SARS-CoV-2 spike(S) anti-human ILA antibody and its use in the treatment of COVID-19

Abstract

The present invention provides antibodies that bind to the SARS-CoV-2 spike (S) protein. The present invention further relates to pharmaceutical compositions, immunotherapy compositions, and methods using the aforementioned antibodies that bind to the SARS-CoV-2 spike (S) protein. The antibodies of this disclosure are interchangeably referred to as anti-SARS-CoV-2 S glycoprotein antibodies and anti-CoV S glycoprotein antibodies. In various embodiments, this specification provides antibodies or fragments thereof that bind to the idiopathic acute respiratory syndrome coronavirus 2 (CoV) spike (S) glycoprotein.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 586,184 (filed September 28, 2023), which is incorporated herein by reference in its entirety.

[0002] Reference to electronic sequence listings This application includes a sequence listing submitted electronically in XML format, the entire sequence listing of which is incorporated herein by reference. The aforementioned XML copy was created on 27 September 2024, named 1450_106WO1_Sequence_Listing_09_27_2024, and has a size of 105,759 bytes.

[0003] This disclosure relates, in general terms, to anti-SARS-CoV-2 spike (S) antibodies and fragments thereof that are useful in treating viral infections. More specifically, anti-SARS-CoV-2 spike (S) antibodies and fragments thereof are used in the treatment of coronavirus 19 disease (COVID-19). [Background technology]

[0004] Infectious diseases remain a global challenge. The outbreak of idiopathic acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has infected more than 640 million people worldwide. Globally, the death toll has exceeded 6.6 million. The SARS-CoV-2 coronavirus belongs to the same viral family as severe acute respiratory syndrome coronavirus (SARS-CoV) and Middle East respiratory syndrome coronavirus (MERS-CoV), which have killed hundreds of people in the past 17 years. SARS-CoV-2 causes the disease known as COVID-19. Mutations in the S spike protein of SARS-CoV-2 allow SARS-CoV-2 variants to evade neutralizing monoclonal antibodies produced by past infection with SARS-CoV-2 or vaccination.

[0005] Therefore, the development of broad-spectrum neutralizing antibodies to treat COVID-19 is desirable. [Overview of the project] [Means for solving the problem]

[0006] This specification provides antibodies or fragments thereof that bind to the SARS-CoV-2 S glycoprotein of the SARS-CoV-2 virus or its variants. The antibodies of this disclosure are interchangeably referred to as anti-SARS-CoV-2 S glycoprotein antibodies and anti-CoV S glycoprotein antibodies. In various embodiments, this specification provides an antibody or fragment thereof that binds to the spike (S) glycoprotein of the coronavirus 2 (CoV) associated with sudden acute respiratory syndrome, wherein the antibody or fragment comprises: (i) a light chain variable complementarity determination region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 9, 12, 15, and 18; (ii) a light chain variable complementarity determination region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 10, 13, 16, and 19; and (iii) a light chain variable complementarity determination region 3 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 11, 14, 17, and 20. (iv) a heavy chain variable complementarity determination region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 21, 24, 27, and 30; (v) a heavy chain variable complementarity determination region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 22, 25, 28, and 31; and (vi) a heavy chain variable complementarity determination region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 23, 26, 29, and 32.In various embodiments, this specification provides antibodies or fragments thereof that bind to the SARS-CoV-2 spike (S) protein, wherein the antibodies or fragments bind to the polypeptides of SEQ ID NOs. 5-8 in amounts of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least (ii) a heavy chain variable (VH) domain containing an amino acid sequence having 99% or 100% identity, and (ii) a light chain variable (VL) domain containing an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs: 1 to 4. [Brief explanation of the drawing]

[0007] [Figure 1] This is an illustration of the SARS-CoV-2 S glycoprotein (SEQ ID NO: 65) used in the method for producing the anti-SARS-CoV-2 S glycoprotein and its fragments described herein. This image shows the mutation of the SARS-CoV-2 S glycoprotein of SEQ ID NO: 65 compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 74.

[0008] [Figure 2]This graph shows the pseudovirus neutralizing activity of antibodies NVX.172.10(A), NVX.205.10(B), NVX.62.12(C), and NVX.324.6(D). The bar graph represents the mAb concentration required to induce 50% pseudovirus neutralization (pVN50). Note that lower values ​​indicate stronger pseudovirus neutralizing activity.

[0009] [Figure 3] This shows the binding kinetics of the monoclonal antibody NVX.205.10 to the SARS-CoV-2 S glycoprotein. Binding curves between this mAb and prototype rS, XBB.1.5 rS, XBB.2.3 rS, XBB.1.16 rS, XBB.1.16.6 rS, EG.5.1 rS, and FL.1.5.1 rS are shown in the binding and dissociation phases.

[0010] [Figure 4] This shows the binding kinetics of the monoclonal antibody NVX.324.6 to the SARS-CoV-2 S glycoprotein. Binding curves between this mAb and prototype rS, XBB.1.5 rS, XBB.2.3 rS, XBB.1.16 rS, XBB.1.16.6 rS, EG.5.1 rS, and FL.1.5.1 rS are shown in the binding and dissociation phases.

[0011] [Figure 5] The binding kinetics of monoclonal antibodies NVX.205.10 and NVX.324.6 to SARS-CoV-2 S RBD and NTD are shown in quantitative analysis using biolayer interferometry. ka represents the binding rate, and kd represents the dissociation rate.

[0012] [Figure 6] These are SDS-PAGE gel and Western blot images showing that antibodies NVX.205.10 and NVX.172.10 specifically bind to the receptor-binding domain (RBD) of the SARS-CoV-2 S glycoprotein.

[0013] [Figure 7] These are SDS-PAGE gel and Western blot images showing that antibodies NVX.62.12 and NVX.324.6 specifically bind to the N-terminal domain (NTD) of the SARS-CoV-2 S glycoprotein.

[0014] [Figure 8A] This shows the 2D classification of Fab generated from mAb NVX.62.12. The 2D classification represents binding to the intact SARS-CoV-2 XBB.1.5 spike trimer on the NTD. [Figure 8B] This shows the 2D classification of Fab generated from mAb NVX.324.6. The 2D classification represents binding to the intact SARS-CoV-2 XBB.1.5 spike trimer on the NTD.

[0015] [Figure 8C] This shows the 2D classification of Fab generated from mAb NVX.172.10 (Figure 8C). The 2D classification represents binding to the intact SARS-CoV-2 XBB.1.5 spike trimer on the RBD. [Figure 8D] This shows the 2D classification of Fab generated from mAb NVX.205.10. The 2D classification represents binding to the intact SARS-CoV-2 XBB.1.5 spike trimer on the RBD.

[0016] [Figure 9] 3D models (side and top views) of 2D class-averaged particles mapped for each binding Fab are shown for NVX.172.10, NVX.205.10, NVX.62.12, and NVX.324.6, respectively.

[0017] [Figure 10]This shows the binding interface between XBB.1.5 NTD and NVX.324.6 Fab. The XBB.1.5 NTD subdomain (gray) and epitope loop (N1-N5) are represented by their respective colors (N1-green, N2-blue, N3-magenta, N4-yellow, N5-orange) in a modeled structure. Similarly, these are labeled on the NTD sequence and secondary structure along with the glycan (dark gray sphere). On the right, a ribbon representation of the XBB.1.5 NTD subdomain bound to an NTD-specific NVX.324.6 Fab fragment (model) is shown (VH-blue, VL-pink, CDR-dark blue).

[0018] [Figure 11] This shows the binding interface between XBB.1.5 RBD and NVX.205.10 Fab. It displays the surface representation of XBB.1.5 RBD (gray) representing epitope classes (1-4), and the ACE2 receptor binding interface with or without subdomain-specific NVX.205.10 Fab fragments (VH-cyan, VL-yellow) (ribbon). [Modes for carrying out the invention]

[0019] definition As used herein and in the accompanying claims, the singular forms “a,” “an,” and “the” include multiple references unless otherwise clearly defined by the context. Thus, for example, a reference to “(one) protein” refers to one protein or a mixture of such proteins, and a reference to “the method” includes references to equivalent steps and methods known to those skilled in the art, etc.

[0020] As used herein, the term “adjuvant” refers to a compound that, when used in combination with an immunogen, enhances, or otherwise modifies or alters the immune response induced to an immunogen. Modification of the immune response includes enhancing or broadening the specificity of either or both the antibody and / or cellular immune responses. As used herein, the terms “about” or “approximately” when preceding a number indicate a value within a range of plus or minus 10%. For example, “about 100” includes 90 and 110.

[0021] As used herein, the terms “immunogen,” “antigen,” and “epitope” refer to substances capable of inducing an immune response (e.g., proteins (including glycoproteins) and peptides).

[0022] As used herein, “substantially” means that the isolation of a substance (e.g., a compound, polynucleotide, or polypeptide) is carried out in such a way that it constitutes the majority of the sample in which the substance is present. For example, in a sample, substantially purified components constitute 85%, preferably 85% to 90%, more preferably at least 95% to 99.5%, and most preferably at least 99% of the sample. If components are substantially replaced, the amount remaining in the sample is about 0.5% to about 10% or less, preferably about 0.5% to about 1.0% or less.

[0023] As used herein, the terms “to treat,” “treatment,” and “to treat” refer to an approach to obtain a beneficial or desired outcome (e.g., a clinical outcome). For the purposes of this disclosure, beneficial or desired outcomes may include inhibiting or suppressing the onset or progression of an infection or disease, alleviating or reducing the onset of symptoms of an infection or disease, or a combination thereof.

[0024] As used herein, “prevention” is interchangeable with “prophylaxis” and may mean the complete prevention of an infection or disease, or the prevention of the onset of symptoms of such infection or disease, the delay of the onset of an infection or disease or symptoms of such infection or disease, or a reduction in the severity of any subsequent infection or disease or symptoms of such infection or disease.

[0025] As used herein, “effective dose” or “effective amount” refers to the amount of antibody sufficient to induce an immune response that reduces at least one symptom of a pathogen infection. The effective dose or effective amount can be quantified by measuring the amount of neutralizing secretion and / or serum antibody, for example, by plaque neutralization, complement fixation, enzyme-linked immunosorbent assay (ELISA), or microneutralization assay.

[0026] As used herein, the term “Subject” includes humans and other animals. Typically, the subject is human. For example, the subject is an adult, teenager, child (2 to 14 years), infant (birth to 2 years), or newborn (2 months or younger). In certain embodiments, the subject is 4 months or younger, or 6 months or younger. In various embodiments, the adult is approximately 65 years of age or older, or an elderly person approximately 60 years of age or older. In various embodiments, the subject is a pregnant woman or a woman intending to become pregnant. In other embodiments, the subject is not human, but a non-human primate, such as a baboon, chimpanzee, gorilla, or macaque. In various embodiments, the subject is a pet (e.g., a dog or cat).

