequence segments. This artificial combination can be achieved through chemical synthesis, or more commonly, through artificial manipulation of the separated nucleic acid segments, for example, through genetic engineering techniques. Recombinant proteins are proteins having a non-naturally occurring sequence or having a sequence artificially combined from two originally separate sequence segments. In several respects, recombinant proteins are encoded by heterologous (e.g., recombinant) nucleic acids that have been introduced into a host cell (e.g., bacteria or eukaryotic cells). For example, the nucleic acid can be introduced into an expression vector having a signal capable of expressing the protein encoded by the introduced nucleic acid, or the nucleic acid can be integrated into the host cell chromosome.

[0112] SARS-CoV-2, also known as the 2019 novel coronavirus, is a positive-sense single-stranded RNA virus belonging to the β-coronavirus genus and has become a highly lethal cause of severe acute respiratory infection. The viral genome is capped, polyadenylated, and covered with nucleocapsid proteins. The SARS-CoV-2 viral particle includes a viral envelope containing a large spike glycoprotein. Like most coronaviruses, the SARS-CoV-2 genome has a common genomic organization, including replicase genes at the 5' two-thirds of the genome and structural genes at the 3' one-third. The SARS-CoV-2 genome encodes a typical set of structural protein genes in the order 5'-spike (S)-enveloping (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. Severe cases can progress to severe pneumonia, multiple organ failure, and death. The time from exposure to symptom onset is approximately 2 to 14 days.

[0113] SARS-CoV-2 infection can be detected using standard methods for detecting viral infection, including, but not limited to, assessing patient symptoms and background, and genetic testing such as reverse transcription polymerase chain reaction (rRT-PCR). This test can be performed on patient samples (e.g., respiratory or blood samples).

[0114] Spike (S) protein (coronavirus): A class I fusion glycoprotein. For SARS-CoV, it is initially synthesized as a precursor protein of approximately 1256 amino acids, and for SARS-CoV-2, as a precursor protein of approximately 1273 amino acids. The individual precursor S polypeptides form a homotrimer and undergo glycosylation in the Golgi apparatus, followed by processing to remove the signal peptide. The cytokines are then cleaved by cellular proteases between approximately positions 679 / 680 of the SARS-CoV protein and approximately positions 685 / 686 of the SARS-CoV-2 protein to generate separate S1 and S2 polypeptide chains. These chains remain associated in the homotrimer as the S1 / S2 progenitors, thus forming a heterodimeric trimer. The S1 subunit is located at the distal end of the viral membrane and contains a receptor-binding domain (RBD), which is believed to mediate viral attachment to its host receptor. The S2 subunit contains fusion protein mechanisms, such as fusion peptides, two heptapeptide repeat sequences (HR1 and HR2), and the central helix, transmembrane domain, and cytoplasmic tail domain typical of fusion glycoproteins.

[0115] Exemplary sequences of the SARS-CoV S protein are provided in GENBANK® GI: 30795145 and were available on February 1, 2022. Exemplary sequences of the HKU1-CoV S protein are provided in GENBANK® GI: 123867264 and were available on February 1, 2022. Exemplary sequences of the OC43-CoV S protein are provided in GENBANK® GI: 744516696 and were available on February 1, 2022. Exemplary sequences of the NL63-CoV S protein are provided in GENBANK® GI: 71153773 and were available on February 1, 2022. Exemplary sequences of the 229E-CoV S protein are provided in GENBANK® GI: 1060650120 and were available on February 1, 2022. The S protein comprises both S1 and S2 domains.

[0116] The numbering used in the publicly available SARS-CoV-2 S protein and its fragments is relative to the SARS-CoV-2 S protein sequence, which is deposited with NCBI reference number YP_009724390.1 and became available on February 1, 2022. It is incorporated herein by reference in its entirety.

[0117] Sequence identity: The similarity between two or more nucleic acid sequences or two or more amino acid sequences, expressed as a percentage of identity. Sequence identity can be measured as a percentage; the higher the percentage, the higher the sequence identity. The V of an antibody that specifically binds to a target antigen... L or V H Homologous and variant forms are typically characterized by having at least about 75% sequence identity, calculated based on a full-length alignment with the amino acid sequence of interest, such as at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity.

[0118] Any suitable method can be used to align sequences for comparison. Non-limiting examples of procedures and alignment algorithms are described in: Smith and Waterman. Adv. Applied Math. 2(4):482-489, 1981; Needleman andWunsch, J. Mol. Biol. 48(3):443-453, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85(8):2444-2448, 1988; Higgins and Sharp, Gene, 73(1):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, details sequence alignment methods and homology calculations. NCBI Basic Local Alignment Search Tool (BLAST) (A Altschul et al. , J. Mol. Biol. (215(3):403-410, 1990) is available from multiple sources, including the National Center for Biotechnology Information (NCBI) and the Internet, for use in conjunction 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 is available on the NCBI website.

[0119] Typically, when two sequences are compared, the number of matches can be determined by counting the number of positions in the two sequences where the same nucleotide or amino acid residues are present. The percentage of sequence identity between two sequences is determined by dividing the number of matches by the length of the sequence listed in the identified sequence, or by the hinge length (e.g., 100 consecutive nucleotide or amino acid residues from the sequence listed in the identified sequence), and then multiplying the result by 100.

[0120] Specific binding: When referring to antibodies, antigen-binding fragments, or bispecific antibodies, this refers to a binding reaction that identifies the presence of a target protein in the presence of heterogeneous protein groups and other biological agents. Therefore, under specified conditions, antibodies preferentially bind to specific target proteins, peptides, or polysaccharides (e.g., antigens present on the surface of pathogens, such as the coronavirus spike protein), without binding significantly to other proteins present in the sample or subject. Regarding spike proteins, epitopes may be present on the spike proteins of more than one type of coronavirus, allowing antibodies to bind to the spike proteins of more than one type of virus, but not to other proteins (e.g., proteins from other viruses or other proteins of coronaviruses (non-spike proteins)). Specific binding can be determined using standard methods. For a description of the forms and conditions of immunoassays that can be used to determine specific immunoreactivity, see Harlow & Lane. Antibodies, A Laboratory Manual , 2 nded., Cold Spring Harbor Publications, New York (2013).

[0121] Regarding antibody-antigen complexes, the K-axis of the specific binding of antigens and antibodies... D Less than about 10 -7 moles, for example less than about 10 -8 mole, 10 -9 moles, even less than about 10 -10 Moore. K D This refers to the dissociation constant of a given interaction, such as peptide-ligand interactions or antibody-antigen interactions. For example, for a bimolecular interaction between an antibody or antigen-binding fragment and an antigen, it is the concentration of each component in the bimolecular interaction divided by the concentration of the complex.

[0122] Antibodies that specifically bind to epitopes (e.g., S domain, RBD domain, or NTD domain) on the coronavirus spike protein can bind to molecules / reagents that include that domain, including viruses, spike protein-attached substrates, or proteins in biological specimens. Of course, it is important to recognize that some degree of non-specific interaction can occur between antibodies and non-targets. Typically, specific binding results in association between the antibody and the spike protein that is much stronger than association between the antibody and other different coronavirus proteins (e.g., E protein, M protein, or N protein) or non-coronavirus proteins. Specific binding typically results in a greater than 2-fold increase (per unit time) in the amount of antibody binding to a protein containing that epitope or to cells or tissues expressing the target epitope compared to proteins or cells or tissues lacking that epitope. Under these conditions, specific binding to the protein requires selection of antibodies that are specific to that particular protein. Various immunoassays are suitable for selecting antibodies or other ligands that have a specific immune response to a specific protein. For example, solid-phase enzyme ELISA is commonly used to select monoclonal antibodies that have a specific immune response to a protein.

[0123] Subjects: Live multicellular vertebrate organisms, including humans and non-human mammals such as non-human primates, pigs, camels, bats, sheep, cows, dogs, cats, rodents, etc. In this example, the subject is a human. In a specific instance, the subject is a human. In another instance, subjects requiring suppression of SARS-CoV-2 infection are selected. For example, subjects who are not infected but at risk of SARS-CoV-2 infection, or who are already infected and require treatment.

[0124] Transformed: Transformed cells are cells into which nucleic acid molecules have been introduced using molecular biology techniques. As used herein, the terms transformed (e.g., transformation, transfection, transduction, etc.) encompass all techniques that can introduce nucleic acid molecules into cells, including viral vector transduction, plasmid vector transformation, and accelerated DNA introduction via electroporation, liposome transfection, and gene guns.

[0125] Vector: An entity containing a nucleic acid molecule (e.g., a DNA or RNA molecule) carrying a promoter operatively linked to and capable of expressing a coding sequence of a protein of interest. Non-limiting examples include naked DNA or packaged (lipid and / or protein) DNA, naked RNA or packaged RNA, a subcomponent of a virus or bacterium or other microorganism that may not be capable of replication, or a virus or bacterium or other microorganism that may be capable of replication. Vectors are sometimes referred to as constructs. A recombinant DNA vector is a vector containing recombinant DNA. A vector may include nucleic acid sequences that allow it to replicate in a host cell, such as an origin of replication. A vector may also include one or more optional marker genes and other genetic elements. A viral vector is a recombinant nucleic acid vector 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 publicly disclosed DVD-immunoglobulin or antigen-binding fragment that specifically binds to and neutralizes the coronavirus spike protein. In some aspects, a viral vector is an adeno-associated virus (AAV) vector.

[0126] Under conditions sufficient to…: a phrase used to describe any environment that allows the desired activity.

[0127] II. Description of Several Aspects This document discloses bispecific antibodies, including DVD-immunoglobulin, comprising two separate monoclonal antibodies or antigen-binding fragments that specifically bind to the coronavirus spike protein. The monoclonal antibody and antigen-binding fragment included in this bispecific antibody specifically bind to the coronavirus spike protein and neutralize SARS-CoV-2 virus as well as at least one other beta-coronavirus or alpha-coronavirus. These antibodies and antigen-binding fragments can be entirely human. The disclosed DVD-immunoglobulin can neutralize coronaviruses, such as, but not limited to, SARS-CoV-2. In some aspects, the disclosed DVD-immunoglobulin can inhibit coronavirus infection in vivo and can be administered before or after infection with a coronavirus (e.g., but not limited to, SARS-CoV-2). Furthermore, this document discloses compositions comprising DVD-immunoglobulin and a pharmaceutically acceptable vector. Nucleic acids encoding DVD-immunoglobulin, and expression vectors (e.g., adeno-associated virus (AAV) viral vectors) containing these nucleic acids are also provided. These DVD-immunoglobulins, nucleic acid molecules, host cells, and compositions can be used for research, diagnostic, therapeutic, and preventative purposes. For example, publicly available DVD-immunoglobulin can be used to diagnose subjects with coronavirus infection, or it can be administered to suppress coronavirus infection in subjects.

[0128] Bispecific antibodies include monoclonal antibodies that specifically bind to the coronavirus spike protein and their antigenic binding. Combined fragments The bispecific antibodies discussed herein refer to molecules comprising two separate monoclonal antibodies or antigen-binding fragments thereof that specifically bind to the coronavirus spike protein. These monoclonal antibodies and antigen-binding fragments include variable domains (or antigen-binding fragments thereof) of the heavy and / or light chains, containing CDR1, CDR2, and / or CDR3 (unless the context otherwise requires) according to the IMGT numbering scheme. The CDR positions in the monoclonal antibodies or antigen-binding fragments included in a disclosed bispecific antibody can be determined using various CDR numbering schemes, such as Kabat, Chothia, or the IMGT numbering scheme. The amino acid sequences of the heavy and light chains of monoclonal antibodies and their CDRs according to the IMGT numbering scheme are merely examples. In some aspects, Kabat localization is used to determine the CDRs.

[0129] In some aspects, a bispecific antibody comprises at least one monoclonal antibody (or an antigen-binding fragment thereof) comprising the heavy and light chain CDRs of the antibodies COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27 described below. In other aspects, a bispecific antibody comprises two monoclonal antibodies (or antigen-binding fragments thereof) comprising the heavy and light chain CDRs of one of the antibodies COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27 described below. The bispecific antibody may be DVD-immunoglobulin.

[0130] In some aspects, the bispecific antibody includes heavy and light chain variable regions of one or both of the following antibodies: COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27. In other aspects, the bispecific antibody includes heavy and light chain CDRs of one or both of the following antibodies: COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27. In still other aspects, the bispecific antibody includes heavy and light chain variable regions of two of the following antibodies: COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27. In other respects, the bispecific antibody comprises the heavy and light chain CDRs of two of the following antibodies: COV44-62, COV30-14, COV72-37, COV89-22, COV44-79, and COV91-27. The bispecific antibody may be DVD-immunoglobulin.

[0131] In some aspects, the bispecific antibody includes at least one monoclonal antibody that binds to the S domain of the coronavirus spike protein. In other aspects, the bispecific antibody includes at least one monoclonal antibody that binds to a stem-helix in the S2 domain of the spike protein, such as LQPELDSFKEELDKYFKNHTS (SEQ ID NO: 118), or LDSFKEELDKYF (SEQ ID NO: 119). In a further aspect, the bispecific antibody includes at least one monoclonal antibody that binds to a fusion peptide epitope in the S2 domain of the spike protein, such as SFIEDLLFNKVTLA (SEQ ID NO: 120).

[0132] In some aspects, the bispecific antibody includes at least one monoclonal antibody that specifically binds to the spike protein of at least three beta coronaviruses selected from the group consisting of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, and OC43. In other aspects, the bispecific antibody includes at least one monoclonal antibody that binds to both beta and alpha coronaviruses. In still other aspects, the bispecific antibody includes at least one monoclonal antibody that binds to the spike protein of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63, and 229E.

[0133] The following monoclonal antibodies or their antigen-binding fragments may be included in publicly available bispecific antibodies (e.g., DVD-immunoglobulin).

[0134] a. Monoclonal antibody COV44-62 In some instances, the antibody or antigen-binding fragment is based on or derived from the COV44-62 antibody and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some instances, the antibody or antigen-binding fragment contains V... H and V L These antibodies contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the COV44-62 antibody, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2. In some respects, the antibody or antigen-binding fragment neutralizes beta-coronaviruses and alpha-coronaviruses. In other respects, the antibody or antigen-binding fragment specifically binds to the spike protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63, and 229E.

[0135] In some respects, antibody or antigen-binding fragments contain V H It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) the same amino acid sequence as shown in SEQ ID NO: 1, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In further respects, the antibody or antigen-binding fragment contains V... L It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 5, and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey independently contain at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 1 and 5, respectively, and specifically bind to and neutralize the coronavirus spike protein. The coronavirus can be SARS-CoV-2.

[0136] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 2, 3, and 4, respectively. H and / or containing V such as LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 6, 7 and 8 respectively. L It binds specifically to the coronavirus spike protein and neutralizes the coronavirus. The coronavirus can be SARS-CoV-2.

[0137] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 2, 3, and 4, respectively. H V, comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 6, 7, and 8 respectively. L V H Contains an amino acid sequence that is at least 90% identical (e.g., 95%, 96%, 97%, 98%, or 99% identical) to SEQ ID NO: 1, and wherein V L It contains an amino acid sequence that is at least 90% identical (e.g., 95%, 96%, 97%, 98%, or 99%) to SEQ ID NO: 5, and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations caused by sequence identification are outside of CDR. The coronavirus can be SARS-CoV-2.

[0138] In some respects, antibody or antigen-binding fragments contain V H It contains the amino acid sequence shown in SEQ ID NO: 1 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In other words, the antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 5 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, the antibody or antigen-binding fragment contains V H and V LThey contain amino acid sequences as shown in SEQ ID NO: 1 and 5, respectively, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2.

[0139] In some respects, publicly available antibodies inhibit viral entry and / or viral replication. These antibodies and antigen-binding fragments can be included in publicly available bispecific antibodies (such as DVD-immunoglobulin).

[0140] b. Monoclonal antibody COV30-14 In some respects, the antibody or antigen-binding fragment is based on or derived from the COV30-14 antibody and specifically binds to the coronavirus spike protein, neutralizing the coronavirus.

[0141] In some instances, the antibody or antigen-binding fragment contains V H and V L These antibodies contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the COV30-14 antibody, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen-binding fragment specifically binds to the spike protein of at least three β-coronaviruses 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 both β-coronaviruses and α-coronaviruses. In some aspects, the antibody or antigen-binding fragment specifically binds to the stem-helix in the S2 domain of the spike protein.

[0142] In some respects, antibody or antigen-binding fragments contain V H It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the same amino acid sequence as the one shown in SEQ ID NO: 9, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In further respects, the antibody or antigen-binding fragment contains V... L It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 13, and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey independently contain at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequences shown in SEQ ID NO:9 and 13, respectively, and specifically bind to the coronavirus spike protein and neutralize the coronavirus. The coronavirus can be SARS-CoV-2.

[0143] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, 11, and 12, respectively. H and / or containing V of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 14, 15 and 16 respectively. L It binds specifically to the coronavirus spike protein and neutralizes the coronavirus. The coronavirus can be SARS-CoV-2.

[0144] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 10, 11, and 12, respectively. H V, comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 14, 15, and 16 respectively. L V H Contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 9 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 9), and wherein V L It contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 13 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations caused by sequence identity are outside the CDR. The coronavirus can be SARS-CoV-2.

[0145] In some respects, antibody or antigen-binding fragments contain V H It contains the amino acid sequence shown in SEQ ID NO: 9 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In other words, the antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 13 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, the antibody or antigen-binding fragment contains V H and V LThey contain amino acid sequences as shown in SEQ ID NO: 9 and 13, respectively, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2.

[0146] In some respects, publicly available antibodies inhibit viral entry and / or viral replication. These antibodies and antigen-binding fragments can be included in publicly available bispecific antibodies (such as DVD-immunoglobulin).

[0147] c. Monoclonal antibody COV72-37 In some respects, the antibody or antigen-binding fragment is based on or derived from the COV72-37 antibody and specifically binds to the coronavirus spike protein, neutralizing the coronavirus.

[0148] In some instances, the antibody or antigen-binding fragment contains V H and V L These contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 of the COV72-37 antibody (e.g., according to IMGT, Kabat, or Chothia), and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen-binding fragment specifically binds to the spike protein of at least three β-coronaviruses 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 both β-coronaviruses and α-coronaviruses. In some aspects, the antibody or antigen-binding fragment specifically binds to the stem-helix in the S2 domain of the spike protein.

[0149] In some respects, antibody or antigen-binding fragments contain V H It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 17, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In further respects, the antibody or antigen-binding fragment contains V... L It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 21, and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey independently contain at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequences shown in SEQ ID NO:17 and 21, respectively, and specifically bind to the coronavirus spike protein and neutralize the coronavirus. The coronavirus can be SARS-CoV-2.

[0150] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively. H The structural domain, and / or containing V of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 22, 23 and 24 respectively. L It binds specifically to the coronavirus spike protein and neutralizes the coronavirus. The coronavirus can be SARS-CoV-2.

[0151] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 18, 19, and 20, respectively. H V, comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 22, 23, and 24 respectively. L V H Contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 17 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 17), and wherein V L It contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 21 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 21), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations caused by sequence identity are outside the CDR. The coronavirus can be SARS-CoV-2.

[0152] In some respects, antibody or antigen-binding fragments contain V H It contains the amino acid sequence shown in SEQ ID NO: 17 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In other words, the antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 21 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, the antibody or antigen-binding fragment contains V H and V LThey contain amino acid sequences as shown in SEQ ID NO: 17 and 21, respectively, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2.

[0153] In some respects, publicly available antibodies inhibit viral entry and / or viral replication. These antibodies and antigen-binding fragments can be included in publicly available bispecific antibodies (such as DVD-immunoglobulin).

[0154] d. Monoclonal antibody COV89-22 In some respects, the antibody or antigen-binding fragment is based on or derived from the COV89-22 antibody and specifically binds to the coronavirus spike protein, neutralizing the coronavirus.

[0155] In some instances, the antibody or antigen-binding fragment contains V H and V L These antibodies contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the COV89-22 antibody, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2. In some aspects, the antibody or antigen-binding fragment specifically binds to the spike protein of at least three β-coronaviruses 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 both β-coronaviruses and α-coronaviruses. In some aspects, the antibody or antigen-binding fragment specifically binds to the stem-helix in the S2 domain of the spike protein.

[0156] In some respects, antibody or antigen-binding fragments contain V H It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the same amino acid sequence as the one shown in SEQ ID NO: 25, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In further respects, the antibody or antigen-binding fragment contains V... L It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 29, and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey independently contain at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the same amino acid sequence as those shown in SEQ ID NO: 25 and 29, and specifically bind to the coronavirus spike protein and neutralize the coronavirus. The coronavirus can be SARS-CoV-2.

[0157] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 26, 27, and 28, respectively. H and / or containing Vi LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 30, 31, and 32, respectively. L It binds specifically to the coronavirus spike protein and neutralizes the coronavirus. The coronavirus can be SARS-CoV-2.

[0158] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 26, 27, and 28, respectively. H V, comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 30, 31, and 32 respectively. L V H Contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 25 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 25), and wherein V L It contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 29 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 29), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations caused by sequence identity are outside the CDR. The coronavirus can be SARS-CoV-2.

[0159] In some respects, antibody or antigen-binding fragments contain V H It contains the amino acid sequence shown in SEQ ID NO: 25 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In further respects, the antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 29 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, the antibody or antigen-binding fragment contains V H and V LThey contain amino acid sequences as shown in SEQ ID NO: 25 and 29, respectively, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2.

[0160] In some respects, publicly available antibodies inhibit viral entry and / or viral replication. These antibodies and antigen-binding fragments can be included in publicly available bispecific antibodies (such as DVD-immunoglobulin).

[0161] e. Monoclonal antibody COV44-79 In some respects, the antibody or antigen-binding fragment is based on or derived from the COV44-79 antibody and specifically binds to the coronavirus spike protein, neutralizing the coronavirus.

[0162] In some instances, the antibody or antigen-binding fragment contains V H and V L These antibodies contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the COV44-79 antibody, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2. In some respects, the antibody or antigen-binding fragment neutralizes beta-coronaviruses and alpha-coronaviruses. In other respects, the antibody or antigen-binding fragment specifically binds to the spike protein from SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63, and 229E.

[0163] In some respects, antibody or antigen-binding fragments contain V H It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) the same amino acid sequence as shown in SEQ ID NO: 33, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In further respects, the antibody or antigen-binding fragment contains V... L It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 37, and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey independently contain at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the same amino acid sequence as those shown in SEQ ID NO: 33 and 37, and specifically bind to the coronavirus spike protein and neutralize the coronavirus. The coronavirus can be SARS-CoV-2.

[0164] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 34, 35, and 36, respectively. H and / or containing VC of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 38, 39 and 40 respectively. L It binds specifically to the coronavirus spike protein and neutralizes the coronavirus. The coronavirus can be SARS-CoV-2.

[0165] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 34, 35, and 36, respectively. H V, comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 38, 39, and 40 respectively. L V H Contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 33 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 33), and wherein V L It contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 37 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 37), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations caused by sequence identity are outside the CDR. The coronavirus can be SARS-CoV-2.

[0166] In some respects, antibody or antigen-binding fragments contain V H It contains the amino acid sequence shown in SEQ ID NO: 33 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In other words, the antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 37 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, the antibody or antigen-binding fragment contains V H and V LThey contain amino acid sequences as shown in SEQ ID NO: 33 and 37, respectively, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2.

[0167] In some respects, publicly available antibodies inhibit viral entry and / or viral replication. These antibodies and antigen-binding fragments can be included in publicly available bispecific antibodies (such as DVD-immunoglobulin).

[0168] f. Monoclonal antibody COV91-27 In some respects, the antibody or antigen-binding fragment is based on or derived from the COV91-27 antibody and specifically binds to the coronavirus spike protein, neutralizing the coronavirus.

[0169] In some instances, the antibody or antigen-binding fragment contains V H and V L These antibodies contain HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3 (e.g., according to IMGT, Kabat, or Chothia) of the COV91-27 antibody, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2. In some respects, the antibody or antigen-binding fragment specifically binds to the spike protein of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63, and 229E. In some respects, the antibody or antigen-binding fragment neutralizes beta coronaviruses and alpha coronaviruses.

[0170] In some respects, antibody or antigen-binding fragments contain V H It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 41, and specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In further respects, the antibody or antigen-binding fragment contains V... L It contains at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the amino acid sequence shown in SEQ ID NO: 45, and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In another aspect, the antibody or antigen-binding fragment contains V... H and V LThey independently contain at least 90% (e.g., at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%) of the same amino acid sequence as those shown in SEQ ID NO: 41 and 45, and specifically bind to the coronavirus spike protein and neutralize the coronavirus. The coronavirus can be SARS-CoV-2.

[0171] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 42, 43, and 44, respectively. H and / or containing VC of LCDR1, LCDR2 and LCDR3 as shown in SEQ ID NO: 46, 47 and 48 respectively. L It binds specifically to the coronavirus spike protein and neutralizes the coronavirus. The coronavirus can be SARS-CoV-2.

[0172] In some respects, the antibody or antigen-binding fragment comprises: V containing HCDR1, HCDR2, and HCDR3 as shown in SEQ ID NO: 42, 43, and 44, respectively. H V, comprising LCDR1, LCDR2, and LCDR3 as shown in SEQ ID NO: 46, 47, and 48 respectively. L V H Contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 41 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 41), and wherein V L It contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 45 (e.g., 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 45), and the antibody or antigen-binding fragment specifically binds to the coronavirus spike protein and neutralizes the coronavirus. In this respect, variations caused by sequence identity are outside the CDR. The coronavirus can be SARS-CoV-2.