[0027] In some embodiments, the subject is immunodeficient. In some embodiments, the immunodeficient subject is administered an immunosuppressive agent. Non-limiting examples of immunosuppressive agents include corticosteroids (e.g., prednisone), alkylating agents (e.g., cyclophosphamide), antimetabolites (e.g., azathioprine or 6-mercaptopurine), transplant-related immunosuppressive drugs (e.g., cyclosporine, tacrolimus, sirolimus, or mycophenolate mofetil), mitoxantrone, chemotherapeutic agents, methotrexate, and tumor necrosis factor (TNF) blockers (e.g., etanercept, adalimumab, infliximab). In some embodiments, the immunodeficient subject is infected with a virus (e.g., human immunodeficiency virus or Epstein-Barr virus). In some embodiments, the virus is a respiratory virus (e.g., respiratory syncytial virus, influenza, parainfluenza, adenovirus, or picornavirus). In some embodiments, the immunocompromised subject has acquired immunodeficiency syndrome (AIDS). In some embodiments, the immunocompromised subject is infected with human immunodeficiency virus (HIV). In some embodiments, the immunocompromised subject is immunocompromised due to a treatment regimen designed to prevent inflammation or transplant rejection. In some embodiments, the immunocompromised subject is a transplant recipient. In some embodiments, the immunocompromised subject has undergone radiotherapy or splenectomy.In various embodiments, the target of immunodeficiency includes cancer, autoimmune diseases, tuberculosis, substance use disorders (e.g., alcohol, opioid, or cocaine use disorder), stroke or cerebrovascular disease, organ or hematopoietic stem cell transplantation, sickle cell disease, thalassemia, autoimmune lymphoproliferative syndrome (ALPS), polyglandular autoimmune syndrome type 1 (APS-1), B-cell proliferation (BENTA) disorder with NF-κB and T-cell anergy, caspase-8 deficiency (CEDS), chronic granulomatous disease (CGD), unclassified immunodeficiency (CVID), congenital neutropenia syndrome, cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4) deficiency, DOCK8 deficiency, GATA2 deficiency, glycosylation disorders with immunodeficiency, and hyperimmune globulin E syndrome (H Diagnosed with IES), hyperimmune globulin M syndrome, diabetes mellitus, type 1 diabetes mellitus, type 2 diabetes mellitus, interferon-gamma deficiency, interleukin-12 deficiency, interleukin-23 deficiency, leukocyte adhesion disorder, lipopolysaccharide-responsive beige-like anchor (LRBA) deficiency, PI3 kinase disorder, PLCG2-related antibody deficiency and immunodysregulation (PLAID), severe combined immunodeficiency (SCID), STAT3 dominant-negative disorder, STAT3 gain-of-function disorder, verrucae-hypogammaglobulinemia-immunocompromise-myelocatexis (WHIM) syndrome, Wiscott-Aldrich syndrome (WAS), X-linked agammaglobulinemia (XLA), X-linked lymphoproliferative disorder (XLP), uremia, malnutrition, or X-Men disease. In various embodiments, immunocompromised subjects are current or former smokers. In various embodiments, the immunocompromised subject has B cell abnormalities, T cell abnormalities, macrophage abnormalities, cytokine abnormalities, phagocytic deficiency, phagocytic dysfunction, complement deficiency, or a combination thereof.

[0028] In some embodiments, the subject is overweight or obese. In some embodiments, the body mass index (BMI) of the overweight subject is 25 kg / m². 2 More than 30kg / m 2 It is less than 30 kg / m². In various embodiments, the BMI of the obese subject is 30 kg / m². 2In various embodiments, the subject is a mental health condition. In various embodiments, the mental health condition is depression, schizophrenia, or anxiety.

[0029] As used herein, the term “pharmaceutically acceptable” means that it is approved by a U.S. federal or state regulatory agency, or that it is listed in the United States Pharmacopoeia, the European Pharmacopoeia, or other widely recognized pharmacopoeias for use in mammals, more specifically in humans. These compositions may be useful as vaccine and / or antigenic compositions for inducing a protective immune response in vertebrates.

[0030] As used herein, the term “modification” when referring to the SARS-CoV-2 spike(S) polypeptide refers to a single-amino acid mutation, deletion, or addition of the CoV S polypeptide. The location of the modification within the CoV S polypeptide can be determined by aligning the polypeptide sequence to SEQ ID NO: 74 (CoV S polypeptide with signal peptide) or SEQ ID NO: 73 (mature CoV S polypeptide without signal peptide).

[0031] The term SARS-CoV-2 "variant" is used herein interchangeably with "heterogeneous SARS-CoV-2 strain" and refers to a SARS-CoV-2 virus containing a CoV S polypeptide having one or more modifications compared to the SARS-CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73. For example, a SARS-CoV-2 variant is a CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73. Compared to Spoly, it may have at least about 2, at least about 3, at least about 4, at least about 5, at least about 6, at least about 7, at least about 8, at least about 9, at least about 10, at least about 11, at least about 12, at least about 13, at least about 14, at least about 15, at least about 16, at least about 17, at least about 18, at least about 19, at least about 20, at least about 21, at least about 22, at least about 23, at least about 24, at least about 25, at least about 26, at least about 27, at least about 28, at least about 29, at least about 30, at least about 31, at least about 32, at least about 33, at least about 34, at least about 35, at least about 36, at least about 37, at least about 38, at least about 39, at least about 40, at least about 41, at least about 42, at least about 43, at least about 44, at least about 45, at least about 46, at least about 47, at least about 48, at least about 49, or at least about 50 modifications. For example, a SARS-CoV-2 variant may have 1 to 100 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73, up to 2, up to 3, up to 4, up to 45, up to 50, up to 55, up to 60, up to 65, up to 70, up to 75, up to 80, up to 85, up to 90, up to 95, or up to 100 modifications.In various embodiments, SARS-CoV-2 variants may have 1 to about 100 modifications, about 2 to about 35 modifications, about 5 to about 10 modifications, about 5 to about 20 modifications, about 10 to about 20 modifications, about 15 to about 25 modifications, about 20 to about 30 modifications, about 20 to about 40 modifications, about 25 to about 45 modifications, about 25 to about 100 modifications, about 25 to about 45 modifications, and about 35 to about 100 modifications compared to the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73.

[0032] In various embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In various embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having at least 70% to about 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In various embodiments, a heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having at least 70% to about 99.5% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In various embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 90% to 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In various embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 90% to 99.8% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In various embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 95% to 99.9% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In various embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide that has approximately 95% to 99.8% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74.In various embodiments, the heterologous SARS-CoV-2 strain is a SARS-CoV-2 virus containing a CoV S polypeptide having approximately 95% to 99% identity with the CoV S polypeptide having the amino acid sequence of SEQ ID NO: 73 or SEQ ID NO: 74. In various embodiments, the World Health Organization (WHO) label for the heterologous SARS-CoV-2 strain is alpha, beta, gamma, delta, epsilon, eta, iota, kappa, zeta, mu, or omicron. In various embodiments, heterologous SARS-CoV-2 strains are selected from the group consisting of B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1. The following document describes Pango phylogenetic nomenclature, which is incorporated herein by reference in its entirety: O'Toole et al. BMC Genomics, 23, 121 (2022).

[0033] In some embodiments, the World Health Organization (WHO) label for heterologous SARS-CoV-2 strains is Omicron. In some embodiments, heterologous SARS-CoV-2 strains with the WHO label Omicron have at least 35 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 73. In some embodiments, heterologous SARS-CoV-2 strains with the WHO label Omicron have 35-55, 35-65, 35-75, 35-85, 35-95, or 35-105 modifications compared to the wild-type SARS-CoV-2 S polypeptide of SEQ ID NO: 73. In various embodiments, the modifications are T6I, T6R, A14S, A54V, V70A, T82I, G129D, H133Q, K134E, W139R, E143G, F144L, Q170E, I197V, L199I, V200E, V200G, G239V, G244S, G326D, G326H, R333T, L355I, S3 58F, S358L, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, V432P, G433S, L43 9R, L439Q, N447K, S464N, T465K, E471A, F473V, F473S, F477S, Q480R, G483S, Q485R, N488 Selected from the group consisting of Y, Y492H, T534K, T591I, D601G, G626V, H642Y, N645S, N666K, P668H, S691L, N751K, D783Y, N843K, Q941H, N956K, L968F, D1186N, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acid 214 and amino acid 215, as well as combinations thereof.

[0034] In various embodiments, the variant CoV S polypeptide includes a combination of modifications selected from the group consisting of: (i)A54V, T82I, G129D, L199I, G326D, S358L, S360P, S362F, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N 751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 56, deletion of amino acid 57, deletion of amino acid 130, deletion of amino acid 131, deletion of amino acid 132, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215; (ii) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (iii) T6R, A14S, T82I, G129D, E143G, L199I, G326D, S358L, S360P, K404N, N427K, G433S, S464N, T465K, E471A, Q480R, G483S, Q485R, N488Y, Y492H, T534K, D601G, H642Y, N666K, P668H, N751K, D783Y, N843K, Q941H, N956K, L968F, deletion of amino acid 144, deletion of amino acid 145, deletion of amino acid 198, and insertion of a tripeptide having the amino acid sequence of EPE between amino acids 214 and 215; (iv) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, K404N, N427K, L439Q, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, S691L, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (v) Deletion of T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, S464N, T465K, E471A, Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (vi) T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, D601G, H642Y, N645S, N666K, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (vii)V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, G626V, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (viii)V3G, T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (ix)T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (x)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, N447K, S464N, T465K, E471A, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (xi)T6I, A14S, G129D, K134E, W139R, F144L, I197V, V200G, G244S, G326H, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, G433S, L439R, N447K, S 464N, T465K, E471A, F473S, Q485R, N488Y, Y492H, T591I, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, D1186N, deletion of amino acid 11, deletion of amino acid 12, and deletion of amino acid 13; (xii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N645S, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xiii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xiv)T6I, A14S, V70A, G129D, H133Q, Q170E, V200E, G239V, G326H, R333T, L355I, S 358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S46 4N, T465K, E471A, F473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, and deletion of amino acid 131; (xv)T6I, A14S, G129D, H133Q, Q170E, V200E, G326H, R333T, L355I, S358F, S360P, S36 2F, T363A, D392N, R395S, K404N, N427K, V432P, G433S, N447K, S464N, T465K, E471A, F 473S, F477S, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131; (xvi)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; (xvii)T6I, A14S, G129D, V200G, G326D, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, K431T, L439R, N447K, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, and deletion of amino acid 57; and (xviii)T6I, A14S, G129D, V200G, G326D, R333T, S358F, S360P, S362F, T363A, D392N, R395S, K404N, N427K, L439R, S464N, T465K, E471A, F473V, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, N956K, deletion of amino acid 11, deletion of amino acid 12, deletion of amino acid 13, deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131; (xix) Deletion of amino acid 56, deletion of amino acid 57, and deletion of amino acid 131, N488Y, A557D, D601G, P668H or P668R, T703I, S969A, and D1105H; (xx)D67A, K404N, E471K, N488Y, D601G, and A688V; (xxi)D67A, D202G, L229H, K404N, E471K, N488Y, D601G, and A688V; (xxii)D67A, D202G, deletion of one, two, or three amino acids from amino acids 228-230, K404N, E471K, N488Y, D601G, and A688V; (xxiii)D67A, L229H, R233I, N488Y, K404N, E471K, D601G, and A688V; (xxiv)L5F, T7N, P13S, D125Y, R177S, K404T, E471K, N488Y, D601G, H642Y, T1014I, and V1163F; (xxv)W139C and L439; (xxvi) Deletion of amino acid 144, deletion of amino acid 145, T6R, E143G, L439R, T465K, D601G, P668R, and D937N; (xxvii) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, L439R, T465K, D601G, P668R, and D937N; (xxviii) Deletion of amino acid 144, deletion of amino acid 145, T6R, T82I, G129D, Y132H, E143G, A209V, K404N, L439R, T465K, D601G, P668R, and D937N; (xxix) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R, and D937N; (xxx) Deletion of amino acid 144, deletion of amino acid 145, T6R, W51H, H53W, G129D, E143G, D200V, L201R, W245I, K404N, N426K, L439R, T465K, E471K, N488Y, D601G, P668R, and D937N; (xxxi) Deletion of amino acid 144, deletion of amino acid 145, T6R, G129D, E143G, K404N, L439R, T465K, E471Q, D601G, P668R, and D937N; (xxxii) Deletion of Q39R, A54V, E471K; D601G, Q664H, F875L, and one, two, three, or four of amino acids 56, 57, 131, and 132; (xxxiii)T82I, D240G, E471K, D601G, and A688V; (xxxiv)L439R, E471Q, D601G, P668R, and Q1058H; (xxxv)G62V, T63I, R233N, L439Q, F477S, D601G, T846N, and deletion of amino acids 1, 2, 3, 4, 5, or 6 from amino acids 234-240; (xxxvi)T82I, Y131S, Y132N, R333K, E471K, N488Y, D601G, P668H, and D937N; and (xxxvii) G129D, G326D, S360P, S362F, K404N, N427K, T465K, E471A or E471K, Q480K or Q480R, Q485R, N488Y, Y492H, D601G, H642Y, N666K, P668H, N751K, D783Y, Q941H, and N953K (The amino acids of the CoV S glycoprotein are numbered relative to the polypeptide having the sequence of SEQ ID NO: 73).

[0035] As used herein, “antibody (singular)” and “antibody (plural)” (immunoglobulin) include monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies formed from at least two intact antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fv(scFv), single-chain antibodies, single-domain antibodies, domain antibodies, Fab fragments, F(ab')2 fragments, antibody fragments exhibiting desired biological activity, disulfide-linked Fv(sdFv), and anti-idiotype (anti-Id) antibodies (e.g., anti-Id antibodies against the antibodies of the present invention), intrabodies, and any of the epitope-binding fragments described above. More specifically, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules (i.e., molecules containing antigen-binding sites). Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), a class (e.g., IgG1, IgG2, IgG3, IgG4, IgAl, and IgA2), or a subclass.

[0036] Native antibodies and immunoglobulins are typically heterotetrameric glycoproteins with approximately 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is attached to a heavy chain by one disulfide covalent bond, although the number of disulfide bonds differs between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has interchain disulfide bridges spaced at regular intervals. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other; the constant domain of this light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Light chains are classified as either lambda chains or kappa chains. The variable domain of the kappa light chain is also referred to herein as VK. The term "variable region" is sometimes used to refer to the variable domains of the heavy or light chain. Certain amino acid residues are thought to form an interface between the light chain variable domain and the heavy chain variable domain. Such antibodies can be derived from any mammal (but not limited to humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc.).

[0037] The term "variable" refers to the fact that in certain parts of the variable domain, the sequence differs significantly between antibodies, and this difference is responsible for the binding specificity of each antibody to a particular antigen. However, variability is not evenly distributed throughout the variable domain of an antibody, but is concentrated in segments called complementarity-determining regions (CDRs) within the variable domains of both the light and heavy chains. The more highly conserved parts of the variable domain are called framework regions (FWs). The native heavy and light chain variable domains each contain four FR regions, mostly employing a β-sheet configuration, connected by three CDRs, which form loops that connect to the β-sheet structure and, in some cases, form part of the β-sheet structure. The CDRs in each chain are held together in close proximity to the FW region and, together with the CDRs from the other chain, contribute to the formation of the antibody's antigen-binding site (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)). The constant domain generally does not directly participate in antigen binding, but it can influence antigen binding affinity and may exhibit various effector functions (e.g., antibody involvement in ADCC, CDC, and / or apoptosis).

[0038] As used herein, the term “hypervariable region” refers to the amino acid residues of an antibody that are involved in binding to an antigen. The hypervariable region consists of amino acid residues from the "complementarity-determining region" or "CDR" (e.g., residues 24-34 (VL CDR1), 50-56 (VL CDR2), and 89-97 (VL CDR3) in the light chain variable domain, and residues 31-35 (VH CDR1), 50-65 (VH CDR2), and 95-102 (VH CDR3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991)) and / or residues from the "hypervariable loop" (e.g., residues 26-32 (VL CDR1), 50-52 (VL CDR2), and 91-96 (VL CDR3) in the light chain variable domain, and residues 26-32 (VH CDR3) in the heavy chain variable domain). This includes CDR1), 53-55 (VH CDR2), and 96-101 (VH CDR3); Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987). A "framework" or "FW" residue is a variable domain residue adjacent to a CDR. FW residues are present in chimeric, humanized, human, domain antibodies, diabodies, vaccibodies, linear antibodies, and bispecific antibodies.

[0039] As used herein, the term “monoclonal antibody” refers to an antibody obtained from a substantially homogeneous antibody population (i.e., the individual antibodies constituting the population are identical except for minor naturally occurring variations that may be present). Monoclonal antibodies are highly specific and target a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations (typically containing different antibodies directed against different determinants (epitopes)), each monoclonal antibody targets a single determinant on an antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they can be synthesized in hybridoma cells that are not contaminated with other immunoglobulin-producing cells. Alternative methods of production are known to those skilled in the art; for example, monoclonal antibodies can be produced by cells that have been stably or transiently transfused with the heavy and light chain genes encoding the monoclonal antibody.

[0040] The modifier “monoclonal” refers to the characteristic of a population antibody obtained from substantially homogeneous antibodies and should not be interpreted as requiring any particular method of antibody manipulation. The term “monoclonal” is used herein to refer to antibodies derived from a clonal population of cells (including any eukaryote, prokaryote, or phage clone), and not to a method of antibody manipulation. For example, monoclonal antibodies used in accordance with the present invention may be prepared by the hybridoma method first described in Kohler et al., Nature, 256:495 (1975), or by any recombinant DNA method (see, e.g., U.S. Patent No. 4,816,567) (including isolation from phage antibody libraries using techniques described in, e.g., Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991)). These methods can be used to generate monoclonal mammals, chimeric antibodies, humanized antibodies, domain antibodies, diabodies, vaccibodies, linear antibodies, and bispecific antibodies.

[0041] The term “chimeric” antibody includes antibodies in which at least a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence of an antibody derived from a particular species or belonging to a particular antibody class or subclass, and at least another portion of the chain is identical or homologous to a corresponding sequence of an antibody derived from another species or belonging to another antibody class or subclass, and further includes fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primatized” antibodies that contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys such as baboons, rhesus macaques, or cynomolgus macaques) and a human constant region sequence (U.S. Patent No. 5,693,780).

[0042] The "humanized" form of a non-human (e.g., mouse) antibody is a chimeric antibody containing a minimal sequence derived from a non-human immunoglobulin. In most cases, a humanized antibody is a human immunoglobulin (recipient antibody) in which native CDR residues are replaced by corresponding CDR residues from a non-human species (donor antibody) (e.g., mouse, rat, rabbit, or non-human primate with desired specificity, affinity, and capabilities). In some examples, FW region residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, a humanized antibody may contain residues not found in either the recipient or donor antibody. Such modifications are made to further refine antibody performance. Generally, the heavy or light chain of a humanized antibody contains substantially all of at least one variable domain, where all or substantially all of the CDRs correspond to those of a non-human immunoglobulin, and all or substantially all of the FWs are from a human immunoglobulin sequence. In certain embodiments, the humanized antibody contains at least a portion of the immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0043] "Human antibodies" can be antibodies derived from humans, or antibodies obtained from transgenic organisms "engineered" to produce specific human antibodies in response to antigen challenge, and can be produced by any method known in the art. In certain techniques, elements of human heavy and light chain loci are introduced into a strain of organism derived from an embryonic stem cell line that includes targeted disruption of endogenous heavy and light chain loci. The transgenic organism can synthesize human antibodies specific to human antigens, and this organism can be used to produce human antibody-secreting hybridomas. Human antibodies may also be antibodies whose heavy and light chains are encoded by nucleotide sequences derived from one or more sources of human DNA. Fully human antibodies can also be constructed by gene or chromosome translocation methods, as well as phage display techniques or in vitro activated B cells, all of which are known in the art.

[0044] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) recognize bound antibodies on target cells, subsequently causing lysis of the target cells. In one embodiment, such cells are human cells. While we do not wish to limit ourselves to any particular mechanism of action, these cytotoxic cells that mediate ADCC generally express Fc receptors (FcRs). NK cells, the main cells that mediate ADCC, express FcγRIII, while monocytes express FcγRI, FcγRII, FcγRIII, and / or FcγRIV. FcR expression in hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991). To evaluate the ADCC activity of a molecule, in vitro ADCC assays such as those described in U.S. Patents 5,500,362 and 5,821,337 can be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the molecule of interest may be evaluated in vivo in animal models, for example, as disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998).

[0045] Complement-dependent cell injury, or CDC, refers to a molecule's ability to initiate complement activation in the presence of complement, thereby lysing its target. The complement activation pathway is initiated when the first component of the complement system (C1q) binds to a molecule (e.g., an antibody) that is complexed with a congener antigen. To assess complement activation, a CDC assay (e.g., described in Gazzano-Santaro et al., J.Immunol.Methods, 202:163 (1996)) can be performed.

[0046] "Effector cells" are white blood cells that express one or more FcRs and perform effector functions. These cells express at least FcγRI, FCγRII, FcγRII, and / or FcγRIV and perform ADCC effector functions. Examples of human white blood cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils.

[0047] The terms “Fc receptor” or “FcR” are used to describe receptors that bind to the Fc region of an antibody. In one embodiment, FcR is a native human FcR. Furthermore, in certain embodiments, FcR is one that binds to an IgG antibody (gamma receptor) and includes the FcγRI, FcγRII, FcγRII, and FcγRIV subclass receptors (including allele variants and alternative splicing forms of these receptors). FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) within its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine inhibitory motif (IT1M) within its cytoplasmic domain. (See Daeron, Annu. Rev. Immunol., 15:203-234 (1997)). FcRs are outlined in Ravetch and Kinet, Annu. Rev. Immunol., 9:457-92 (1991); Capel et al., Immunomethods, 4:25-34 (1994); and de Haas et al., J. Lab. Clin. Med., 126:330-41 (1995). Other FcRs (including those to be identified in the future) are also included in the term “FcR” as used herein. This term also includes the fetal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al., Immunol., 117:587 (1976) and Kim et al., J. Immunol., 24:249 (1994)).

[0048] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This region consists of a dimer of one heavy chain and one light chain variable domain, tightly bonded non-covalently or covalently. In this configuration, the three CDRs of each variable domain interact to define the antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of Fv containing only the three antigen-specific CDRs) has the ability to recognize and bind to the antigen, although with lower affinity than the entire binding site.

[0049] The “affinity” of an antibody against an epitope used in the therapeutic(s) described herein is a term well understood in the art and refers to the degree or strength of binding of the antibody to the epitope. Affinity can be measured and / or expressed by several methods known in the art (including, but not limited to, equilibrium dissociation constant (KD or Kd), apparent equilibrium dissociation constant (KD' or Kd'), and IC50 (the amount required to produce 50% inhibition in a competitive assay)). For the purposes of this invention, affinity should be understood as the average affinity to a given population of antibodies that bind to the epitope. The KD' values ​​reported herein in mg IgG / mL or mg / mL represent mg ​​Ig per serum mL, although plasma may also be used. When antibody affinity is used as a basis for administering or selecting the therapeutic(s) described herein, antibody affinity can be measured before and / or during treatment, and the obtained values ​​can be used by clinicians to assess whether a human patient is a suitable candidate for treatment.

[0050] As used herein, the term "avidity" is a measure of the overall binding strength (i.e., both antibody arms) of an antibody to an antigen. Avidity depends on three factors: (i) the affinity of the antibody to the epitope on the antigen, (ii) the valencies of both the antibody and the antigen, and (iii) the structural arrangement of the interacting moieties. Antibody avidity can be quantified by measuring the dissociation of antigen-antibody binding in antigen excess using any means known in the art (e.g., by modification of an indirectly fluorescent antibody, as described in Gray et al., J. Virol. Meth., 44:11-24. (1993), for example).

[0051] As used herein, the term “neutralizing antibody” refers to an antibody that reduces the ability of a pathogen to initiate or maintain infection within a host. A neutralizing anti-CoV S glycoprotein antibody is an antibody that reduces the ability of the SARS-CoV-2 virus or its variants to initiate or maintain infection within a host.

[0052] "Epitope" is a well-understood term in this art, referring to any chemical portion that specifically binds to an antibody. "Antigen" is the portion or molecule that contains the epitope, and therefore also specifically binds to an antibody.

[0053] As used herein, the term “antibody half-life” refers to the pharmacokinetic property of an antibody, which is a measure of the mean survival time of an antibody molecule after administration. Antibody half-life can be expressed, for example, in measurements in serum or plasma (i.e., circulating half-life) or other tissues, as the time required to eliminate 50 percent of a known amount of immunoglobulin from a patient’s body or a particular compartment of that body. Half-life can vary depending on the immunoglobulin or class of immunoglobulin. Generally, an increase in antibody half-life corresponds to an increase in the mean residence time (MRT) of the administered antibody in circulation.