[0173] In some respects, antibody or antigen-binding fragments contain V H It contains the amino acid sequence shown in SEQ ID NO: 41 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In other words, the antibody or antigen-binding fragment contains V... L It contains the amino acid sequence shown in SEQ ID NO: 45 and specifically binds to the coronavirus spike protein, neutralizing the coronavirus. In some respects, the antibody or antigen-binding fragment contains V H and V LThey contain amino acid sequences as shown in SEQ ID NO: 41 and 45, respectively, and specifically bind to the coronavirus spike protein, neutralizing the coronavirus. The coronavirus can be SARS-CoV-2.

[0174] In some respects, publicly available antibodies inhibit viral entry and / or viral replication. These antibodies and antigen-binding fragments can be included in publicly available bispecific antibodies (such as DVD-immunoglobulin).

[0175] Further descriptions of antibodies, antigen-binding fragments, and DVD-immunoglobulins Bispecific antibodies can be designed and generated using any suitable method, such as cross-linking two or more antibodies of the same or different types, or antigen-binding fragments (e.g., scFv). Exemplary methods for preparing multispecific antibodies (e.g., bispecific antibodies) include the methods described in PCT Publication No. WO2013 / 163427, which is incorporated herein by reference in its entirety. Non-limiting examples of suitable cross-linking agents include heterobifunctional cross-linking agents having two distinct reactive groups separated by a suitable spacer (e.g., m-maleimide benzoyl-N-hydroxysuccinimide ester), or homobifunctional cross-linking agents (e.g., disuccinimide octanoate).

[0176] Multispecific antibodies (e.g., bispecific antibodies) can have any suitable form that allows binding to the coronavirus spike protein via the antibody or antigen-binding fragments provided herein. Bispecific single-chain antibodies can be encoded by a single nucleic acid molecule. Non-limiting examples of bispecific single-chain antibodies and methods for constructing such antibodies are provided in U.S. Patent 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, and 6,723,538. Further 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 et al. , Proc. Natl. Acad. Sci. USA , 92(15):7021-7025, 1995; Kufer et al. , Cancer Immunol. Immunother. , 45(3-4):193-197, 1997; Löffler et al. , Blood , 95(6):2098-2103, 2000; and Brühl et al., J. Immunol. , 166(4):2420-2426,2001. The generation of bispecific Fab-scFv (“bibody”) molecules is described, for example, by 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 dimers, the scFv molecule can be fused with one of the VL-CL(L) or VH-CH1 chains, for example, to produce a dimer in which scFv is fused to the C-terminus of the Fab chain.

[0177] The bispecific antibodies disclosed herein may include antigen-binding fragments, such as Fab, F(ab')2, and Fv, which comprise both heavy and light chains and specifically bind to the coronavirus spike protein. These antibody fragments retain the ability to selectively bind to antigens and are “antigen-binding” fragments, and therefore can be included in bispecific antibodies. Non-limiting examples of such fragments include: (1) Fab, which is a fragment containing a monovalent antigen-binding fragment of an antibody molecule, can be produced by digesting an intact antibody with papain to obtain a complete light chain and a portion of a heavy chain; (2) Fab' is a fragment of the antibody molecule, which can be obtained by treating the whole antibody with pepsin and then reducing it to obtain the whole light chain and a part of the heavy chain. (3) (Fab')2, which is an antibody fragment that can be obtained by treating an intact antibody with pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments linked together by two disulfide bonds; (4) Fv, which contains V L Genetically engineered fragments, and V L Expressed as two chains; and (5) Single-chain antibodies (e.g., scFv) are defined as containing V linked by a suitable peptide linker. H and V L Genetically engineered molecules, as single-stranded molecules for gene fusion (see, for example, Ahmad) et al. , Clin. Dev. Immunol. ,2012, doi:10.1155 / 2012 / 980250; Marbry and Snavely, IDrugs , 13(8):543-549,2010). scFv in V H - Domains and V L- The intramolecular orientation of the domain is not decisive for the provided antibody (e.g., for the provided multispecific antibody). Therefore, it is possible to use domains with two possible arrangements (V H -Structural Domain-Joint Structural Domain-V L - Domain; V L -Structural Domain-Joint Structural Domain-V H - scFv of the structural domain.

[0178] (6) Single-chain antibody dimer (scFV2), defined as a dimer of scFV. It is also known as a "miniantibody".

[0179] Any suitable method can be used to generate the antigen-binding fragments discussed above. Harlow and Lane, Antibodies: A Laboratory Manual , 2 nd An unrestricted example is provided in Cold Spring Harbor Laboratory, New York, 2013.

[0180] Antigen-binding fragments can be prepared by proteolytic hydrolysis of antibodies or by expressing DNA encoding the fragment in host cells (e.g., *E. coli* cells). Antigen-binding fragments can also be obtained by digesting intact antibodies with pepsin or papain using conventional methods. For example, antigen-binding fragments can be generated by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment called F(ab')2. This fragment can be further cleaved using a thiol reducing agent and optionally with a blocking group of the thiol group generated by disulfide bond cleavage to produce a 3.5S Fab' monovalent fragment.

[0181] Other methods of antibody cleavage can also be used (e.g., separating the heavy chain to form a monovalent light chain-heavy chain fragment), further cleavage of the fragment, or other enzymatic, chemical, or genetic techniques, as long as the resulting fragment binds to the antigen recognized by the intact antibody.

[0182] The antibody or antigen-binding fragments included in the bispecific antibodies disclosed herein may be human antibodies or fragments thereof; these antibodies or fragments thereof may be used in the bispecific antibodies disclosed herein. Chimeric antibodies are also provided and may be used in bispecific antibodies. The antibody or antigen-binding fragment may include any suitable frame region, such as (but not limited to) human frame regions from other sources, or optimized frame regions. Alternatively, the heavy or light chain of the antibody may include heterologous frame regions, such as, but not limited to, mouse or monkey frame regions. These molecules may also be included in the disclosed bispecific antibodies.

[0183] Bispecific antibodies, such as DVD-immunoglobulin, can be any isotype. Bispecific antibodies, or DVD-immunoglobulins, can be, for example, IgA, IgM, or IgG antibodies, such as IgG1, IgG2, IgG3, or IgG4. The class of antibody, bispecific antibody, or DVD-immunoglobulin that specifically binds to the coronavirus spike protein can be switched to another. On one hand, the separation encoding V... L or V H The nucleic acid molecule is designed so that it does not contain any nucleic acid sequences that encode either the light chain or the heavy chain constant region. Then, the sequence encoding V... L or V H The nucleic acid molecules can be operatively linked to C-cells encoding different classes of immunoglobulin molecules. L Or C H The nucleic acid sequence. For example, a sequence containing C can be used. L Or C H This is achieved through carriers or nucleic acid molecules of the chain. For example, an antibody that specifically binds to the spike protein, initially IgG, can be classified as IgA. Class conversion can be used to transform one IgG subtype into another, such as from IgG1 to IgG2, IgG3, or IgG4.

[0184] DVD-immunoglobulins can be derivatized or linked to other molecules (e.g., other peptides or proteins). Typically, the antibody or antigen-binding fragment in a bispecific antibody is derivatized so that this derivatization or labeling does not adversely affect binding to the spike protein. For example, the antibody or antigen-binding fragment can be functionally linked (through chemical coupling, gene fusion, non-covalent association, or other means) to one or more other molecular entities, such as another antibody (e.g., another bispecific antibody or dimer to create a multispecific antibody), a detectable biomarker, an effector molecule, or a protein or peptide that can mediate association between the antibody or antibody fragment and other molecules (e.g., the streptavidin core region or a polyhistidine tag).

[0185] In several respects, each antibody or antigen-binding fragment included in DVD-immunoglobulin specifically binds to the coronavirus spike protein, with affinity (e.g., via K) D (Measurement) not higher than 1.0 × 10 -8 M, not higher than 5.0 × 10 -8 M, not higher than 1.0 × 10 -9 M, not higher than 5.0 × 10 -9 M, not higher than 1.0 × 10 - ¹ 0 M, not higher than 5.0 × 10 - ¹ 0 M or not higher than 1.0 × 10- ¹¹ M. For example, K D It can be measured by a radiolabeled antigen binding assay (RIA) using a Fab version of the antibody of interest and its antigen. In one assay, it is performed by using a very low concentration of ( ) in the presence of a series of titrated unlabeled antigens. 125 I) Labeled antigens are balanced with Fab, and then the bound antigens are captured using a plate coated with anti-Fab antibody to measure the solution-binding affinity of Fab to the antigen (see, for example, Chen). et al. , J. Mol. Biol. 293(4):865-881,1999). To establish assay conditions, MICROTITER® multiwell plates (Thermo Scientific) were coated overnight with 5 μg / ml capture anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate (pH 9.6), followed by blocking with 2% (w / v) bovine serum albumin in PBS at room temperature (approximately 23°C) for two to five hours. In non-adsorption plates (NUNC™ catalog number #269620), 100 μM or 26 pM [ 125 I]-Antigens were mixed with the Fab of interest at serial dilutions (e.g., with Presta). et al. , Cancer Res. (The evaluation of the anti-VEGF antibody Fab-12 in 57(20):4593-4599, 1997) was consistent. The Fab of interest was then incubated overnight; however, to ensure equilibrium was reached, the incubation could be extended (e.g., about 65 hours). The mixture was then transferred to a capture plate for incubation at room temperature (e.g., for one hour). The solution was then removed and the plate was washed eight times with 0.1% polysorbate 20 (TWEEN-20®) in PBS. After the plate was dried, 150 μl / well of scintillation buffer (MICROSCINT™-20; PerkinElmer) was added and the plate was counted for several tens of minutes on a TOPCOUNT™ γ counter (PerkinElmer). The concentration at which each Fab produced less than or equal to 20% maximum binding was selected for competitive binding assays.

[0186] In another assay, surface plasmon resonance assays can be used, employing a BIACORE®-2000 or BIACORE®-3000 (BIAcore, Inc., Piscataway, NJ), measuring K at ~10 response units (RU) using an immobilized antigen CM5 chip at 25°C. DIn short, following the supplier's instructions, the carboxymethylated dextran biosensor chip (CM5, BIACORE®, Inc.) was activated with N-ethyl-N'-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen was diluted to 5 μg / ml (~0.2 μM) with 10 mM sodium acetate (pH 4.8) and then injected at a flow rate of 5 l / min to achieve approximately 10 response units (RU) of conjugate protein. After antigen injection, 1 M ethanolamine was injected to block unreacted groups. For kinetic measurements, Fab was serially diluted twice (0.78 nM to 500 nM) and injected at approximately 25 l / min into PBS (PBST) containing 0.05% polysorbate 20 (TWEEN-20™) surfactant at 25°C. Using a simple one-to-one Langmuir binding model (BIACORE® evaluation software version 3.2), the association rate (k) was calculated by simultaneously fitting association and dissociation sensor maps. on ) and dissociation rate (k off Equilibrium dissociation constant (K) D ) is calculated as k off / k on Ratio. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the binding rate measured by the above surface plasmon resonance assay exceeds 10... 6 M - ¹ s - ¹, the binding rate can be determined using fluorescence quenching techniques, which measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm; emission = 340 nm, 16 nm bandwidth) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25 °C in the presence of increasing concentrations of antigen, as measured in a spectrophotometer, such as an Aviv Instruments spectrophotometer equipped with a flow-stop device or a ThermoSpectronic 8000 Series SLM-AMINCO™ spectrophotometer with a stirring cuvette. Affinity can also be measured using Carterra LSA via high-throughput SPR. In some respects, multispecific antibodies, such as bispecific antibodies, for example, DVD-immunoglobulin, are provided, which contain an antibody or antigen-binding fragment that specifically binds to the coronavirus spike protein with a specific affinity.

[0187] Bispecific tetravalent immunoglobulin, also known as DVD-immunoglobulin or DVD-IG™, is disclosed in Wu et al., MAbs. 2009;1:339–47, doi: 10.4161 / mabs.1.4.8755, which is incorporated herein by reference. See also Nat Biotechnol. 2007 Nov;25(11):1290-7. doi: 10.1038 / nbt1345. Epub 2007 Oct14., which is also incorporated herein by reference. The DVD-immunoglobulin molecule comprises two heavy chains and two light chains. However, unlike IgG, both the heavy and light chains of the DVD-immunoglobulin molecule contain additional variable domains (VDs), which are located via the V domains of existing monoclonal antibodies (mAbs). H and V L The N-terminal linker sequence connects the heavy and light chains. Therefore, upon binding, the resulting DVD-immunoglobulin molecule contains four antigen recognition sites (see Jakob et al.). Mabs 5: 358-363, 2013, which is incorporated herein by reference; see Jakob et al. for schematic diagrams and space-filling diagrams. Figure 1 DVD-immunoglobulin molecules have the ability to bind to two different antigens on each DFab simultaneously.

[0188] The outermost or N-terminal variable domain is called VD1, and the innermost variable domain is called VD2; VD2 is located near the C-terminus CH1 or CL. As disclosed by Jakob et al. above, DVD-immunoglobulin molecules can be mass-produced and purified to a homogeneous state, possessing pharmacological properties similar to conventional IgG1, and exhibiting in vivo efficacy.