[0054] The term "isotype" refers to the classification of the heavy and light chain constant regions of antibodies. The constant domain of an antibody does not participate in antigen binding but exhibits various effector functions. Depending on the amino acid sequence of the heavy chain constant region, a given human antibody or immunoglobulin is classified into one of five major immunoglobulin classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes can be further classified into subclasses (isotypes) (e.g., IgG1 (gamma 1), IgG2 (gamma 2), IgG3 (gamma 3), and IgG4 (gamma 4), as well as IgA1 and IgA2). The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional structures of various classes of immunoglobulins are well known. Of the various human immunoglobulins, only IgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. Human IgG1 and IgG3 are known to mediate ADCC in humans. The human light chain constant region is classified into two major classes: kappa and lambda.

[0055] As used herein, the term "immunogenic" means that a compound is capable of inducing an immune response (stimulation of the production of specific antibodies and / or the proliferation of specific T cells).

[0056] As used herein, the term “broad-spectrum neutralizing antibody” refers to an antibody or fragment thereof that binds to multiple heterologous SARS-CoV-2 S glycoproteins. In various embodiments, the broad-spectrum neutralizing antibody binds to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, at least fifteen, at least sixteen, at least seventeen, at least eighteen, at least nineteen, or at least twenty heterologous SARS-CoV-2 S glycoproteins. In various embodiments, the broad-spectrum neutralizing antibody binds to 2 to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 heterologous SARS-CoV-2 S glycoproteins. In various embodiments, the broad-spectrum neutralizing antibody binds to 2 to 10 heterologous SARS-CoV-2 S glycoproteins.

[0057] Antibodies that bind to SARS-CoV-2 spike polypeptide The present invention relates to SARS-CoV-2 spike polypeptides and antibodies (anti-CoV S glycoprotein antibodies) that bind to variants thereof, and compositions comprising such antibodies. The SARS-CoV-2 spike polypeptide ("CoV S glycoprotein") may contain the following amino acid sequence.

[0058]

[0059] In various embodiments, the CoV S glycoprotein contains an N-terminal signal peptide, which has the amino acid sequence of SEQ ID NO: 74. The signal peptide is underlined.

[0060] [ka] [ka]

[0061] The CoV S glycoprotein (SEQ ID NO: 73) is divided into an S1 subunit (amino acids 1-672 of SEQ ID NO: 73) and an S2 subunit (amino acids 673-1260 of SEQ ID NO: 73). The S1 subunit is further divided into an N-terminal domain (NTD, amino acids 1-318 of SEQ ID NO: 73), a receptor-binding domain (RBD, amino acids 318-514 of SEQ ID NO: 739), subdomains 1 and 2 (SD1 / 2, amino acids 529-668 of SEQ ID NO: 73), and a furin cleavage site (amino acids 669-672 of SEQ ID NO: 73). The S2 subunit includes an HR1 domain (amino acids 889-971 of SEQ ID NO: 73), an HR2 domain (amino acids 1150-1200 of SEQ ID NO: 73), a transmembrane domain (TM, amino acids 1201-1224 of SEQ ID NO: 73), and a cytoplasmic domain (CD, amino acids 1225-1260 of SEQ ID NO: 73). In various embodiments, the anti-CoV S glycoprotein antibody binds to the S1 subunit, S2 subunit, NTD, RBD, furin cleavage site, HR1 domain, TM domain, CD, or a combination thereof of the SARS-CoV 2 S glycoprotein.

[0062] In various embodiments, the CoV S glycoprotein is up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, It has the modifiers 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0063] Exemplary modifications to the CoV S glycoprotein are shown in the table below. [Table 3-1] [Table 3-2] [Table 3-3] [Table 3-4] [Table 3-5] [Table 3-6] [Table 3-7] [Table 3-8] [Table 3-9] [Table 3-10] [Table 3-11] [Table 3-12] [Table 3-13]

[0064] In various embodiments, the SARS-CoV-2 S glycoprotein has a sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of sequence numbers 65-72.

[0065] In various embodiments, anti-SARS-CoV-2 spike (S) glycoprotein antibodies can mediate antigen-dependent cell-mediated cytotoxicity (ADCC). In various embodiments, the present invention relates to anti-CoV S glycoprotein antibodies of the IgG1, IgG2, IgG3, IgG4, or IgG5 isotype. In various embodiments, the antibodies mediate human ADCC, CDC, and / or apoptosis.

[0066] In one embodiment, the anti-SARS-CoV-2 glycoprotein antibody comprises a variable heavy chain (VH) and a variable light chain (VL). In various embodiments, the anti-SARS-CoV-2 glycoprotein antibody comprises a VL having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-4. In various embodiments, the anti-SARS-CoV-2 glycoprotein antibody comprises a VH having an amino acid sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any five of SEQ ID NOs: 5-8, or at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-8. In various embodiments, the anti-SARS-CoV-2 glycoprotein antibody comprises VL of SEQ ID NO: 1, or VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 1, and VH of SEQ ID NO: 5, or VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 5. In various embodiments, the anti-SARS-CoV-2 glycoprotein antibody comprises VL of SEQ ID NO: 2, or VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 2, and VH of SEQ ID NO: 6, or VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 6.In various embodiments, the anti-SARS-CoV-2 glycoprotein antibody comprises VL of SEQ ID NO: 3, or VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 3, and VH of SEQ ID NO: 7, or VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 7. In various embodiments, the anti-SARS-CoV-2 glycoprotein antibody comprises VL of SEQ ID NO: 4, or VL that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 4, and VH of SEQ ID NO: 8, or VH that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 8.

[0067] In some embodiments, the VL of SEQ ID NOs: 1-4 includes an N-terminal leader sequence. Up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids can be removed from any one of the N-terminal leader sequences of SEQ ID NOs: 1-4. In some embodiments, this specification provides antibodies containing VL without an N-terminal leader sequence. In some embodiments, the VH of SEQ ID NOs: 5-8 includes an N-terminal leader sequence. Up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids can be removed from any one of the N-terminal leader sequences of SEQ ID NOs: 5-8. In some embodiments, this specification provides antibodies containing VH without an N-terminal leader sequence. In some embodiments, the antibodies described herein contain up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids from the N-terminal leader sequence of VH or VL.

[0068] Antibodies NVX.62.12, NVX.172.10, NVX.205.10, and NVX 324.6 were identified from hybridomas generated from mice immunized with a two-dose primary series of XBB.1.5 rS.

[0069] In various embodiments, VL and VH are selected from Table 1 below. [Table 1]

[0070] In some embodiments, the anti-CoV S glycoprotein antibody includes a heavy chain variable complementarity determination region 1 (VH CDR1) having one of the amino acid sequences of SEQ ID NOs. 21, 24, 27, and 30. In some embodiments, the anti-CoV S glycoprotein antibody includes a heavy chain variable complementarity determination region 2 (VH CDR2) having one of the amino acid sequences of SEQ ID NOs. 22, 25, 28, and 31. In some embodiments, the anti-CoV S glycoprotein antibody includes a heavy chain variable complementarity determination region 3 (VH CDR3) having one of the amino acid sequences of SEQ ID NOs. 23, 26, 29, and 32. In some embodiments, the anti-CoV S glycoprotein antibody includes a light chain variable complementarity determination region 1 (VL CDR1) having one of the amino acid sequences of SEQ ID NOs. 9, 12, 15, and 18. In some embodiments, the anti-CoV S glycoprotein antibody includes a light chain variable complementarity determination region 1 (VL CDR2) having any two amino acid sequences from SEQ ID NOs. 10, 13, 16, and 19. In some embodiments, the anti-CoV S glycoprotein antibody includes a light chain variable complementarity determination region 1 (VL CDR3) having any three amino acid sequences from SEQ ID NOs. 11, 14, 17, and 20.

[0071] In various embodiments, this specification provides anti-CoV S glycoprotein antibodies comprising VL CDR1 selected from the group consisting of SEQ ID NOs: 9, 12, 15, and 18; VL CDR2 selected from the group consisting of SEQ ID NOs: 10, 13, 16, and 19; VL CDR3 selected from the group consisting of SEQ ID NOs: 11, 14, 17, and 20; VH CDR1 selected from the group consisting of SEQ ID NOs: 21, 24, 27, and 30; VH CDR2 selected from the group consisting of SEQ ID NOs: 22, 25, 28, and 31; and VH CDR3 selected from the group consisting of SEQ ID NOs: 23, 26, 29, and 32.

[0072] In various embodiments, VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2, and VL CDR3 are independently selected from Table 2. [Table 2]

[0073] In various embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 21, VH CDR2 of SEQ ID NO: 22, and VH CDR3 of SEQ ID NO: 23. In various embodiments, the anti-CoV S glycoprotein antibody includes VL CDR1 of SEQ ID NO: 9, VL CDR2 of SEQ ID NO: 10, and VL CDR3 of SEQ ID NO: 11. In various embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 21, VH CDR2 of SEQ ID NO: 22, and VH CDR3 of SEQ ID NO: 23, as well as VL CDR1 of SEQ ID NO: 9, VL CDR2 of SEQ ID NO: 10, and VL CDR3 of SEQ ID NO: 11.

[0074] In some embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 24, VH CDR2 of SEQ ID NO: 25, and VH CDR3 of SEQ ID NO: 26. In some embodiments, the anti-CoV S glycoprotein antibody includes VL CDR1 of SEQ ID NO: 12, VL CDR2 of SEQ ID NO: 13, and VL CDR3 of SEQ ID NO: 14. In some embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 24, VH CDR2 of SEQ ID NO: 25, and VH CDR3 of SEQ ID NO: 26, as well as VL CDR1 of SEQ ID NO: 12, VL CDR2 of SEQ ID NO: 13, and VL CDR3 of SEQ ID NO: 14.

[0075] In some embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 27, VH CDR2 of SEQ ID NO: 28, and VH CDR3 of SEQ ID NO: 29. In some embodiments, the anti-CoV S glycoprotein antibody includes VL CDR1 of SEQ ID NO: 15, VL CDR2 of SEQ ID NO: 16, and VL CDR3 of SEQ ID NO: 17. In some embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 27, VH CDR2 of SEQ ID NO: 28, and VH CDR3 of SEQ ID NO: 29, as well as VL CDR1 of SEQ ID NO: 15, VL CDR2 of SEQ ID NO: 16, and VL CDR3 of SEQ ID NO: 17.

[0076] In some embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 30, VH CDR2 of SEQ ID NO: 31, and VH CDR3 of SEQ ID NO: 32. In some embodiments, the anti-CoV S glycoprotein antibody includes VL CDR1 of SEQ ID NO: 18, VL CDR2 of SEQ ID NO: 19, and VL CDR3 of SEQ ID NO: 20. In some embodiments, the anti-CoV S glycoprotein antibody includes VH CDR1 of SEQ ID NO: 30, VH CDR2 of SEQ ID NO: 31, and VH CDR3 of SEQ ID NO: 32, as well as VL CDR1 of SEQ ID NO: 18, VL CDR2 of SEQ ID NO: 19, and VL CDR3 of SEQ ID NO: 20.

[0077] The present invention encompasses antibodies that bind to the CoV S glycoprotein and include derivatives of the VH domain, VH CDR1, VH CDR2, VH CDR3, VL domain, VL CDR1, VL CDR2, or VL CDR3 described herein, which can bind to the SARS-CoV 2 S glycoprotein or its variants. In various embodiments, the anti-CoV S glycoprotein antibody binds to the CoV S glycoprotein of a SARS-CoV-2 strain having a PANGO lineage selected from the group consisting of B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, B.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1.