[0189] In some respects, the publicly available bispecific antibodies are in the form of DVD-immunoglobulin. Figures 2-9 An exemplary amino acid sequence of DVD-immunoglobulin is listed. In this nomenclature, the outer variable domain (VD1) is listed first, followed by the inner domain (VD2), and the linker is listed third. Constant domains (such as IgG or IgM constant domains) are linked to the inner domains using linkers. The following linkers can be used: GS connector.

[0190] GGGGSGGGGSGGGG (SEQ ID NO: 121) There are three other EL connectors: one for heavy chains, one with an outer κ, and one with an outer λ.

[0191] EL connector (when the external V is a heavy chain) ASTKGPSVFPLAP (SEQ ID NO: 122) EL connector (when external V is κ) TVAAPSVFIFPP (SEQ ID NO: 123) EL connector (when external V is λ) QPKAAPSVTLFPP (SEQ ID NO: 124) 1. Exemplary DVD-Immunoglobulin Exemplary amino acid sequences of the VH and VL of bispecific antibodies (particularly DVD-immunoglobulin) are provided as in SEQ ID NO: 49-117 and 125-127, and are... Figures 2-9 As shown in the image. The following discloses other exemplary bispecific antibodies: A.COV44-62_COV30-14_GS and COV44-62_COV30-14_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV44-62. H and V L As VD1, and DVD-immunoglobulin VD2 includes COV30-14 V H and V L The above discloses the V of these antibodies. H and V L Multiple aspects of the domain; any one of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0192] In other respects, the heavy chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: a first V containing SEQ ID NO:2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 6, 7, and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 14, 15 and 16 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0193] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 1. HStructural domain, connector, second V containing SEQ ID NO: 9 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 5 L Structural domain, connector, and second V containing SEQ ID NO: 13 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0194] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 125. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 126. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 125, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 126.

[0195] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 127. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 49. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 127, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 49.

[0196] B.COV30-14_COV44-62_GS and COV30-14_COV44-62_EL In some respects, DVD-immunoglobulin VD1 includes V of COV30-14. H and V L As VD1, and DVD-immunoglobulin VD2 including COV44-62 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0197] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 HThe light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 14, 15, and 16. L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0198] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 9. H Structural domain, connector, second V containing SEQ ID NO: 1 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 13 L Structural domain, connector, and second V containing SEQ ID NO: 5 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0199] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 50. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 51. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 50, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 51.

[0200] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 52. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 53. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 52, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 53.

[0201] C: COV44-62_COV72-37_GS and COV44-62_COV72-37_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV44-62. H and V L As VD1, and DVD-immunoglobulin VD2 including COV72-37 V Hand V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0202] In other respects, the heavy chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: a first V containing SEQ ID NO:2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 6, 7, and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 22, 23 and 24 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0203] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 1. H Structural domain, connector, second V containing SEQ ID NO: 17 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 5 L Structural domain, connector, and second V containing SEQ ID NO: 21 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0204] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 54. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 55. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 54, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 55.

[0205] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 56. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 57. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 56, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 57.

[0206] D: COV72-37_COV44-62_GS and COV72-37_COV44-62_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV72-37. H and V L As VD1, and DVD-immunoglobulin VD2 including COV44-62 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0207] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V containing SEQ ID NO:18, 19, and 20. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 22, 23, and 24. L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0208] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 17. H Structural domain, connector, second V containing SEQ ID NO: 1 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 21 L Structural domain, connector, and second V containing SEQ ID NO: 5 LThe linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0209] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 58. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 59. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 58, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 59.

[0210] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 60. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 61. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 60, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 61.

[0211] E: COV44-62_COV89-22_GS and COV44-62_COV89-22_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV44-62. H and V L As VD1, and DVD-immunoglobulin VD2 includes V of COV89-22 H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0212] In other respects, the heavy chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: a first V containing SEQ ID NO:2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 6, 7, and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 30, 31 and 32 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0213] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 1. H Structural domain, connector, second V containing SEQ ID NO: 25 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 5 L Structural domain, connector, and second V containing SEQ ID NO: 29 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0214] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 62. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 63. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 62, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 63.

[0215] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 64. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 65. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 64, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 65.

[0216] F: COV89-22_COV44-62_GS and COV89-22_COV44-62_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV89-22. H and V L As VD1, and DVD-immunoglobulin VD2 including COV44-62 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0217] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 26, 27 and 28. HStructural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: V... comprising SEQ ID NO: 30, 31, and 32. L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0218] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 25. H Structural domain, connector, second V containing SEQ ID NO: 1 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 29 L Structural domain, connector, and second V containing SEQ ID NO: 5 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0219] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 66. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 67. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 66, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 67.

[0220] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 68. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 69. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 68, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 69.

[0221] G: COV44-79_COV30-14_GS and COV44-79_COV30-14_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV44-79. H and V LAs VD1, and DVD-immunoglobulin VD2 includes COV30-14 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0222] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V comprising SEQ ID NO:34, 35, and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NOs: 38, 39, and 40. L Structural domain, connector, containing the second V of SEQ ID NO: 14, 15 and 16 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0223] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 33. H Structural domain, connector, second V containing SEQ ID NO: 9 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 37 L Structural domain, connector, and second V containing SEQ ID NO: 13 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0224] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 70. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 71. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 70, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 71.

[0225] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 72. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 73. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 72, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 73.

[0226] H: COV30-14_COV44-79_GS and COV30-14_COV44-79_EL In some respects, DVD-immunoglobulin VD1 includes V of COV30-14. H and V L As VD1, and DVD-immunoglobulin VD2 including COV44-79 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0227] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 14, 15, and 16. L Structural domain, connector, containing the second V of SEQ ID NO: 38, 39 and 40 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0228] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 9. H Structural domain, connector, second V containing SEQ ID NO: 33 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 13 L Structural domain, connector, and second V containing SEQ ID NO: 37 LThe linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0229] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 74. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 75. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 74, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 75.

[0230] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 76. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 77. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 76, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 77.

[0231] I: COV44-79_COV72-37_GS and COV44-79_COV72-37_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV44-79. H and V L As VD1, and DVD-immunoglobulin VD2 including COV72-37 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0232] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V comprising SEQ ID NO:34, 35, and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO: 38, 39, 40. L Structural domain, connector, containing the second V of SEQ ID NO: 22, 23 and 24 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0233] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 33. H Structural domain, connector, second V containing SEQ ID NO: 17 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 37 L Structural domain, connector, and second V containing SEQ ID NO: 21 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0234] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 78. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 79. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 78, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 79.

[0235] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 80. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 81. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 80, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 81.

[0236] J: COV72-37_COV44-79_GS and COV72-37_COV44-79_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV72-37. H and V L As VD1, and DVD-immunoglobulin VD2 including COV44-79 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0237] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V containing SEQ ID NO:18, 19, and 20. HStructural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 22, 23, and 24. L Structural domain, connector, containing the second V of SEQ ID NO: 38, 39 and 40 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0238] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 17. H Structural domain, connector, second V containing SEQ ID NO: 33 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 21 L Structural domain, connector, and second V containing SEQ ID NO: 37 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0239] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 82. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 83. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 82, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 83.

[0240] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 84. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 85. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 84, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 85.

[0241] K: COV44-79_COV89-22_GS and COV44-79_COV89-22_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV44-79. H and VL As VD1, and DVD-immunoglobulin VD2 includes V of COV89-22 H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0242] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V comprising SEQ ID NO:34, 35, and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NOs: 38, 39, and 40. L Structural domain, connector, containing the second V of SEQ ID NO: 30, 31 and 32 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0243] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 33. H Structural domain, connector, second V including SEQ ID 25 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO:37. L Structural domain, connector, and second V containing SEQ ID NO: 29 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0244] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 86. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 87. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 86, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 87.

[0245] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 88. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 89. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 88, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 89.

[0246] L: COV89-22_COV44-79_GS and COV89-22_COV44-79_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV89-22. H and V L As VD1, and DVD-immunoglobulin VD2 including COV44-79 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0247] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 26, 27 and 28. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: V... comprising SEQ ID NO: 30, 31, and 32. L Structural domain, connector, containing the second V of SEQ ID NO: 38, 39 and 40 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0248] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO25. H Structural domain, connector, second V containing SEQ ID NO: 33 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO:29 L Structural domain, connector, and second V containing SEQ ID NO: 37 LThe linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0249] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 90. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 91. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 90, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 91.

[0250] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 92. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 93. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 92, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 93.

[0251] M: COV91-27_COV30-14_GS and COV91-27_COV30-14_EL In some respects, DVD-immunoglobulin VD1 includes V of COV91-27. H and V L As VD1, and DVD-immunoglobulin VD2 includes COV30-14 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0252] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V containing SEQ ID NO:42, 43, and 44. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 46, 47, and 48. L Structural domain, connector, containing the second V of SEQ ID NO: 14, 15 and 16 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0253] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 41. H Structural domain, connector, second V containing SEQ ID NO: 9 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 45 L Structural domain, connector, and second V containing SEQ ID NO: 13 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0254] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 94. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 95. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 94, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 95.

[0255] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 96. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 97. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 96, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 97.

[0256] N: COV30-14_COV91-27_GS and COV30-14_COV91-27_EL In some respects, DVD-immunoglobulin VD1 includes V of COV30-14. H and V L As VD1, and DVD-immunoglobulin VD2 including COV91-27 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0257] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 10, 11 and 12. HStructural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 14, 15, and 16. L Structural domain, connector, containing the second V of SEQ ID NO: 46, 47 and 48 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0258] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 9. H Structural domain, connector, second V containing SEQ ID NO: 41 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 13 L Structural domain, connector, and second V containing SEQ ID NO: 45 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0259] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 98. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 99. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 98, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 99.

[0260] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 100. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 101. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 100, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 101.

[0261] O: COV91-27_COV72-37_GS and COV91-27_COV72-37_EL In some respects, DVD-immunoglobulin VD1 includes V of COV91-27. H and VL As VD1, and DVD-immunoglobulin VD2 including COV72-37 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0262] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V containing SEQ ID NO:42, 43, and 44. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 46, 47, and 48. L Structural domain, connector, containing the second V of SEQ ID NO: 22, 23 and 24 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0263] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO41. H Structural domain, connector, second V containing SEQ ID NO: 17 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO:45. L Structural domain, connector, and second V containing SEQ ID NO: 21 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0264] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 102. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 103. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 102, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 103.

[0265] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 104. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 105. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 104, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 105.

[0266] P: COV72-37_COV91-27_GS and COV72-37_COV91-27_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV72-37. H and V L As VD1, and DVD-immunoglobulin VD2 including COV91-27 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0267] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V containing SEQ ID NO:18, 19, and 20. H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 22, 23, and 24. L Structural domain, connector, containing the second V of SEQ ID NO: 46, 47 and 48 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0268] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 17. H Structural domain, connector, second V containing SEQ ID NO: 41 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 21 L Structural domain, connector, and second V containing SEQ ID NO: 45 LThe linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0269] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 106. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 107. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 106, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 107.

[0270] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 108. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 109. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 108, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 109.

[0271] Q: COV91-27_COV89-22_GS and COV91-27_COV89-22_EL In some respects, DVD-immunoglobulin VD1 includes V of COV91-27. H and V L As VD1, and DVD-immunoglobulin VD2 includes V of COV89-22 H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0272] In other respects, the heavy chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: a first V containing SEQ ID NO:42, 43, and 44. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus, V containing SEQ ID NO: 46, 47, and 48. L Structural domain, connector, containing the second V of SEQ ID NO: 30, 31 and 32 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0273] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 41. H Structural domain, connector, second V containing SEQ ID NO: 25 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 45 L Structural domain, connector, and second V containing SEQ ID NO: 29 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0274] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 110. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 111. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 110, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 111.

[0275] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 112. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 113. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 112, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 113.

[0276] R: COV89-22_COV91-27_GS and COV89-22_COV91-27_EL In some respects, the VD1 of DVD-immunoglobulin includes the V of COV89-22. H and V L As VD1, and DVD-immunoglobulin VD2 including COV91-27 V H and V L The above discloses the V of these antibodies. H and V L The domains are multifaceted; any of these aspects can be used for DVD-immunoglobulin bispecific antibodies.

[0277] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 26, 27 and 28.H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody, in the order from N-terminus to C-terminus, includes: V... comprising SEQ ID NO: 30, 31, and 32. L Structural domain, connector, containing the second V of SEQ ID NO: 46, 47 and 48 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0278] In other respects, the heavy chain of the bispecific antibody comprises, in the order from N-terminus to C-terminus, a first V containing SEQ ID NO: 25. H Structural domain, connector, second V containing SEQ ID NO: 41 H The light chain contains a structural domain, as well as a heavy chain constant structural domain, such as the IgG or IgA constant structural domain. The light chain of the bispecific antibody includes, in the order from N-terminus to C-terminus: V containing SEQ ID NO: 29 L Structural domain, connector, and second V containing SEQ ID NO: 45 L The linker includes structural domains, as well as light chain constant domains, such as IgG or IgA constant domains. In some respects, the linker can be a GS linker. In other respects, the linker can be an EL linker.