[0078] Modifications (e.g., additions, deletions, and / or substitutions) can be introduced into the nucleotide sequence encoding the antibody using standard techniques known to those skilled in the art, such techniques include, for example, site-directed mutagenesis and PCR-mediated mutagenesis, which are commonly used to generate amino acid substitutions. In another embodiment, the VH and / or VK CDR derivatives may have conserved amino acid substitutions (see, for example, above) made at one or more predicted non-essential amino acid residues (i.e., amino acid residues not determinant to the specific binding of the antibody to the SARS-CoV-2 S glycoprotein). Mutations can also be introduced randomly along all or part of the VH and / or VL CDR coding sequence, for example by saturation mutagenesis, and the biological activity of the resulting mutants can be screened to identify mutants that retain activity. After mutagenesis, the coding antibody can be expressed, and the antibody activity can be quantified.

[0079] The present invention further includes antibodies that bind to the SARS-CoV-2 S glycoprotein, wherein the antibody or antibody fragment comprises one or more CDRs, the CDRs comprising an amino acid sequence that is at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequences of one or more CDRs described herein. The percentage of identity of the two amino acid sequences can be quantified by any method known to those skilled in the art (including, but not limited to, BLAST protein lookup).

[0080] Framework area of ​​anti-CoV S glycoprotein antibodies

[0081] In various embodiments, the anti-CoV S glycoprotein antibody comprises VL and VH, each containing four framework regions (FW1, FW2, FW3, and FW4). In various embodiments, FW1, FW2, FW3, and FW4 of VL are independently selected from Table 4. In various embodiments, FW1, FW2, FW3, and FW4 of VH are independently selected from Table 5. [Table 4] [Table 5]

[0082] Kabat numbering is based on the important work product entitled Kabat et al. (1991) Sequences of Proteins of Immunological Interest, Publication No. 91-3242 (3 volumes in total, published by National Institutes of Health, National Technical Information Service) (hereinafter referred to as "Kabat"). Kabat provides multiple sequence alignments of immunoglobulin chains from antibody isotypes of numerous species. The aligned sequences are numbered according to the single numbering system, the Kabat numbering system. The Kabat sequences have been updated since their 1991 publication and are available as an electronic sequence database (the latest downloadable version is 1997). Any immunoglobulin sequence can be numbered according to Kabat by performing an alignment with the Kabat reference sequence. Accordingly, the Kabat numbering system provides a unified framework for the numbering of immunoglobulin chains. Unless otherwise indicated, all immunoglobulin amino acid sequences described herein are numbered according to the Kabat numbering system. Similarly, all single amino acid positions referred to herein are numbered according to the Kabat numbering system.

[0083] In another embodiment, the affinity constant or K of the anti-CoV S glycoprotein antibody of the present invention a (k on / k off ) is at least 10 2 M -1 , at least 5×10 2 M -1 , at least 10 3 M -1 , at least 5×10 3 M -1 , at least 10 4 M -1 , at least 5×10 4 M -1 , at least 10 5 M -1, at least 5 × 10 5 M -1 , at least 10 6 M -1 , at least 5 × 10 6 M -1 , at least 10 7 M -1 , at least 5 × 10 7 M -1 , at least 10 8 M -1 , at least 5 × 10 8 M -1 , at least 10 9 M -1 , at least 5 × 10 9 M -1 , at least 10 10 M -1 , at least 5 × 10 10 M -1 , at least 10 11 M -1 , at least 5 × 10 11 M -1 , at least 10 12 M -1 , at least 5 × 10 12 M -1 , at least 10 13 M -1 , at least 5 × 10 13 M -1 , at least 10 14 M -1 , at least 5 × 10 14 M -1 , at least 10 15 M -1 , or at least 5 × 10 15 M -1 This may be the case. In various embodiments, the dissociation constant or K of the anti-CoV S glycoprotein antibody of the present invention d (k off / k on ) was evaluated using the method described herein or a method known to those skilled in the art (e.g., BIAcore assay, ELISA), with a value of 5 × 10 -2 Less than M, 10 -2 Less than M, 5 x 10 -3 Less than M, 10 -3 Less than M, 5 x 10-4 less than M, 10 -4 less than M, 5×10 -5 less than M, 10 -5 less than M, 5×10 -6 less than M, 10 -6 less than M, 5×10 -7 less than M, 10 -7 less than M, 5×10 -8 less than M, 10 -8 less than M, 5×10 -9 less than M, 10 -9 less than M, 5×10 -10 less than M, 10 -10 less than M, 5×10 -11 less than M, 10 -11 less than M, 5×10 -12 less than M, 10 -12 less than M, 5×10 -13 less than M, 10 -13 less than M, 5×10 -14 less than M, 10 -14 less than M, 5×10 -15 less than M, or 10 -15 less than M.

[0084] The present invention further provides a polynucleotide comprising a nucleotide sequence encoding the anti-CoV S glycoprotein antibody or fragment thereof described herein. The present invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions (e.g., as defined herein) to a polynucleotide encoding an anti-CoV S glycoprotein antibody.

[0085] Stringent hybridization conditions include, but are not limited to, hybridizing the filter-bound DNA in 6× sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by one or more washes in 0.2× SSC / 0.1% SDS at approximately 50-65°C, highly stringent conditions (e.g., hybridizing the filter-bound DNA in 6× SSC at approximately 45°C, followed by one or more washes in 0.1× SSC / 0.2% SDS at approximately 60°C), or any other stringent hybridization conditions known to those skilled in the art (e.g., see Ausubel, FM et al., eds. 1989 Current Protocols in Molecular Biology, vol. 1, Green Publishing Associates, Inc. and John Wiley and Sons, Inc., NY (pp. 6.3.1-6.3.6 and 2.10.3)).

[0086] Polynucleotides can be obtained by any method known in the art, and the nucleotide sequence of the polynucleotide can be determined. For example, if the nucleotide sequence of an antibody is known, the polynucleotide encoding the antibody can be assembled from chemically synthesized oligonucleotides (as described, for example, in Kutmeier et al., BioTechniques 17:242 (1994)). This method, briefly described, involves the synthesis of overlapping oligonucleotides containing a portion of the antibody-encoding sequence, annealing and ligation of these oligonucleotides, and amplification of the ligated oligonucleotides by PCR.

[0087] Antibody-encoding polynucleotides can also be generated from nucleic acids from suitable sources. If a clone containing nucleic acid encoding a particular antibody is unavailable, but the sequence of the antibody molecule is known, the nucleic acid encoding the immunoglobulin can be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from any tissue or cell expressing the antibody (e.g., hybridoma cells selected to express the antibody), or nucleic acids isolated therefrom (preferably polyA+RNA)) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence, or by cloning using oligonucleotide probes specific to a particular gene sequence, for example, by identifying a cDNA clone from an antibody-encoding cDNA library. The amplified nucleic acid generated by PCR can then be cloned into a replicable cloning vector using any method well known in the art.

[0088] Furthermore, the present invention also provides polynucleotide sequences encoding the VH and VL framework regions and CDR of the antibodies described herein, as well as expression vectors for efficiently expressing them in mammalian cells.

[0089] In one embodiment, the anti-CoV S glycoprotein antibody described herein mediates antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cell-mediated cytotoxicity (CDC), and / or apoptosis. In one embodiment, the anti-CoV S glycoprotein antibody of the present invention mediates antibody-dependent cell-mediated cytotoxicity (ADCC) and / or apoptosis. In one embodiment, the anti-CoV S glycoprotein antibody of the present invention enhances antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the anti-CoV S glycoprotein antibody of the present invention includes a variant Fc region that mediates the enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC). In one embodiment, the anti-CoV S glycoprotein antibody of the present invention includes an Fc region having a complex N-glycosidic glycan linked to Asn297, where fucose is not bound to N-acetylglucosamine at the reducing end, and the Fc region mediates the enhancement of antibody-dependent cell-mediated cytotoxicity (ADCC).

[0090] Production of humanized anti-CoV S glycoprotein antibodies The humanized antibodies described herein can be produced using a variety of techniques known in the art, including, but not limited to, CDR grafting (see, e.g., European Patent No. 239,400; International Publication No. WO91 / 09967; and U.S. Patents No. 5,225,539, 5,530,101, and 5,585,089 (each of which is incorporated herein by reference in whole)), veneering, or resurfacing (see, e.g., European Patents No. EP592,106 and EP519,596; Padlan, 1991, Molecular Immunology 28(4 / 5):489-498; Studnicka et al., 1994, Protein Engineering, 7(6):805-814; and Roguska et al.) See, for example, al., 1994, Proc. Natl. Acad. Sci., 91:969-973 (each of these in whole is incorporated herein by reference), chain shuffling (see, for example, U.S. Patent No. 5,565,332 (each of these in whole is incorporated herein by reference)), as well as, for example, U.S. Patent No. 6,407,213, U.S. Patent No. 5,766,886, International Publication No. WO9317105, Tan et al., J. Immunol., 169:1119-25 (2002), Caldas et al., Protein Eng., 13(5):353-60 (2000), Morea et al., Methods, 20(3):267-79 (2000), Baca et al. Examples include the techniques disclosed in al., J. Biol. Chem., 272(16):10678-84 (1997), Roguska et al., Protein Eng., 9(10):895-904 (1996), Couto et al., Cancer Res., 55(23 Supp):5973s-5977s (1995), Couto et al., Cancer Res., 55(8):1717-22 (1995), Sandhu JS, Gene, 150(2):409-10 (1994), and Pedersen et al., J. Mol. Biol., 235(3):959-73 (1994) (each of these in whole is incorporated herein by reference).In various embodiments, humanized antibodies are generated using phage display, framework homologous germline-based humanization, or germline humanization that preserves the vernier zone. Often, antigen binding is modified, preferably improved, by substituting FW residues within the FW region with corresponding residues from the CDR donor antibody. These FW substitutions are identified by methods well known in the art, for example, by modeling the interaction between CDR and FW residues to identify FW residues important for antigen binding, or by sequence comparison to identify abnormal FW residues at specific locations (see, e.g., Queen et al., U.S. Patent No. 5,585,089, and Riechmann et al., 1988, Nature, 332:323 (which is incorporated herein by reference)).

[0091] Humanized anti-CoV S glycoprotein antibodies have one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as “import” residues and are typically obtained from “import” variable domains. Thus, humanized antibodies contain one or more CDRs derived from non-human immunoglobulin molecules and a human-derived framework region. Antibody humanization is well known in the art and is essentially the method of Winter and his collaborators (Jones et al. Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al.). Humanized chimeric antibodies can be created by substituting a rodent CDR or CDR sequence with a corresponding sequence in a human antibody, i.e., by CDR grafting (EP239,400; PCT Publication No. WO91 / 09967; and U.S. Patents No. 4,816,567; No. 6,331,415; No. 5,225,539; No. 5,530,101; No. 5,585,089; No. 6,548,640; the contents of these are incorporated herein by reference in their entirety). In such humanized chimeric antibodies, a domain significantly smaller than the intact human variable domain is substituted with a corresponding sequence derived from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues, and possibly some FW residues, are substituted with residues from similar sites in a rodent antibody. Humanization of anti-CoV S glycoprotein antibodies can also be achieved by veneering or resurfacing (EP592,106;EP519,596;Padlan,1991,Molecular Immunology 28(4 / 5):489-498;Studnicka et al.,Protein Engineering,7(6):805-814(1994);and Roguska et al.,Proc.Natl.Acad.Sci.,91:969-973(1994)) or chain shuffling (U.S. Patent No. 5,565,332), the entirety of which is incorporated herein by reference.

[0092] The selection of human variable domains (both light and heavy chains) used for humanized antibody production is intended to reduce antigenicity. According to the so-called "best-fit" method, the variable domain sequences of rodent antibodies are screened against an entire library of known human variable domain sequences. Next, the human sequences most closely related to the rodent sequences are screened for the presence of specific residues that may be important for antigen binding, proper structural formation, and / or the stability of the intended humanized mAb (Sims et al., J.Immunol., 151:2296 (1993); Chothia et al., J.Mol.Biol., 196:901 (1987); the contents of these are incorporated herein by reference in their entirety). The resulting FW sequences that match the desired criteria are used as the human donor FW regions for the humanized antibodies.

[0093] Alternatively, a specific FW (Forward Fluctuation) is used, derived from the consensus sequence of all human antibodies in a particular subgroup of the light or heavy chain. The same FW can be used for several different humanized anti-CoV S glycoprotein antibodies (Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta et al., J. Immunol., 151:2623 (1993); these contents are incorporated herein by reference in their entirety).

[0094] Anti-CoV S glycoprotein antibodies can be humanized while retaining high affinity for SARS-CoV-2 S glycoprotein and other desirable biological properties. According to one aspect of the present invention, humanized antibodies are prepared by an analytical process of the parent sequence and humanized products of various concepts using three-dimensional models of the parent and humanized sequences. Three-dimensional immunoglobulin models are generally available and well known to those skilled in the art. Computer programs are available that illustrate and display the possible three-dimensional structures of selected candidate immunoglobulin sequences. By examining these representations, it is possible to analyze the roles that residues are thought to play in the function of the candidate immunoglobulin sequence, i.e., the residues that affect the ability of the candidate immunoglobulin to bind to SARS-CoV-2 S glycoprotein. In this way, FW residues can be selected and combined from the recipient sequence and import sequence so that the desired antibody properties, such as affinity for SARS-CoV-2 S glycoprotein, are achieved. Generally, CDR residues have the most direct and significant impact on antigen binding.

[0095] Humanized antibodies can retain the same antigen specificity as the original antibody, i.e., the ability to bind to the SARS-CoV-2 S glycoprotein in this invention. However, using a specific method of humanization, the affinity and / or specificity of the antibody binding to the SARS-CoV-2 S glycoprotein can be altered using a method of "directional evolution" as described in Wu et al., J.Mol.Biol, 294:151 (1999) (the entire text of which is incorporated herein by reference).

[0096] The humanized anti-CoV S glycoprotein antibodies described herein can be constructed by selecting different human framework regions for implantation of 239.12, 322.3, 425.6, and 35.13 CDR as described herein.

[0097] Monoclonal anti-CoV S glycoprotein antibody Monoclonal anti-CoV S glycoprotein antibodies exhibit binding specificity to the SARS-CoV-2 antigen and can mediate human ADCC, CDC, and / or apoptotic mechanisms. Such antibodies can be produced using a wide variety of techniques known in the art, including the use of hybridomas, recombinants, and phage display techniques, or combinations thereof. The antibodies are highly specific and target a single antigenic site. Manipulated anti-CoV S glycoprotein antibodies can be produced by any means known in the art (including, but not limited to, the techniques described below and improvements thereof). Large-scale, high-yield production typically involves culturing host cells that produce manipulated anti-CoV S glycoprotein antibodies and recovering the anti-CoV S glycoprotein antibodies from the host cell cultures.

[0098] Hybridoma Technique Monoclonal antibodies can be purified using hybridoma techniques, which are known in the art and are taught, for example, in Harlow et al., Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); and Hammerling et al., in Monoclonal Antibodies and T Cell Hybridomas, 563-681 (Elsevier, NY, 1981) (the aforementioned references are incorporated herein by reference in their entirety). For example, in the hybridoma method, mice or other suitable host animals (e.g., hamsters or macaques) are immunized to produce antibodies that specifically bind to the proteins used for immunization, or to induce lymphocytes capable of producing such antibodies. Lymphocytes can also be immunized in vitro. Next, lymphocytes are fused with myeloma cells using a suitable fusion agent (e.g., polyethylene glycol) to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0099] The hybridoma cells prepared in this manner are seeded and grown in a suitable culture medium containing one or more substances that inhibit the growth or survival of unfused parent myeloma cells. For example, if parent myeloma cells do not contain the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibit the growth of HGPRT-deficient cells.

[0100] In specific embodiments, myeloma cells are used that efficiently fuse, support stable high-level antibody production by selected antibody-producing cells, and are sensitive to culture media such as HAT medium. These myeloma cell lines include mouse myeloma strains (e.g., those derived from MOPC-21 and MPC-11 mouse tumors available from Salk Institute Cell Distribution Center, San Diego, CA, USA, and SP-2 or X63-Ag8.653 cells available from American Type Culture Collection, Rockville, MD, USA). Human myeloma and mouse-human heterozygous myeloma cell lines have also been described for human monoclonal antibody production (Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

[0101] The production of monoclonal antibodies against SARS-CoV-2 S glycoprotein is assayed in a culture medium in which hybridoma cells are growing. The binding specificity of the monoclonal antibodies produced by hybridoma cells can be quantified by immunoprecipitation or in vitro binding assay (e.g., radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)).

[0102] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity, and / or activity, the clones can be subcloned using limiting dilution procedures and grown using standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable media for this purpose include, for example, D-MEM or RPMI1640 medium. Furthermore, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0103] Monoclonal antibodies secreted by subclones can be suitably separated from culture media or serum by conventional immunoglobulin purification procedures, such as protein A-Sepharose chromatography, hydroxyl apatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0104] Recombinant DNA techniques The DNA encoding the anti-CoV S glycoprotein antibody described herein can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the anti-CoV S glycoprotein antibody). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed in an expression vector and then transfused into host cells (e.g., E. coli cells that do not separately produce immunoglobulin proteins, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells) to synthesize the anti-CoV S glycoprotein antibody in recombinant host cells.

[0105] In phage display, a functional antibody domain is displayed on the surface of a phage particle that has the polynucleotide sequence encoding it. For more details, see V H and V L The DNA sequence encoding the domain is amplified from an animal cDNA library (e.g., a human or mouse cDNA library from the affected tissue).H and V L The DNA encoding the domain is recombined together with the scFv linker by PCR, and cloned into a phagemid vector. The vector is electroporated into E. coli, and the E. coli is infected with helper phage. Phages used in these methods are typically filamentous phages including fd and M13, and V II domain and V L domain are usually recombinantly fused to either phage gene III or gene VIII. Phages expressing an antigen-binding domain that binds to a specific antigen can be selected or identified with the antigen (e.g., using labeled antigen or antigen that is bound or captured to a solid surface or beads). Examples of phage display methods that can be used to generate the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods, 182:41-50; Ames et al., 1995, J. Immunol. Methods, 184:177-186; Kettleborough et al., 1994, Eur. J. Immunol., 24:952-958; Persic et al., 1997, Gene, 187:9-18; Burton et al., 1994, Advances in Immunology, 57:191-280; International Application No. PCT / GB91 / 01134; International Publication Nos. WO90 / 02809, WO91 / 10737, WO92 / 01047, WO92 / 18619, WO93 / 11236, WO95 / 15982, WO95 / 20401, and WO97 / 13844; as well as U.S. Patent Nos. 5,698,426, 5,223,409, 5,403,484, 5,580,717, 5,427,908, 5,750,753, 5,821,047, 5,571,698, 5,427,908, 5,516,637, 5,780,225, 5,658,727, 5,733,743, and 5,969,108 (each of which is incorporated herein by reference in its entirety).

[0106] As described in the references above, after phage selection, the antibody-coding region from the phage can be isolated and used to generate a whole antibody (including human antibodies) or any other desired antigen-binding fragment, which can then be used to express any desired host (including mammalian cells, insect cells, plant cells, yeast, and bacteria) as described below, for example. Alternatively, techniques for recombinantly generating Fab, Fab', and F(ab')2 fragments can be employed using methods known in the art (e.g., methods disclosed in PCT Publication WO92 / 22324; Mullinax et al. 1992, BioTechniques, 12(6):864-869; Sawai et al., 1995, AJRI, 34:26-34; and Better et al., 1988, Science, 240:1041-1043 (the aforementioned references are incorporated in their entirety by reference)).

[0107] Antibodies can be isolated from antibody phage libraries generated using the technique described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J.Mol.Biol., 222:581-597 (1991) describe the isolation of mouse and human antibodies, respectively, using phage libraries. Chain shuffling can be used to generate high-affinity (nM range) human antibodies (Marks et al., Bio / Technology, 10:779-783 (1992)), and can also be used for combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al., Nuc.Acids.Res, 21:2265-2266 (1993)). Therefore, these techniques represent a promising alternative to conventional monoclonal antibody hybridoma techniques for the isolation of anti-CoV S glycoprotein antibodies.

[0108] To generate the whole antibody, the VH or VL sequence in the scFv clone can be amplified using PCR primers containing the VH or VL nucleotide sequence, a restriction site, and a flanking sequence to protect the restriction site. Using cloning techniques known to those skilled in the art, the PCR-amplified VH domain can be cloned into a vector expressing a heavy chain constant region, e.g., the human gamma-4 constant region, and the PCR-amplified VL domain can be cloned into a vector expressing a light chain constant region (e.g., the human kappa or lambda constant region). The vector for expressing the VH or VL domain may include an EF-la promoter, a secretion signal, a cloning site for a variable domain, a constant domain, and a selection marker (e.g., neomycin). Alternatively, the VH and VL domains may be cloned into a single vector expressing the required constant region. The heavy chain and light chain conversion vectors are then simultaneously transfected into a cell line to generate a stable or transient cell line expressing the full-length antibody (e.g., IgG) using techniques known to those skilled in the art.

[0109] Furthermore, DNA can also be modified, for example, by substituting the coding sequences of human heavy and light chain constant domains with homologous mouse sequences (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently bonding all or part of the coding sequence of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0110] Chimeric antibodies The anti-CoV S glycoprotein antibodies described herein explicitly include “chimeric” antibodies (immunoglobulins) in which a portion of the heavy chain and / or light chain is identical or homologous to a corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and further include fragments of such antibodies insofar as they exhibit the desired biological activity (U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include “primatized” antibodies that contain a variable domain antigen-binding sequence derived from a non-human primate (e.g., Old World monkeys such as baboons, rhesus macaques, or cynomolgus macaques) and a human constant region sequence (U.S. Patent No. 5,693,780).

[0111] In various embodiments, the K of the anti-CoV S glycoprotein antibody described herein D , or those of SARS-CoV-2 S glycoprotein, may be ≤50 nM, ≤10 nM, ≤1 nM, ≤0.5 nM, ≤0.1 nM, ≤0.05 nM, ≤0.01 nM, or ≤0.001 nM. Methods and reagents suitable for quantifying such binding properties of the antibodies of the present invention, or their modified / mutant derivatives, are known in the art and / or commercially available (see above, as well as, for example, U.S. Patents 6,849,425, 6,632,926, 6,294,391, and 6,143,574 (each of these in whole is incorporated herein by reference)). Furthermore, instruments and software designed for such kinetic analysis are commercially available (e.g., Biacore® A100 and Biacore® 2000 instruments; Biacore International AB, Uppsala, Sweden).

[0112] Sequence identity or similarity is defined herein as the percentage of amino acid residues in a candidate sequence that are identical (i.e., the same residues) or similar (i.e., amino acids from the same group based on common side-chain properties, see below) to the anti-CoV S glycoprotein antibody after the sequences have been aligned and gaps introduced where necessary to achieve the maximum sequence identity percentage. N-terminus, C-terminus, or internal extensions, deletions, or insertions into the antibody sequence other than the variable domain are not considered to affect sequence identity or similarity.

[0113] Methods for comparing the identity of two or more sequences are well known in the art. The identity percentage is calculated using a tool called CLUSTALW2, which is available online. The following default parameters can be used for CLUSTALW2 pairwise alignment: Protein Weight Matrix=Gonnet; Gap Open=10; Gap Extension=0.1. Unless otherwise stated, this specification uses the CLUSTALW2 tool to calculate the identity percentage.