[0279] In one aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 114. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 115. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 114, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 115.

[0280] In another aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 116. In another aspect, the light chain of DVD-immunoglobulin comprises SEQ ID NO: 117. In a further aspect, the heavy chain of DVD-immunoglobulin comprises SEQ ID NO: 116, and the light chain of DVD-immunoglobulin comprises SEQ ID NO: 117.

[0281] 2. Variations In some respects, amino acid sequence variants of DVD-immunoglobulin are provided. For example, it may be desirable to enhance the binding affinity and / or other biological properties of DVD-immunoglobulin. This can be achieved by introducing appropriate modifications into the encoding antibody V. H Domain and / or V L The nucleotide sequence of the domain, or through peptide synthesis, is used to prepare amino acid sequence variants of the antibody. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the antibody amino acid sequence. The final construct can be obtained by any combination of deletions, insertions, and substitutions, as long as the final construct possesses the desired properties (e.g., antigen binding).

[0282] In some respects, variants with one or more amino acid substitutions are provided. Sites of interest for substitution mutations include the CDR and framework regions of DVD-immunoglobulins. Amino acid substitutions can be introduced into antibodies of interest, and products can be screened for desired activities, such as maintained / enhanced antigen binding, reduced immunogenicity, or enhanced ADCC or CDC. These can be included in DVD-immunoglobulins.

[0283] Variant usually retains V H District and V L The correct folding and stabilization of the amino acid residues between regions will preserve the charge properties of these residues, thus maintaining the molecule's low pI and low toxicity. This can be achieved in V... H District and V L Amino acid substitutions are made in the region to increase yield. In some aspects, the bispecific antibody is DVD-immunoglobulin and includes up to 10 (e.g., up to 1, 2, 3, 4, 5, 6, 7, 8, or 9) amino acid substitutions in SEQ ID NO: 49-120. In other aspects, DVD-immunoglobulin includes at least 95% (e.g., 96%, 97%, 98%, or 99%) the same heavy chain domain and / or light chain domain as in one of SEQ ID NO: 49-120, wherein any alterations are located in the frame region (rather than the CDR region).

[0284] In some respects, substitutions, insertions, or deletions may occur within one or more CDRs, as long as these changes do not substantially reduce the ability of DVD-immunoglobulins to bind antigens when included in bispecific antibodies. For example, conserved changes that do not substantially reduce binding affinity can be made in CDRs (e.g., the conserved substitutions provided herein). In the variant V provided above... H and V L In some aspects of the sequence, each CDR remains unchanged, or contains no more than one, two, or three amino acid substitutions. In the variant V provided above...H and V L Some aspects of the sequence are modified only for framework residues, so the CDR remains unchanged.

[0285] To enhance the binding affinity of DVD-immunoglobulin, V can be targeted. L and V H Random mutations are performed on segments, such as within the HCDR3 or LCDR3 regions, a process similar to the in vivo somatic mutation process responsible for antibody affinity maturation during the innate immune response. Therefore, V can be amplified using PCR primers complementary to HCDR3 or LCDR3, respectively. H and V L The primers are spigoted at specific positions to achieve in vitro affinity maturation. During this process, a random mixture of four nucleotide bases is spigoted into the primers at specific locations, resulting in a PCR product encoding V. H and V L The segment in which random mutations have been introduced into V H and / or V L The CDR3 region. This allows for the analysis of these randomly mutated V... H and V L The segments were tested to determine their binding affinity to the spike protein.

[0286] In some respects, DVD-immunoglobulins are altered to increase or decrease the degree of glycosylation of antibody or antigen-binding fragments. The addition or deletion of glycosylation sites can be conveniently achieved by altering the amino acid sequence, thus creating or removing one or more glycosylation sites.

[0287] In cases where bispecific antibodies (such as DVD-immunoglobulin) include an Fc region, the carbohydrates attached to it can be altered. Naturally occurring antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually attached to Asn297 of the CH2 domain of the Fc region via N-linked bonds. See, for example, Wright… et al. Trends Biotechnol. 15(1):26-32, 1997. Oligosaccharides can include a variety of carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as fucose attached to the GlcNAc in the "backbone" of the biantennary oligosaccharide structure. In some respects, oligosaccharides in DVD-immunoglobulins can be modified to create variants with certain improved properties.

[0288] On one hand, publicly disclosed variants of DVD-immunoglobulins are provided, which have carbohydrate structures lacking (directly or indirectly) fucose attached to the Fc region. For example, the amount of fucose in such antibodies can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the glycan chain at Asn297 relative to the sum of all glycan structures attached to Asn 297 (e.g., complexes, mixtures, and high-mannose structures), as measured by MALDI-TOF mass spectrometry, for example, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue located approximately at position 297 in the Fc region; however, due to small sequence variations in antibodies, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucoidylated variants may possess enhanced ADCC function. See, for example, U.S. Patent Publication Nos. 2003 / 0157108 (Presta, L.); and 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; US2002 / 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; WO2005 / 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 Lec 13 CHO cells (Ripka) lacking protein fucosylation. et al. , Arch. Biochem. Biophys.249(2):533-545, 1986; US Patent Application Nos. US 2003 / 0157108 and WO2004 / 056312, particularly Example 11), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, for example, Yamane-Ohnuki et al. , Biotechnol. Bioeng. ,87(5): 614-622,2004;Kanda et al. , Biotechnol. Bioeng. 94(4):680-688, 2006; and WO2003 / 085107).

[0289] Further provided are DVD-immunoglobulin variants with bimeric oligosaccharides, for example, wherein the biantennary oligosaccharide attached to the Fc region of the antibody is bimeric by GlcNAc. Such antibody variants may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described, for example, WO 2003 / 011878 (Jean-Mairet). et al. ); US Patent No. 6,602,684 (Umana) et al. ); and US 2005 / 0123546 (Umana) et al. Antibody variants having 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, for example, in WO 1997 / 30087; WO 1998 / 58964; and WO 1999 / 22764.

[0290] In several respects, the constant region of DVD-immunoglobulins includes one or more amino acid substitutions to optimize the antibody's in vivo half-life. The serum half-life of IgG antibodies is regulated by the nascent Fc receptor (FcRn). Therefore, in several respects, bispecific antibodies contain amino acid substitutions that enhance binding to the FcRn. Non-limiting examples of such substitutions include substitutions at the constant region of the following IgG antibodies: T250Q and M428L (see, e.g., Hinton). et al., J Immunol., 176(1):346-356, 2006); M428L and N434S (“LS” mutations, see, for example, Zalevsky, et al. , Nature Biotechnol. 28(2):157-159 , 2010); N434A (see, for example, Petkova) et al., Int. Immunol.,18(12):1759-1769, 2006); T307A, E380A and N434A (see, for example, Petkova et al., Int. Immunol ., 18(12):1759-1769, 2006); and M252Y, S254T and T256E (see, for example, Dall'Acqua et al., J. Biol. Chem ., 281(33):23514-23524, 2006). The disclosed DVD-immunoglobulin may be linked to or include an Fc polypeptide that includes any of the substitutions listed above, for example, the Fc polypeptide may include M428L and N434S substitutions.

[0291] In some respects, the DVD-immunoglobulin provided herein can be further modified to contain additional non-protein moieties. Suitable moieties for bispecific antibody derivatization 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), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), and dextran or poly(N-vinylpyrrolidone) polyethylene glycol, propropylene glycol homopolymers, prolypropylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in the production process due to its stability in water. The polymer can be of any molecular weight and can 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. Typically, the number and / or type of polymer used for derivatization can be determined based on considerations, including but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in a given application under specific conditions, etc.

[0292] Conjugate As disclosed herein, DVD-immunoglobulin, which specifically binds to the coronavirus spike protein, can be conjugated to reagents, such as effector molecules or detectable biomarkers. Covalent and non-covalent attachment methods can be used. A variety of effector molecules and detectable biomarkers can be used, including (but not limited to) toxins and radioactive agents, such as... 125 I,32 P, 14 C 3 H and 35 S, as well as other markers, targeting moieties, enzymes, and ligands. The selection of specific effector molecules or detectable biomarkers depends on the specific target molecule or cell and the desired biological effect.

[0293] The procedure for attaching a detectable biomarker to an antibody varies depending on the chemical structure of the effector. Peptides typically contain multiple functional groups, such as carboxyl (-COOH), free amino (-NH2), or thiol (-SH) groups, which can react with suitable functional groups on the peptide, resulting in binding to the effector molecule or detectable biomarker. Alternatively, DVD-immunoglobulin can be derivatized to expose or attach additional reactive functional groups. Derivatization can involve the attachment of any suitable adapter molecule. The adapter is capable of forming a covalent bond with the antibody or antigen-binding fragment and with the effector molecule or detectable biomarker. Suitable adapters include, but are not limited to, straight-chain or branched-chain carbon adapters, heterocyclic carbon adapters, or peptide adapters. In the case where DVD-immunoglobulin and the effector molecule or detectable biomarker are peptides, the adapter can be linked to the constituent amino acids via its side chain (e.g., by forming a disulfide bond with cysteine) or α-carbon, or via the amino and / or carboxyl groups of the terminal amino acid.

[0294] Given the numerous methods already reported for attaching various radiodiagnostic compounds, radiotherapy compounds, labels (e.g., enzymes or fluorescent molecules), toxins, and other reagents to antibodies, a suitable method for attaching a given reagent to DVD-immunoglobulin can be determined.

[0295] DVD-immunoglobulin can be conjugated to detectable biomarkers; for example, detectable biomarkers that can be detected by ELISA, spectrophotometry, flow cytometry, microscopy, or diagnostic imaging techniques such as CT, computed axial computed tomography (CAT), MRI, magnetic resonance imaging (MTR), ultrasound, fiber optics, and laparoscopy. Specific, non-limiting examples of detectable biomarkers include fluorophores, chemiluminescent agents, enzyme-linked compounds, radioisotopes, and heavy metals or compounds (e.g., superparamagnetic iron oxide nanocrystals for MRI detection). For example, available detectable biomarkers include fluorescent compounds, including luciferin, fluorescein isothiocyanate, rhodamine, 5-dimethylamino-1-naphthalenesulfonyl chloride, phycoerythrin, lanthanide phosphors, and so on. Bioluminescent biomarkers are also available, such as luciferase, green fluorescent protein (GFP), and yellow fluorescent protein (YFP). Antibodies and antigen-binding fragments (such as DVD-immunoglobulin) can also be conjugated to detectable enzymes (such as horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase, glucose oxidase, etc.). When DVD-immunoglobulin is conjugated to a detectable enzyme, it can be detected by adding another reagent that utilizes the enzyme to generate a recognizable reaction product. For example, in the presence of the reagent horseradish peroxidase, the addition of hydrogen peroxide and diaminobenzidine results in a visually detectable colored reaction product. DVD-immunoglobulin can also be conjugated to biotin and detected by indirectly measuring avidin or streptavidin binding. Notably, avidin itself can be conjugated to enzymes or fluorescent labels.

[0296] DVD-immunoglobulin can be conjugated with paramagnetic reagents (e.g., gadolinium). Paramagnetic reagents, such as superparamagnetic iron oxide, can also be used as labels. DVD-immunoglobulin can also be conjugated with lanthanides (e.g., europium and dysprosium) and manganese. ADVD-immunoglobulin can also be labeled with predetermined peptide epitopes that can be recognized by secondary reporters (e.g., leucine zipper pairs, secondary antibody binding sites, metal-binding domains, epitope tags).

[0297] DVD-immunoglobulin can also be conjugated with radiolabeled amino acids, for example, for diagnostic purposes. For instance, radiolabeling can be used to detect coronaviruses via radiography, emission spectroscopy, or other diagnostic techniques. Examples of peptide labeling include, but are not limited to, the following radioisotopes: 3 H, 14 C 35 S, 90 Y、 99m Tc, 111 In、 125 I, 131I. Radioactive labels can be detected, for example, using photographic film or a scintillation counter; fluorescent labels can be detected using a photodetector to detect the emitted light. Enzyme labels are typically detected by providing a substrate to the enzyme and detecting the reaction products produced by the enzyme's action on the substrate; colorimetric labels can be detected simply by visualizing the colored label.

[0298] In the conjugate, the average number of detectable marker moieties per DVD-immunoglobulin can range from, for example, 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 (e.g., DVD-immunoglobulin) in the conjugate ranges from about 1 to about 2, about 1 to about 3, about 1 to about 8; about 2 to about 6; about 3 to about 5; or about 3 to about 4. The loading of the conjugate (e.g., the effector molecule ratio per antibody) can be controlled in various ways, for example, by: (i) limiting the molar excess of the effector molecule-linker intermediate or linker reagent relative to the antibody; (ii) limiting the conjugation reaction time or temperature; (iii) partially or limiting the reduction conditions used for cysteine ​​thiol modification; (iv) engineering the amino acid sequence of the antibody by recombinant technology such that the number and position of cysteine ​​residues are modified to control the number or position of linker-effect molecule attachments.

[0299] Polynucleotides and expression As disclosed herein, nucleic acid molecules (e.g., cDNA or RNA molecules) encoding amino acid sequences of DVD-immunoglobulins and conjugates that specifically bind to the coronavirus spike protein are provided. Using the amino acid sequences provided herein (e.g. Figures 2-9 The CDR sequence shown, V H and V L Sequences, including DVD-immunoglobulin sequences, sequences available in the art (e.g., frame region or constant region sequences), and the genetic code, can readily generate nucleic acids encoding these molecules. In several respects, nucleic acid molecules can encode the V of two antibodies in a bispecific antibody. H V L , or V H and V L Both (e.g., in bicistronic expression vectors). In several ways, nucleic acid molecules can be expressed in host cells (e.g., mammalian cells) to produce publicly available antibody or antigen-binding fragments.

[0300] The genetic code can be used to construct multiple functionally equivalent nucleic acid sequences, such as those with different sequences but encoding the same antibody sequence, or nucleic acids encoding conjugates or fusion proteins including DVD-immunoglobulins.

[0301] Nucleic acid molecules encoding DVD-immunoglobulins and conjugates that specifically bind to the coronavirus spike protein can be prepared by any suitable method, such as cloning the appropriate sequence or direct chemical synthesis using standard methods. Chemical synthesis yields single-stranded oligonucleotides. These can be converted into double-stranded DNA by hybridization with complementary sequences or by polymerization with DNA polymerase using a single strand as a template.

[0302] Exemplary nucleic acids can be prepared using 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).

[0303] Nucleic acids can also be prepared through amplification methods. Amplification methods include polymerase chain reaction (PCR), ligase chain reaction (LCR), transcription-based amplification systems (TAS), and autonomous sequence replication systems (3SR).

[0304] Nucleic acid molecules can be expressed in recombinant engineered cells, such as bacterial, plant, yeast, insect, and mammalian cells. Antibodies, antigen-binding fragments, and conjugates can be expressed as individual proteins, including V... H and / or V L (As needed, it can be linked to an effector molecule or a detectable marker), or it can be expressed as a fusion protein. Any suitable expression and purification method can be used for the antibody and antigen-binding fragment; Al-Rubeai (Ed.), Antibody Expression and Production Non-restrictive examples are provided in (Dordrecht; New York: Springer, 2011). Nucleic acid sequences may optionally encode a leader sequence.

[0305] To create an scFv, you can encode V. H and V L The DNA fragment can be operatively ligated to another fragment encoding a flexible adapter (e.g., encoding the amino acid sequence (Gly4-Ser)3), enabling V H and V L The sequence can be expressed as a continuous single-chain protein, where V L and V H The structural domains are connected by flexible joints (see, for example, Bird). et al., Science ,242(4877):423-426, 1988; Huston et al., Proc. Natl. Acad. Sci. USA , 85(16):5879-5883,1988; McCafferty et al., Nature , 348:552-554, 1990; Kontermann and Dübel (Eds.), Antibody Engineering Vols. 1-2, 2 nd ed., Springer-Verlag, 2010; Greenfield(Ed.), Antibodies: A Laboratory Manual , 2 nd (ed. New York: Cold Spring Harbor Laboratory Press, 2014). Optionally, the adapter may include a cleavage site, such as a furin cleavage site.

[0306] If only a single V is used H and V L Then, a single-chain antibody can be monovalent; if two V's are used... H and V L Then the single-chain antibody can be bivalent; or if more than two V's are used... H and V L Therefore, single-chain antibodies can be multivalent. Bispecific or multivalent antibodies that specifically bind to the coronavirus spike protein and another antigen can be generated. The encoded V... H and V L Optionally, it may include locations located in V H and V L Frin protease cleavage sites between domains. Linkers can also be encoded, for example, when nucleic acid molecules encode bispecific antibodies in the form of DVD-immunoglobulins.

[0307] One or more DNA sequences encoding DVD-immunoglobulins or conjugates can be expressed in vitro via DNA transfer to suitable host cells. Cells can be prokaryotic or eukaryotic. A variety of expression systems are available for protein expression, including *E. coli*, other bacterial hosts, yeast, and various higher eukaryotic cell lines such as COS, CHO, HeLa, and myeloma cell lines, which can be used to express the disclosed antibodies and antigen-binding fragments. Stable transfer methods can be used, meaning the foreign DNA can be persistently retained in the host. This disclosure also covers hybridomas expressing antibodies of interest.

[0308] The expression of nucleic acids encoding bispecific antibodies (e.g., DVD-immunoglobulin antibodies) described herein can be achieved by operatively linking DNA or cDNA to a promoter (which may be constitutive or inducible) and subsequently integrating it into an expression cassette. The promoter can be any promoter of interest, including cytomegalovirus promoters. Optionally, an enhancer, such as a cytomegalovirus enhancer, is included in the construct. The cassette is adaptable for replication and integration in prokaryotes or eukaryotes. A typical expression cassette contains specific sequences that can be used to regulate the expression of the protein-coding DNA. For example, the expression cassette may include a suitable promoter, enhancer, transcription and translation terminators, a start sequence, a start codon (i.e., ATG) preceding the protein-coding gene, splicing signals for introns, sequences for maintaining the correct reading frame of the gene to allow correct mRNA translation, and a stop codon. The vector may encode optional biomarkers, such as biomarkers encoding drug resistance (e.g., ampicillin or tetracycline resistance).

[0309] To achieve high-level expression of cloning genes, it is desirable to construct expression cassettes containing, for example, a strong promoter to guide transcription, a ribosome binding site (e.g., an internal ribosome binding sequence) for translation initiation, and a transcription / translation terminator. For *E. coli*, this may include a promoter, such as a T7, trp, lac, or lambda promoter, a ribosome binding site, and preferably a transcription termination signal. For eukaryotic cells, the control sequences may include promoters and / or enhancers derived from, for example, immunoglobulin genes, HTLV, SV40, or cytomegalovirus, and polyadenylated sequences, and may further include splice donor and / or acceptor sequences (e.g., CMV and / or HTLV splice acceptor and donor sequences). The cassette can be transferred to selected host cells by any suitable method, such as transformation or electroporation for *E. coli*, and calcium phosphate treatment, electroporation, or liposome transfection for mammalian cells. Cells transformed by the cassette can be selected based on antibiotic resistance conferred by the genes contained within the cassette (e.g., amp, gpt, neo, and hyg genes).

[0310] The nucleic acids encoding the polypeptides described herein can be modified without diminishing their biological activity. Modifications can be made to facilitate the cloning, expression, or integration of the target molecule into the fusion protein. These modifications include, for example, stop codons, sequences for creating restriction sites that facilitate localization, sequences for adding methionine to the N-terminus to provide a start site, or additional amino acids (e.g., polyhis) to aid in purification steps.

[0311] Once expressed, DVD-immunoglobulin and its conjugates can be purified according to standard procedures in the art, including ammonium sulfate precipitation, affinity column chromatography, column chromatography, etc. (see Simpson's). et al. (Eds.), Basic methods in Protein Purification and Analysis: A Laboratory Manual (New York: Cold SpringHarbor Laboratory Press, 2009). DVD-immunoglobulins and conjugates do not need to be 100% pure. If used for preventative purposes, the peptides should be substantially free of endotoxins after purification (partial purification or homogenization as needed).

[0312] Methods for expressing DVD-immunoglobulins and conjugates from mammalian cells and bacteria (e.g., *Escherichia coli*) have been described, and these methods are applicable to the antibodies disclosed herein. See, for example, 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.

[0313] Methods and compositions 1. Inhibit coronavirus infection This article discloses methods for suppressing coronavirus infection in subjects. These methods involve administering to subjects an effective amount (i.e., an amount that effectively suppresses infection in the subject) of a disclosed DVD-immunoglobulin, or a nucleic acid encoding such a DVD-immunoglobulin. Combinations of these DVD-immunoglobulins are also useful.

[0314] Publicly available methods may include administering an effective amount (i.e., an amount that effectively suppresses infection in the subject) of a publicly available DVD-immunoglobulin, or a nucleic acid encoding such a DVD-immunoglobulin, to a subject at risk of or already infected with coronavirus. These methods may be used before or after exposure.

[0315] The effectiveness of the method does not require the complete elimination or suppression of the infection. For example, compared to untreated coronavirus infection, the method can reduce the infection by a desired degree, such as 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). In some respects, subjects can also be treated with an effective amount of another agent (e.g., an antiviral agent).

[0316] In some respects, administration of an effective amount of DVD-immunoglobulin or a nucleic acid molecule encoding DVD-immunoglobulin inhibited the occurrence of infection and / or subsequent disease progression in subjects, which may include any statistically significant reduction in the activity (e.g., growth or invasion) of coronavirus infection or symptoms in subjects.

[0317] This article discloses methods for inhibiting coronavirus replication in subjects. These methods involve administering an effective amount (i.e., an amount that effectively inhibits replication in the subject) of a publicly disclosed DVD-immunoglobulin, or a nucleic acid encoding such a DVD-immunoglobulin, to subjects at risk of or with coronavirus infection. These methods can be used before or after exposure.

[0318] Methods for treating coronavirus infection in subjects are disclosed. Methods for preventing coronavirus infection in subjects are also disclosed. These methods include administering to subjects one or more disclosed DVD-immunoglobulins, or nucleic acid molecules encoding one or more DVD-immunoglobulins, or compositions comprising such molecules.

[0319] DVD-immunoglobulin can be administered via intravenous infusion. Dosage may vary, but typically ranges from about 0.5 mg / kg to about 50 mg / kg, for example, doses 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 instances, the dosage of DVD-immunoglobulin may be from about 0.5 mg / kg to about 5 mg / kg, for example, doses of about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, or about 5 mg / kg. DVD-immunoglobulin is administered according to a dosing schedule determined by a medical practitioner. In some instances, DVD-immunoglobulin is administered weekly, every two weeks, every three weeks, or every four weeks.

[0320] In some respects, methods for suppressing infection in subjects further include administering one or more additional agents to the subjects. Additional agents of interest include, but are not limited to, antiviral agents such as hydroxychloroquine, arbidol, remdesivir, favipiravir, baricitinib, lopinavir / ritonavir, zinc ions, and interferon beta-1b, or combinations thereof.

[0321] In some aspects, the method includes administering the first and second DVD-immunoglobulins disclosed herein. In some aspects, two, three, four, five, six, seven, eight, nine, or ten of the disclosed DVD-immunoglobulins may be administered to the subject. Nucleic acid molecules are also useful in these aspects. Combinations of these nucleic acid molecules encoding the disclosed DVD-immunoglobulins (e.g., one, two, three, four, or five of these nucleic acid molecules) may be administered to the subject.

[0322] In some aspects, the administration of DNA or RNA encoding the disclosed DVD-immunoglobulin to a subject can provide in vivo antibody production, for example, using the subject's cellular mechanisms. Any suitable nucleic acid administration method can be used; non-limiting examples are provided in U.S. Patent Nos. 5,643,578, 5,593,972, and 5,817,637. U.S. Patent No. 5,880,103 describes several methods for delivering nucleic acids encoding proteins to an organism. One method of administering nucleic acids is the direct administration of plasmid DNA, for example, using mammalian expression plasmids. The nucleotide sequence encoding the disclosed DVD-immunoglobulin can be placed under the control of a promoter to enhance expression. These methods include liposome delivery of nucleic acids. Such methods can be applied to generate bispecific antibodies. In some aspects, the pVRC8400 vector (described in Barouch) is used. et al. , J. Virol .,79(14), 8828-8834, 2005 (which is incorporated herein by reference) expresses publicly known DVD-immunoglobulin in subjects.

[0323] In several respects, an effective amount of an AAV viral vector containing one or more nucleic acid molecules encoding a publicly disclosed DVD-immunoglobulin can be administered to a subject (e.g., a subject at risk of or already infected with coronavirus). The AAV viral vector is engineered to express a nucleic acid molecule encoding a publicly disclosed bispecific antibody, and administration of an effective amount of the AAV viral vector to the subject results in the expression of an effective amount of DVD-immunoglobulin in the subject. Non-limiting examples of AAV viral vectors that can be used to express a publicly disclosed DVD-immunoglobulin in a subject include Johnson. et al., Nat. Med., 15(8):901-906, 2009 and Gardner et al. , Nature Those provided in , 519(7541):87-91, 2015, are each incorporated herein by reference in their entirety.

[0324] On one hand, nucleic acids encoding publicly available DVD-immunoglobulins can be directly introduced into tissues. For example, nucleic acids can be loaded onto gold microspheres using standard methods and introduced into the skin using a device such as Bio-Rad's HELIOS™ gene gun. The nucleic acids can be "naked," consisting of plasmids controlled by strong promoters.

[0325] DNA is typically injected into muscle, but it can also be injected directly into other sites. The injection dose is typically from about 0.5 μg / kg to about 50 mg / kg, and typically from about 0.005 mg / kg to about 5 mg / kg (see, for example, U.S. Patent No. 5,589,466).