[0114] To generate modified antibodies, one or more amino acid modifications (e.g., substitutions) are introduced into one or more of the hypervariable regions of a species-dependent antibody. Modifications (e.g., substitutions) of one or more framework region residues can also be introduced into anti-CoV S glycoprotein antibodies, resulting in improved binding affinity of the antibody variant to an antigen from a second mammalian species. Examples of framework region residue modifications include direct non-covalent binding to the antigen (Amit et al., Science, 233:747-753 (1986)), interaction / effect on the conformation of the CDR (Chothia et al., J.Mol.Biol., 196:901-917 (1987)), and / or V L- V HOne example is interface involvement (EP 239 440B1). In certain embodiments, modifications of one or more such framework region residues result in enhanced antibody binding affinity to antigens derived from a second mammalian species. For example, in this embodiment of the present invention, about 1 to about 5 framework residues may be modified. In some cases, this may be sufficient to obtain antibody variants suitable for use in preclinical trials, even if no residues in the hypervariable region are modified. However, modified antibodies typically involve further modifications (may be more) to the hypervariable region.

[0115] The hypervariable region residues to be modified may be changed randomly, especially if the starting binding affinity of the anti-CoV S glycoprotein antibody to the antigen of the second mammalian species allows for easy screening of such randomly generated modified antibodies.

[0116] One useful procedure for generating such modified antibodies is called "alanine scanning mutagenesis" (Cunningham and Wells, Science, 244:1081-1085 (1989)). Here, one or more hypervariable region residues are replaced with alanine or polyalanine residues to affect the amino acid interaction with an antigen from a second mammalian species. In the case of hypervariable region residues that are functionally sensitive to substitution, the substitution site is refined by introducing additional or other mutations at or to the substitution site. Thus, the site for introducing amino acid sequence variations is predetermined, while the nature of the mutation itself does not need to be predetermined. The Ala variants thus generated are then screened for their biological activity as described herein.

[0117] Another procedure for generating such modified antibodies involves affinity maturation using phage display (Hawkins et al., J.Mol.Biol., 254:889-896 (1992) and Lowman et al., Biochemistry, 30(45):10832-10837 (1991)). Briefly, several hypervariable regions (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent form from filamentous phage particles as fusions with the M13 gene 111 product packaged within each particle. The biological activity (e.g., binding affinity) of the phage-displayed variants is then screened as disclosed herein.

[0118] Mutations in antibody sequences include substitutions, deletions (including internal deletions), additions (including additions resulting in fusion proteins), or conservative substitutions of amino acid residues within and / or adjacent to the amino acid sequence. As a result, these changes are "silent" in that they produce functionally equivalent anti-CoV S glycoprotein antibodies. Conservative amino acid substitutions can be made based on the similarity of polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphiphilicity of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and charged (acidic) amino acids include aspartic acid and glutamic acid. Furthermore, glycine and proline are residues that can affect chain orientation. Non-conservative substitutions involve exchanging members of one class for members of another. Additionally, if desired, non-classical amino acids or chemical amino acid analogs can be introduced into the antibody sequence as substitutions or additions. Non-classical amino acids include, but are not limited to, D-isomers of common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids, designer amino acids such as β-methylamino acids, Cα-methylamino acids, Nα-methylamino acids, and general amino acid analogs.

[0119] In another embodiment, the site selected for modification is affinity-matured using phage display (see above).

[0120] Individual nucleotides in a DNA sequence can be modified using any mutagenesis technique known in the art, for the purpose of making amino acid substitutions(s) within an antibody sequence, or to create / delete restriction sites to facilitate further manipulation. Such techniques are not limited to, but include chemical mutagenesis, in vitro site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA, 82:488 (1985); Hutchinson, C. et al., J. Biol. Chem., 253:6551 (1978)), oligonucleotide-directed mutagenesis (Smith, Ann. Rev. Genet., 19:423-463 (1985); Hill et al., Methods Enzymol., 155:558-568 (1987)), PCR-based overlap extension (Ho et al., Gene, 77:51-59 (1989)), and PCR-based megaprimer mutagenesis (Sarkar et al., Biotechniques, 8:404-407 (1990)). Modifications can be confirmed by double-stranded dideoxyDNA sequencing.

[0121] In certain embodiments of the present invention, an anti-CoV S glycoprotein antibody can be modified to produce a fusion protein (i.e., an antibody or fragment fused with a heterologous protein, polypeptide, or peptide).

[0122] Additional fusion proteins can be generated by techniques such as gene shuffling, motif shuffling, exon shuffling, and / or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling can be used to modify the activity of anti-CoV S glycoprotein antibodies (e.g., antibodies or fragments thereof with high affinity and low dissociation rate). See, in general, U.S. Patents No. 5,605,793; No. 5,811,238; No. 5,830,721; No. 5,834,252; and No. 5,837,458, as well as Patent et al., 1997, Curr. Opinion Biotechnol., 8:724-33; Harayama, 1998, Trends Biotechnol, 16(2):76-82; Hansson et al., 1999, J. Mol. Biol., 287:265-76; and Lorenzo and Blasco, 1998, Biotechniques 24(2):308-313 (each of these patents and publications in their entirety is incorporated herein by reference). The antibody may also be a binding domain immunoglobulin fusion protein, as described in U.S. Publication No. 20030118592, U.S. Publication No. 200330133939, and PCT Publication No. WO02 / 056910 (all against Ledbetter et al., which are incorporated herein by reference in their entirety).

[0123] Domain antibody The anti-CoV S glycoprotein antibody of the composition and method of the present invention is a human antibody heavy chain (V H ) or light chain (V LThis may be a domain antibody (e.g., an antibody containing a small functional binding unit) corresponding to the variable region of ). Examples of domain antibodies include, but are not limited to, therapeutic target-specific antibodies available from Domantis Limited (Cambridge, UK) and Domantis Inc. (Cambridge, MA, USA) (see, for example, W004 / 058821, W004 / 003019, U.S. Patents 6,291,158, 6,582,915, 6,696,245, and 6,593,081).

[0124] Diabody In certain embodiments of the present invention, the anti-CoV S glycoprotein antibody is a "diabody." The term "diabody" refers to a small antibody fragment having two antigen-binding sites, which are the same polypeptide chain (V H -V L ) contains a light chain variable domain (V L ) connected to a heavy chain variable domain (V H ) includes. By using a linker that is too short to allow the two domains to pair on the same chain, these domains are forced to pair with complementary domains on another chain to produce two antigen-binding sites. The diabody is described in more detail, for example, EP404,097;WO93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0125] linear antibody In a particular embodiment of the present invention, the anti-CoV S glycoprotein antibody is a linear antibody. The linear antibody has a pair of tandem Fd segments (V) that form a pair of antigen-binding regions. H -C H1 -V H -C H1 ) are included. Linear antibodies may be bispecific or monospecific. See Zapata et al., Protein Eng, 8(10):1057-1062 (1995).

[0126] Antibody fragment An "antibody fragment" is a fragment containing a portion of a full-length antibody, generally including its antigen-binding region or variable region. Examples of antibody fragments include Fab, Fab ’ , F(ab ’ Examples include multispecific antibodies formed from )2, Fv fragments, diabodies, linear antibodies, single-chain antibody molecules, and antibody fragments.

[0127] Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods, 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be generated directly by recombinant host cells. For example, antibody fragments can be isolated from the antibody phage library described above. Fab'-SH fragments can also be recovered directly from E. coli and chemically bound to form F(ab')2 fragments (Carter et al., Bio / Technology, 10:163-167 (1992)). Alternatively, F(ab')2 fragments can be isolated directly from recombinant host cell cultures. Other techniques for antibody fragment generation will be apparent to those skilled in the art. In other embodiments, the antibody of choice is a single-chain Fv fragment (scFv). See, for example, WO93 / 16185. In certain embodiments, the antibody is not a Fab fragment.

[0128] bispecific antibody A bispecific antibody is an antibody that has binding specificity to at least two different epitopes.

[0129] Methods for producing bispecific antibodies are known in the art (e.g., Millstein et al., Nature, 305:537-539 (1983); Traunecker et al., EMBO J., 10:3655-3659 (1991); Suresh et al., Methods in Enzymology, 121:210 (1986); Kostelny et al., J.Immunol., 148(5):1547-1553 (1992); Hollinger et al., Proc. Natl Acad. Sci. USA, 90:6444-6448 (1993); Gruber et al. al., J.Immunol., 152:5368 (1994); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,81; 95,731,168; 4,676,980; and 4,676,980; WO94 / 04690; WO91 / 00360; WO92 / 200373; WO93 / 17715; WO92 / 08802; and EP03089).

[0130] In one embodiment, if the anti-CoV S glycoprotein antibody of the composition and method of the present invention is bispecific, the anti-CoV S glycoprotein antibody may be human or humanized and may have specificity for SARS-CoV-2 S glycoprotein and epitopes on T cells, or may bind to human effector cells (e.g., monocytes / macrophages and / or natural killer cells) and induce cell death.

[0131] In one embodiment, the anti-CoV S glycoprotein antibody of the present invention is a bispecific antibody capable of specifically binding to a first and a second antigen, wherein the first antigen is SARS-CoV-2 S glycoprotein and the second antigen is an Fc gamma receptor selected from the group consisting of FcγRI, FcγRIIA, FcγRIIB, Fcγ, RIIIA, and / or FcγRIV. In a further embodiment, the anti-CoV S glycoprotein antibody of the present invention is a bispecific antibody capable of specifically binding to SARS-CoV-2 and FcγRIIB. In another embodiment, the anti-CoV S glycoprotein antibody of the present invention is a bispecific antibody capable of specifically binding to SARS-CoV-2 S glycoprotein and human FcγRIIB.

[0132] Variant Fc region The present invention provides an anti-CoV S glycoprotein antibody having a variant Fc domain, namely, a non-natural Fc region, for example, an Fc region containing one or more non-natural amino acid residues. The variant Fc region of the present invention also includes Fc regions containing amino acid deletions, additions, and / or modifications.

[0133] As used herein, the Fc region will be understood to include a polypeptide comprising the constant region of an antibody, excluding the first constant region immunoglobulin domain. Thus, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains. In the case of IgA and IgM, Fc may include the J chain. In the case of IgG, Fc includes the immunoglobulin domains C-gamma 2 and C-gamma 3 (Cγ2 and Cγ3) and the hinge between C-gamma 1 (Cγ1) and C-gamma 2 (Cγ2). Although the boundaries of the Fc region may differ, the human IgG heavy chain Fc region is typically defined as encompassing residues C226 or P230 to their carboxyl terminus (numbering follows the EU index in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA)). "Kabat's EU index" refers to the residue numbering of human IgG1 EU antibodies as described by Kabat et al. (see above). Fc may refer to this region alone, or to this region in the context of an antibody, antibody fragment, or Fc fusion protein. An Fc variant protein may be an antibody, an Fc fusion, or any protein or protein domain containing an Fc region (including, but not limited to, proteins containing variant Fc regions that are non-natural variants of Fc). Note: Polymorphisms have been observed at multiple Fc locations (including, but not limited to, Kabat 270, 272, 312, 315, 356, and 358), so there may be slight differences between the presented sequence and the prior art sequence.

[0134] This invention encompasses anti-CoV S glycoprotein antibodies having a variant Fc domain. The variant Fc domain may exhibit altered binding properties to Fc ligands (e.g., Fc receptors, Clq) compared to equivalent molecules (e.g., proteins with the same amino acid sequence except for the presence of a wild-type Fc region). Examples of binding properties, but not limited to, include binding specificity and equilibrium dissociation constant (K). D ), dissociation and binding rates (k each) off and k on ), binding affinity and / or avidity are examples. D Binding molecules with a lower K (e.g., Fc variant proteins such as antibodies) D It is generally understood that a high K may be preferable to a high K binding molecule. However, in some cases, the icon or koff value may be better. D This value may be more important than others. A person skilled in the art can determine which kinetic parameters are most important for a given antibody application.