[0326] Depending on the patient's needs and tolerable dosage and frequency, a single or multiple administration of a composition comprising publicly available DVD-immunoglobulin or a nucleic acid molecule encoding such DVD-immunoglobulin may be given. The dose may be administered once, or it may be applied periodically until the desired effect is achieved or until side effects necessitate discontinuation of treatment. Typically, the dose is sufficient to suppress coronavirus infection without causing unacceptable toxicity to the patient.

[0327] Data obtained from cell culture assays and animal studies can be used to formulate dosage ranges for human use. Dosages typically include ED. 50 Within the cyclic concentration range, it exhibits little to no toxicity. The dosage can vary within this range depending on the formulation and route of administration. Effective dosage can be determined through cell culture assays and animal studies.

[0328] Coronavirus spike protein-specific DVD-immunoglobulin, or nucleic acid molecules encoding such molecules, or compositions comprising such molecules, can be administered to subjects in a variety of ways, including local and systemic administration, such as by subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. In one aspect, DVD-immunoglobulin, or nucleic acid molecules encoding DVD-immunoglobulin, or compositions comprising such molecules, can be administered once daily by a single subcutaneous, intravenous, intra-arterial, intraperitoneal, intramuscular, intradermal, or intrathecal injection. DVD-immunoglobulin, or nucleic acid molecules encoding DVD-immunoglobulin, or compositions comprising such molecules, can also be administered by direct injection at or near the site of disease. Further methods of administration include use of an osmotic pump (e.g., an Alzet pump) or a micropump (e.g., an Alzet microosmotic pump), which allows for controlled, continuous, and / or sustained-release delivery of DVD-immunoglobulin, or nucleic acid molecules encoding DVD-immunoglobulin, or compositions comprising such molecules, over a predetermined time period. Osmotic pumps or micropumps can be implanted subcutaneously or near the target site.

[0329] 2. Composition Compositions are provided that comprise one or more of the DVD-immunoglobulins disclosed herein, or nucleic acid molecules encoding such DVD-immunoglobulins, in a pharmaceutically acceptable carrier. In some aspects, the compositions comprise two, three, four, or more DVD-immunoglobulins. The compositions can be used, for example, to inhibit or detect coronavirus infections, such as, but not limited to, SARS-CoV-2 infection. In some aspects, the compositions can be used to inhibit alpha coronavirus or beta coronavirus infection. In other aspects, the compositions can be used to inhibit SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, or OC43 infection. In further aspects, the compositions can be used to inhibit SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63, or 229E infection.

[0330] The composition can be prepared in unit dosage forms, such as in a kit, for administration to a subject. The amount and timing of administration are determined by the attending physician to achieve the desired purpose. DVD-immunoglobulin or nucleic acid molecules encoding DVD-immunoglobulin can be formulated for systemic or local administration. In one example, DVD-immunoglobulin or nucleic acid molecules encoding DVD-immunoglobulin are formulated for parenteral administration, such as intravenous administration.

[0331] In some aspects, the DVD-immunoglobulin or nucleic acid molecules included in the composition are at least 70% (e.g., 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% (e.g., less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or even lower) of macromolecular contaminants (e.g., other mammalian (e.g., human) proteins).

[0332] Compositions for administration may include solutions of DVD-immunoglobulin, conjugates, or nucleic acid molecules encoding DVD-immunoglobulin dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers may be used, such as buffered saline solutions. These solutions are sterile and generally free of undesirable substances. These compositions may be sterilized using any suitable technique. Compositions may contain pharmaceutically acceptable excipients to mimic physiological conditions, such as pH adjusters and buffers, toxicity modifiers, etc., for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of DVD-immunoglobulin or nucleic acid molecules in these formulations may vary considerably and is selected primarily based on fluid volume, viscosity, body weight, etc., depending on the specific route of administration and the needs of the subject.

[0333] Typical compositions for intravenous administration contain approximately 0.01 mg / kg to approximately 30 mg / kg of DVD-immunoglobulin (or a corresponding dose of a conjugate comprising an antibody or antigen-binding fragment) per subject per day. Administerable compositions can be prepared using any suitable method; publications Remington: The Science and Practice of Pharmacy, 22 nd ed. Non-limiting examples are provided in *Pharmaceutical Press, London, UK, 2013*. In some aspects, the composition may be a liquid formulation comprising one or more DVD-immunoglobulins in concentrations ranging from about 0.1 mg / ml to about 20 mg / ml, or about 0.5 mg / ml to about 20 mg / ml, or about 1 mg / ml to about 20 mg / ml, or about 0.1 mg / ml to about 10 mg / ml, or about 0.5 mg / ml to about 10 mg / ml, or about 1 mg / ml to about 10 mg / ml.

[0334] DVD-immunoglobulin or the nucleic acid encoding DVD-immunoglobulin can be provided in lyophilized form and rehydrated with sterile water prior to administration, although they can also be provided as a sterile solution of known concentrations. The solution containing DVD-immunoglobulin or the nucleic acid encoding DVD-immunoglobulin can then be added to an infusion bag containing 0.9% sodium chloride (USP) and administered typically at a dose of 0.5 mg / kg body weight to 15 mg / kg body weight. There is considerable experience in the art regarding the administration of antibody drugs, which have been marketed in the United States since rituximab was approved in 1997. DVD-immunoglobulin or the nucleic acid encoding DVD-immunoglobulin can be administered by slow infusion rather than by intravenous bolus or bolus injection. In one instance, a higher loading dose is administered, followed by a lower level of maintenance dose. For example, an initial loading dose of 4 mg / kg can be infused over a period of approximately 90 minutes, followed by a maintenance dose of 2 mg / kg infused weekly over 30-minute periods for 4–8 weeks, provided the initial dose has been well tolerated.

[0335] Controlled-release parenteral formulations can be formulated as implants, oil-based injectables, or granular 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 an active protein agent (such as a cytotoxin or drug) as a core. In microspheres, the active protein agent is dispersed throughout the particle. Particles, microspheres, and microcapsules smaller than about 1 μm are generally referred to as nanoparticles, nanospheres, and nanocapsules, respectively. Capillaries have a diameter of about 5 μm, therefore nanoparticles are only administered intravenously. Microparticles typically have a diameter of about 100 μm 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 Ticeand Tabibi, Treatise on Controlled Drug Delivery: Fundamentals, Optimization, Applications , A. Kydonieus (Ed.), New York, NY: Marcel Dekker, Inc., pp. 315-339, 1992.

[0336] Polymers can be used for the ion-controlled release of the compositions disclosed herein. Any suitable polymer can be used, such as degradable or non-degradable polymer matrices designed for controlled drug delivery. Alternatively, hydroxyapatite has been used as a microcarrier for the controlled release of proteins. On another front, liposomes have been used for the controlled release of lipid-encapsulated drugs and for drug targeting.

[0337] 2. Detection and Diagnostic Methods Methods for detecting the presence of the coronavirus spike protein in vitro or in vivo are also provided. In one instance, the presence of the coronavirus spike protein was detected in a biological sample from a subject and can be used to identify an infected subject.

[0338] In some aspects, the method detects the presence of at least one coronavirus in a biological sample. In some aspects, the method detects the presence of alpha or beta coronaviruses in a biological sample. In other aspects, the method detects the presence of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, or OC43 in a biological sample. In a further aspect, the method detects the presence of SARS-CoV-2, SARS-CoV, MERS-CoV, HKU1, OC43, NL63, or 229E in a biological sample. In a further aspect, the method detects the presence of at least SARS-CoV-2 in a biological sample.

[0339] The sample can be any type of sample, including but not limited to biopsy tissue, autopsy tissue, and pathological specimens. Biological samples also include tissue sections, such as frozen sections for histological purposes. Biological samples further include bodily fluids, such as blood, serum, plasma, sputum, cerebrospinal fluid, or urine. The detection method may include, under conditions sufficient to form an immune complex, contacting cells or a sample with an antibody that specifically binds to the coronavirus spike protein, an antigen-binding fragment (e.g., DVD-immunoglobulin), or a conjugate thereof (e.g., a conjugate including a detectable biomarker), and detecting the immune complex (e.g., by detecting a detectable biomarker conjugated to the antibody or antigen-binding fragment).

[0340] On the one hand, DVD-immunoglobulin is directly labeled with a detectable marker. On the other hand, DVD-immunoglobulin is unlabeled and can be detected using a secondary antibody or other molecules that can bind to primary DVD-immunoglobulin. The secondary antibody is selected if it can specifically bind to a primary antibody of a particular species and class. For example, if the primary bispecificity includes human IgG, then the secondary antibody could be anti-human IgG. Other molecules that can bind to DVD-immunoglobulin include, but are not limited to, protein A and protein G, both of which are commercially available. Suitable markers for DVD-immunoglobulin or secondary antibodies are known and, as described above, include a variety of enzymes, prosthetic groups, fluorescent substances, luminescent substances, magnetic substances, and radioactive substances.

[0341] In some respects, publicly disclosed DVD-immunoglobulins are used to test vaccines. For example, testing whether a vaccine composition comprising a coronavirus spike protein or a fragment thereof presents a conformation including a publicly disclosed DVD-immunoglobulin epitope. Therefore, this document provides a method for testing vaccines, wherein the method comprises contacting a sample containing a vaccine (e.g., a coronavirus spike protein immunogen) with a publicly disclosed DVD-immunoglobulin under conditions sufficient to form immune complexes, and detecting the immune complexes to detect a vaccine in the sample that includes an epitope of interest. In one instance, the detection of immune complexes in a sample indicates that the vaccine component (e.g., the immunogen) presents a conformation capable of binding DVD-immunoglobulins.

[0342] Example Bispecific antibody VH and Vλ / Vκ sequences were constructed using specific mAb sequences of fusion peptides (COV44-62, COV44-79, COV91-27) and stem-spirals (COV89-22, COV30-14, and COV72-37). The paired bispecific antibody VH and Vλ / Vκ sequences were then commercially cloned into a plasmid in DVD-Ig form and expressed as recombinant antibodies via transient transfection of Expi293 cells. The supernatant containing the expressed recombinant antibody was mixed with multiplex beads conjugated to the SARS-CoV-2 fusion peptide, SARS-CoV-2 stem-spiral antigen, and CD4 (negative control). The beads and supernatant were incubated at room temperature, washed with buffer, and the binding of the expressed bispecific antibody to both the fusion peptide and stem-spiral antigen was detected using anti-human IgG Alexa Fluor 647 secondary antibody. Samples were collected using an iQue Screener Plus flow cytometer, and data were analyzed using FlowJo. AUC analysis was performed using GraphPad Prism and reported after correction for AUCs using the negative control CD4 group.

[0343] Heatmaps were generated showing the binding of DVD-immunoglobulin to peptides representing fusion peptides and stem-helix residues, which are highly conserved in the spike proteins of SARS-CoV-1, MERS-CoV, HCoV-HKU1, HCoV-OC43 (β-coronaviruses), and HCoV-NL63 and HCoV-229E (α-coronaviruses). Fusion peptides (COV44-62, COV44-79, COV91-27), stem-helices (COV89-22, COV30-14, and COV72-37), and anti-HIV-1 spike protein mAbs (VRC01-specific mAbs) were included as controls for mAb binding assays. Figure 1 The area under the curve (AUC) value for each antigen is shown after subtracting the CD4 value from the negative control antigen. Of the expressed antibodies, 30 were confirmed to be bispecific because they were able to bind both the fusion peptide and the stem-helix simultaneously (unlike the control mAb, which only binds the stem-helix or the fusion peptide). The broad-spectrum and neutralizing potency of the bispecific antibodies against SARS-CoV-2, SARS-CoV-1, MERS-CoV, HCoV-OC43, and HCoV-NL63 were evaluated.

[0344] To evaluate the function of bispecific antibodies, recombinant expressed antibodies of various titers were co-incubated with pseudoviruses expressing the SARS-CoV-2 spike protein (USA-WA1 2020 strain) and the firefly luciferase reporter gene. HeLa cells expressing the SARS-CoV-2 receptor ACE2 (HeLa-ACE2) were co-incubated with the antibody-pseudovirus mixture, and luciferase activity was measured at 48 h post-infection (pi) to assess the level of HeLa-ACE2 infection. The 50% neutralizing titer (NT) was calculated using a 5-parameter nonlinear regression. 50 All parental stem-spiral 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 SARS-CoV-2 pseudoviruses within the tested concentration ranges, with COV89-22 being the most potent SH mAb (NT). 50 = 1.15 µg / mL), and COV44-62 is the most potent FPmAb (NT). 50 = 0.82 µg / mL). Of the 27 bispecific antibodies tested, n=15 achieved at least 50% neutralization of SARS CoV-2 pseudovirus within the tested concentration range, of which NT 50 The titer ranged from 0.28 µg / mL to 30.25 µg / mL. Due to low recovery rates in the transfection supernatant, the NT values ​​of the seven bispecific antibodies were not determined.50 ( Figures 11A-11C ).

[0345] Figures 12A-12B This table shows the neutralization of SARS-CoV-2 pseudovirus (USA-WA1 2020 strain) infection in HeLa cells expressing ACE2 by bispecific antibodies. Under the conditions of testing, a 50% neutralizing titer (NT) was achieved. 50 The value represents the antibody concentration at which 50% neutralization of the infection is achieved, calculated using a 5-parameter nonlinear regression. For antibodies that do not reach 50% neutralization, the highest concentration tested is shown.

[0346] Given that the principles of our invention can be applied to many possible aspects, it should be understood that the aspects shown are merely examples of the invention and should not be considered as limiting the scope of the invention. Rather, the scope of the invention is defined by the following claims. Therefore, we claim that everything within the scope and spirit of these claims is our invention.

Claims

1. A dual variable domain immunoglobulin (DVD-Ig) that specifically binds to and neutralizes the coronavirus spike protein, wherein the (DVD) immunoglobulin comprises a heavy chain and a light chain, and wherein: a) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 34, 35 and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V containing SEQ ID NO: 38, 39 and 40. L Structural domain, connector, second V including SEQ ID NO:30, 31 and 32 L The structural domain, as well as the light chain constant structural domain (COV44-79_COV89-22_GS and COV44-79_COV89-22_EL). b) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO: 2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V containing SEQ ID NO: 6, 7 and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 22, 23 and 24 L The structural domain, as well as the light chain constant structural domains (COV44-62_COV72-37_GS and COV44-62_COV72-37_EL). c) The heavy chain comprises, in the order from the amino (N) terminus to the carboxyl (C) terminus, the first V comprising SEQ ID NO: 2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V containing SEQ ID NO: 6, 7 and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 14, 15 and 16 L The structural domain, as well as the heavy chain constant structural domains (COV44-62_COV30-14_GS and COV44-62_COV30-14_EL). d) The heavy chain of the bispecific antibody comprises, in order from N-terminus to C-terminus, a first V comprising SEQ ID NO: 2, 3 and 4. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V containing SEQ ID NO: 6, 7 and 8. L Structural domain, connector, containing the second V of SEQ ID NO: 30, 31 and 32 L The structural domain, as well as the light chain constant structural domain (COV44-62_COV89-22_GS and COV44-62_COV89-22_EL). e) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus: V comprising SEQ ID NO: 14, 15 and 16 L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The structural domain, as well as the light chain constant structural domain (COV30-14_COV44-62_GS and COV30-14_COV44-62_EL). f) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO: 18, 19 and 20. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V including SEQ ID NO: 22, 23 and 24. L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The structural domain, as well as the light chain constant structural domains (COV72-37_COV44-62_GS and COV72-37_COV44-62_EL). g) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO: 26, 27 and 28. H Structural domain, connector, containing the second V of SEQ ID NO: 2, 3 and 4 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V including SEQ ID NO: 30, 31 and 32. L Structural domain, connector, containing the second V of SEQ ID NO: 6, 7 and 8 L The structural domain, as well as the light chain constant structural domain (COV89-22_COV44-62_GS and COV89-22_COV44-62_EL). h) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO: 34, 35 and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V containing SEQ ID NO: 38, 39 and 40. L Structural domain, connector, containing the second V of SEQ ID NO:14, 15 and 16 L The structural domain, as well as the light chain constant structural domain (COV44-79_COV30-14_GS and COV44-79_COV30-14_EL). i) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus: V comprising SEQ ID NO: 14, 15 and 16 L Structural domain, connector, containing the second V of SEQ ID NO:38, 39 and 40 L The structural domain, as well as the light chain constant structural domain (COV30-14_COV44-79_GS and COV30-14_COV44-79_EL). j) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO: 34, 35 and 36. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The structural domain, and the heavy chain constant structural domain; and the light chain contains, in order from N-terminus to C-terminus: V containing SEQ ID NO: 38, 39 and 40. L Structural domain, connector, second V including SEQ ID NO:22, 23 and 24 L The structural domain, as well as the light chain constant structural domain (COV44-79_COV72-37_GS and COV44-79_COV72-37_EL). k) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO: 18, 19 and 20. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V including SEQ ID NO: 22, 23 and 24. L Structural domain, connector, containing the second V of SEQ ID NO:38, 39 and 40 L The structural domain, as well as the light chain constant structural domain (COV72-37_COV44-79_GS and COV72-37_COV44-79_EL). l) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 26, 27 and 28. H Structural domain, connector, containing the second V of SEQ ID NO: 34, 35 and 36 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V including SEQ ID NO: 30, 31 and 32. L Structural domain, connector, containing the second V of SEQ ID NO:38, 39 and 40 L The structural domain, as well as the light chain constant structural domain (COV89-22_COV44-79_GS and COV89-22_COV44-79_EL). m) The heavy chain of the bispecific antibody comprises, in order from N-terminus to C-terminus, a first V comprising SEQ ID NO: 42, 43 and 44. H Structural domain, connector, containing the second V of SEQ ID NO: 10, 11 and 12 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V containing SEQ ID NO: 46, 47 and 48. L Structural domain, connector, containing the second V of SEQ ID NO: 14, 15 and 16 L The structural domain, as well as the light chain constant structural domain (COV91-27_COV30-14_GS and COV91-27_COV30-14_EL). n) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 10, 11 and 12. H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus: V comprising SEQ ID NO: 14, 15 and 16 L Structural domain, connector, containing the second V of SEQ ID NO:46, 47 and 48 L The structural domain, as well as the light chain constant structural domain (COV30-14_COV91-27_GS and COV30-14_COV91-27_EL). o) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 42, 43 and 44. H Structural domain, connector, containing the second V of SEQ ID NO: 18, 19 and 20 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V containing SEQ ID NO: 46, 47 and 48. L Structural domain, connector, second V including SEQ ID NO:22, 23 and 24 L The structural domain, as well as the light chain constant structural domain (COV91-27_COV72-37_GS and COV91-27_COV72-37_EL). p) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO: 18, 19 and 20. H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V including SEQ ID NO: 22, 23 and 24. L Structural domain, connector, containing the second V of SEQ ID NO:46, 47 and 48 L The structural domain, as well as the light chain constant structural domain (COV72-37_COV91-27_GS and COV72-37_COV91-27_EL). q) The heavy chain comprises, in order from N-terminus to C-terminus, a first V including SEQ ID NO: 42, 43 and 44. H Structural domain, connector, containing the second V of SEQ ID NO: 26, 27 and 28 H The light chain of the bispecific antibody comprises, in order from N-terminus to C-terminus, a V-terminus, and a heavy chain constant domain; and the light chain of the bispecific antibody comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO: 46, 47 and 48. L Structural domain, connector, containing the second V of SEQ ID NO: 30, 31 and 32 L The structural domain, and the light chain constant structural domains (COV91-27_COV89-22_GS and COV91-27_COV89-22_EL); or r) The heavy chain comprises, in order from N-terminus to C-terminus, the first V containing SEQ ID NO: 26, 27 and 28. H Structural domain, connector, containing the second V of SEQ ID NO: 42, 43 and 44 H The structural domain, and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, V including SEQ ID NO: 30, 31 and 32. L Structural domain, connector, containing the second V of SEQ ID NO:46, 47 and 48 L The structural domain, as well as the light chain constant structural domain (COV89-22_COV91-27_GS and COV89-22_COV91-27_EL).

2. The DVD-immunoglobulin according to claim 1, wherein: a) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

33. H Structural domain, connector, second V containing SEQ ID NO: 25 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus containing SEQ ID NO:

37. L Structural domain, connector, second V containing SEQ ID NO: 29 L Structural domains and light chain constant structural domains; b) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

1. H Structural domain, connector, second V containing SEQ ID NO: 17 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

5. L Structural domain, connector, and second V containing SEQ ID NO: 21 L Structural domains, and constant structural domains in light chains; c) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

1. H Structural domain, connector, second V containing SEQ ID NO: 9 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

5. L Structural domain, connector, and second V containing SEQ ID NO: 13 L Structural domains, and constant structural domains in light chains; d) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

1. H Structural domain, connector, second V containing SEQ ID NO: 25 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

5. L Structural domain, connector, and second V containing SEQ ID NO: 29 L Structural domains, and constant structural domains in light chains; e) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

9. H Structural domain, connector, second V containing SEQ ID NO: 1 H The light chain contains a structural domain and a heavy chain constant structural domain, such as an IgG or IgA constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

13. L Structural domain, connector and second V containing SEQ ID NO:5 L Structural domains, and constant structural domains in light chains; f) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

17. H Structural domain, connector, second V containing SEQ ID NO: 1 H The structural domain and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus: V containing SEQ ID NO: 21 L Structural domain, connector, and second V containing SEQ ID NO: 5 L Structural domains, and constant structural domains in light chains; g) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

25. H Structural domain, connector, second V containing SEQ ID NO: 1 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

29. L Structural domain, connector, and second V containing SEQ ID NO: 5 L Structural domains, and constant structural domains in light chains; h) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

33. H Structural domain, connector, second V containing SEQ ID NO: 9 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus containing SEQ ID NO:

37. L Structural domain, connector, and second V containing SEQ ID NO: 13 L Structural domains, and constant structural domains in light chains; i) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

9. H Structural domain, connector, second V containing SEQ ID NO: 33 H The bispecific antibody contains a structural domain and a heavy chain constant structural domain; and the light chain of the bispecific antibody comprises, in order from the N-terminus to the C-terminus, a V-terminus comprising SEQ ID NO:

13. L Structural domain, connector, and second V containing SEQ ID NO: 37 L Structural domains, and constant structural domains in light chains; j) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

33. H Structural domain, connector, second V containing SEQ ID NO: 17 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus containing SEQ ID NO:

37. L Structural domain, connector, and second V containing SEQ ID NO: 21 L Structural domains, and constant structural domains in light chains; k) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

17. H Structural domain, connector, second V containing SEQ ID NO: 33 H The structural domain and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus: V containing SEQ ID NO: 21 L Structural domain, connector, and second V containing SEQ ID NO: 37 L Structural domains, and constant structural domains in light chains; l) The heavy chain comprises, in order from N-terminus to C-terminus: a first V containing SEQ ID NO 25 H Structural domain, connector, second V containing SEQ ID NO: 33 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

29. L Structural domain, connector, and second V containing SEQ ID NO: 37 L Structural domains, and constant structural domains in light chains; m) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

41. H Structural domain, connector, second V containing SEQ ID NO: 9 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

45. L Structural domain, connector, and second V containing SEQ ID NO: 13 L Structural domains, and constant structural domains in light chains; n) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

9. H Structural domain, connector, second V containing SEQ ID NO: 41 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

13. L Structural domain, connector, and second V containing SEQ ID NO: 45 L Structural domains, and constant structural domains in light chains; o) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

41. H Structural domain, connector, second V containing SEQ ID NO: 17 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

45. L Structural domain, connector, and second V containing SEQ ID NO: 21 L Structural domains, and constant structural domains in light chains; p) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

17. H Structural domain, connector, second V containing SEQ ID NO: 41 H The structural domain and the heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus: V containing SEQ ID NO: 21 L Structural domain, connector, and second V containing SEQ ID NO: 45 L Structural domains, and constant structural domains in light chains; q) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

41. H Structural domain, connector, second V containing SEQ ID NO: 25 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

45. L Structural domain, connector, and second V containing SEQ ID NO: 29 L structural domains, and constant structural domains of light chains; or r) The heavy chain comprises, in order from N-terminus to C-terminus, a first V containing SEQ ID NO:

25. H Structural domain, connector, second V containing SEQ ID NO: 41 H The light chain comprises a structural domain and a heavy chain constant structural domain; and the light chain comprises, in order from N-terminus to C-terminus, a V-terminus comprising SEQ ID NO:

29. L Structural domain, connector, and second V containing SEQ ID NO: 45 L Structural domains, and constant structural domains in light chains.

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

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

5. The DVD-immunoglobulin according to 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: 127 and 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: 125 and 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 (COV30-14_COV44-62_GS); h) The heavy chain comprises SEQ ID NO: 52 and the light chain comprises SEQ ID NO: 53 (COV30-14_COV44-62_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). The heavy chain (ac) 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). The heavy chain (ae) 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). The heavy chain of ah) 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 The heavy chain (ak) comprises SEQ ID NO: 116 and the light chain comprises SEQ ID NO: 117 (COV89-22_COV91-27_EL).

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

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

8. The DVD-immunoglobulin according to any one of claims 1-7, wherein it is linked to a detectable biomarker.

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

10. The nucleic acid molecule of claim 9, operatively 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 any one of claims 1-7, the nucleic acid molecule of claim 9 or 10, or the carrier of claim 11 or 12; and a pharmaceutically acceptable carrier.

15. A method for generating antibodies or antigen-binding fragments that specifically bind to SARS-CoV proteins, comprising: Expressing one or more nucleic acid molecules encoding the DVD-immunoglobulin according to any one of claims 1-7 in host cells; and Purify the antibody or antigen-binding fragment.

16. A method for detecting the presence of SARS-CoV or SARS-CoV-2 in biological samples from a subject, comprising: Under conditions sufficient to form immune complexes, the biological sample is contacted with an effective amount of DVD-immunoglobulin according to any one of claims 1-7; and The presence of the immune complex in the biological sample is detected, wherein the presence of the immune complex in the biological sample indicates the presence of SARS-CoV or SARS-CoV-2 in the sample.

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

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

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