[0135] The affinity and binding properties of the Fc domain to its ligand can be quantified by various in vitro assays (biochemical or immunological-based assays) known in the art for quantifying Fc-FcγR interactions, i.e., specific binding between the Fc region and FcγR. Such methods include, but are not limited to, equilibrium methods (e.g., enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay (RIA)), or kinetic methods (e.g., BIACORE® analysis), as well as other methods (e.g., indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration)). These and other methods may utilize labeling of one or more of the components being investigated and / or various detection methods (including, but not limited to, colorimetric, fluorescence, luminescence, or isotopic labeling). A detailed explanation of binding affinity and kinetics can be found in Paul, WE, ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0136] In one embodiment, an anti-CoV S glycoprotein antibody having a variant Fc domain exhibits enhanced binding to one or more Fc ligands compared to an equivalent molecule. In another embodiment, an anti-CoV S glycoprotein antibody having a variant Fc domain exhibits at least 2-fold, 3-fold, 5-fold, 7-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or 200-fold higher affinity for Fc ligands compared to an equivalent molecule. In a specific embodiment, an anti-CoV S glycoprotein antibody having a variant Fc domain exhibits enhanced binding to the Fc receptor. In another specific embodiment, an anti-CoV S glycoprotein antibody having a variant Fc domain exhibits enhanced binding to the Fc receptor FcγRIIIA. In a more specific embodiment, an anti-CoV S glycoprotein antibody having a variant Fc domain exhibits enhanced binding to the Fc receptor FcγRIIB. In yet another specific embodiment, an anti-CoV S glycoprotein antibody having a variant Fc domain exhibits enhanced binding to the Fc receptor FcRn. In yet another specific embodiment, an anti-CoV S glycoprotein antibody having a variant Fc domain exhibits enhanced binding to Clq compared to an equivalent molecule.

[0137] In one embodiment, the anti-CoV S glycoprotein antibody of the present invention comprises a variant Fc domain, the variant Fc domain having enhanced binding affinity to Fc gamma receptor IIB compared to an equivalent non-variant Fc domain. In a further embodiment, the anti-CoV S glycoprotein antibody of the present invention comprises a variant Fc domain, the variant Fc domain having at least 2 times, or at least 3 times, or at least 5 times, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times, affinity to Fc gamma receptor IIB compared to an equivalent non-variant Fc domain.

[0138] The serum half-life of proteins containing an Fc region can be enhanced by increasing the binding affinity of the Fc region to FcRn. In one embodiment, an antibody containing a variant Fc domain has an enhanced serum half-life compared to an equivalent molecule.

[0139] Antibody-dependent cell-mediated injury (ADCC) is a form of cytotoxicity in which secreted IgG globulin, bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages), specifically binds to antigen-carrying target cells, enabling these cytotoxic effector cells to subsequently kill the target cells with cytotoxins. Specific, high-affinity IgG antibodies targeting the surface of target cells "arm" the cytotoxic cells and are absolutely necessary for such killing. Lysis of target cells occurs extracellularly, requires direct intercellular contact, and does not involve complement.

[0140] The ability of antibodies containing variant Fc domains to mediate ADCC-mediated lysis of target cells can be assayed. To evaluate ADCC activity, the Fc variant protein of interest is added to target cells in combination with immunoeffector cells. The immunoeffector cells are activated by the antigen-antibody complex, resulting in cell lysis of the target cells. Cell lysis is typically detected by the release of a label (e.g., radioactive substrate, fluorescent dye, or native intracellular protein) from the lysed cells. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Specific examples of in vitro ADCC assays are described in Wisecarver et al., 1985 79:277-282; Bruggemann et al., 1987, J Exp Med 166:1351-1361; Wilkinson et al., 2001, J Immunol Methods 258:183-191; and Patel et al., 1995 J Immunol Methods 184:29-38. Furthermore, the ADCC activity of Fc variant proteins of interest can also be evaluated in vivo using animal models, such as those disclosed in Clynes et al., 1998, Proc. Natl. Acad. Sci. USA 95:652-656.

[0141] In one embodiment, an antibody having a variant Fc domain exhibits enhanced ADCC activity compared to an equivalent molecule. In a specific embodiment, an antibody having a variant Fc domain exhibits at least 2-fold, 3-fold, 5-fold, 10-fold, 50-fold, or 100-fold higher ADCC activity than an equivalent molecule. In another specific embodiment, an antibody having a variant Fc domain exhibits enhanced binding to the Fc receptor FcγRIIIA and enhanced ADCC activity compared to an equivalent molecule. In yet another embodiment, an antibody having a variant Fc domain exhibits enhanced ADCC activity and serum half-life compared to an equivalent molecule.

[0142] In one embodiment, an antibody having a variant Fc domain exhibits reduced ADCC activity compared to an equivalent molecule. In a specific embodiment, the Fc variant protein exhibits ADCC activity at least twice, three times, five times, ten times, fifty times, or even 100 times lower than that of an equivalent molecule. In another specific embodiment, an antibody having a variant Fc domain exhibits reduced binding to the Fc receptor FcγRIIIA and reduced ADCC activity compared to an equivalent molecule. In yet another embodiment, an antibody having a variant Fc domain exhibits reduced ADCC activity and serum half-life compared to an equivalent molecule.

[0143] "Complement-dependent cell injury" and "CDC" refer to the lysis of target cells in the presence of complement. The complement activation pathway is initiated when the first component of the complement system (C1q) binds to a molecule complexed with a congener antigen, such as an antibody. To evaluate complement activation, a CDC assay (e.g., an assay as described in Gazzano-Santoro et al., 1996, J. Immunol., Methods, 202:163) can be performed. In one embodiment, an antibody having a variant Fc domain exhibits enhanced CDC activity compared to an equivalent molecule. In a specific embodiment, the Fc variant protein exhibits at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold higher CDC activity than an equivalent molecule. In another embodiment, an antibody having a variant Fc domain exhibits enhanced CDC activity and serum half-life compared to an equivalent molecule.

[0144] In one embodiment, an antibody having a variant Fc domain has reduced binding to one or more Fc ligands compared to an equivalent molecule. In another embodiment, an antibody having a variant Fc domain has an affinity for Fc ligands that is at least 2 times, or at least 3 times, or at least 5 times, or at least 7 times, or at least 10 times, or at least 20 times, or at least 30 times, or at least 40 times, or at least 50 times, or at least 60 times, or at least 70 times, or at least 80 times, or at least 90 times, or at least 100 times, or at least 200 times lower than an equivalent molecule. In a specific embodiment, an antibody having a variant Fc domain has reduced binding to an Fc receptor. In another specific embodiment, an antibody having a variant Fc domain has reduced binding to the Fc receptor FcγRIIIA. In a further specific embodiment, the antibody having the variant Fc domain described herein has an affinity for the Fc receptor FcγRIIIA that is at least about 5 times lower than that of an equivalent molecule, and the antibody having the variant Fc domain has an affinity for the Fc receptor FcγRIIB that is within about 2 times that of an equivalent molecule. In yet another specific embodiment, the Fc variant protein has reduced binding to the Fc receptor FcRn. In yet another specific embodiment, the antibody having the variant Fc domain has reduced binding to Clq compared to an equivalent molecule.

[0145] In one embodiment, the present invention relates to an antibody having a variant Fc domain, wherein the Fc region is numbered 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269 in the EU index numbering as described in Kabat. The present invention provides an antibody containing a non-natural amino acid residue at one or more positions selected from the group consisting of 279, 280, 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443. Optionally, the Fc region may contain non-natural amino acid residues in additional and / or alternative positions known to those skilled in the art (e.g., U.S. Patent Nos. 5,624,821, 6,277,375, 6,737,056; PCT Patent Publications WO01 / 58957, WO02 / 06919, WO04 / 016). See issues 750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925, and WO06 / 020114.

[0146] In one embodiment, the present invention relates to a formulation wherein the Fc region is numbered 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 251, 252, 254, 255, 256, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280 in the EU index numbering as described in Kabat. The present invention provides a formulation containing a non-natural amino acid residue at one or more positions selected from the group consisting of 284, 292, 296, 297, 298, 299, 305, 313, 316, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 440, and 443. Optionally, the Fc region may contain non-natural amino acid residues in additional and / or alternative positions known to those skilled in the art (e.g., U.S. Patent Nos. 5,624,821, 6,277,375, 6,737,056; PCT Patent Publications WO01 / 58957, WO02 / 06919, WO04 / 016). See issues 750, WO04 / 029207, WO04 / 035752, WO04 / 074455, WO04 / 099249, WO04 / 063351, WO05 / 070963, WO05 / 040217, WO05 / 092925, and WO06 / 020114.

[0147] In a specific embodiment, the present invention relates to an antibody having a variant Fc domain, wherein the Fc region is numbered according to the EU index described in Kabat as 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A , 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 2 44H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 2621, 262A , 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 2 64M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T , 2661, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 2 80A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H , 269G, 297S, 297D, 297E, 298H, 2981, 298T, 298F, 2991, 299L, 299A, 299S, 2 99V, 299H, 299F, 299E, 3051, 313F, 316D, 325Q, 325L, 3251, 325D, 325E, 325A , 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 3 28Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T , 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 33 1F, 331W, 331K, 331Q, 331E, 331S, 331V, 3311, 331C, 331Y, 331H, 331R, 331N,The present invention provides an antibody containing at least one non-natural amino acid residue selected from the group consisting of 331D, 331T, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y, and 434W. Optionally, the Fc region may contain additional / alternative non-natural amino acid residues known to those skilled in the art (see, for example, U.S. Patents 5,624,821, 6,277,375, 6,737,056; PCT Patent Publications WO01 / 58957, WO02 / 06919, WO04 / 016750; WO04 / 029207; WO04 / 035752; and WO05 / 040217).

[0148] In a specific embodiment, the present invention relates to an antibody having a variant Fc domain, wherein the Fc region is numbered according to the EU index described in Kabat as 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235F, 236E, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A , 240T, 240M, 241W, 241L, 241Y, 241E, 241R, 243W, 243L, 243Y, 243R, 243Q, 2 44H, 245A, 247L, 247V, 247G, 251F, 252Y, 254T, 255L, 256E, 256M, 2621, 262A , 262T, 262E, 2631, 263A, 263T, 263M, 264L, 2641, 264W, 264T, 264R, 264F, 2 64M, 264Y, 264E, 265G, 265N, 265Q, 265Y, 265F, 265V, 2651, 265L, 265H, 265T , 2661, 266A, 266T, 266M, 267Q, 267L, 268E, 269H, 269Y, 269F, 269R, 270E, 2 80A, 284M, 292P, 292L, 296E, 296Q, 296D, 296N, 296S, 296T, 296L, 2961, 296H , 269G, 297S, 297D, 297E, 298H, 2981, 298T, 298F, 2991, 299L, 299A, 299S, 2 99V, 299H, 299F, 299E, 3051, 313F, 316D, 325Q, 325L, 3251, 325D, 325E, 325A , 325T, 325V, 325H, 327G, 327W, 327N, 327L, 328S, 328M, 328D, 328E, 328N, 3 28Q, 328F, 3281, 328V, 328T, 328H, 328A, 329F, 329H, 329Q, 330K, 330G, 330T , 330C, 330L, 330Y, 330V, 3301, 330F, 330R, 330H, 331G, 331A, 331L, 331M, 33 1F, 331W, 331K, 331Q, 331E, 331S, 331V, 3311, 331C, 331Y, 331H, 331R, 331N,The present invention provides an antibody containing at least one non-natural amino acid residue selected from the group consisting of 331D, 3311, 332D, 332S, 332W, 332F, 332E, 332N, 332Q, 332T, 332H, 332Y, 332A, 339T, 370E, 370N, 378D, 392T, 396L, 416G, 419H, 421K, 440Y, and 434W. Optionally, the Fc region may contain additional / alternative non-natural amino acid residues known to those skilled in the art (see, for example, U.S. Patents 5,624,821, 6,277,375, 6,737,056; PCT Patent Publications WO01 / 58957, WO02 / 06919, WO04 / 016750; WO04 / 029207; WO04 / 035752; and WO05 / 040217).

[0149] In another embodiment, the present invention provides an antibody having a variant Fc domain, wherein the Fc region contains at least one non-natural amino acid at one or more positions selected from the group consisting of 239, 330, and 332 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region contains at least one non-natural amino acid selected from the group consisting of 239D, 330L, and 332E in the EU index numbering described in Kabat. Optionally, the Fc region may further contain additional non-natural amino acids at one or more positions selected from the group consisting of 252, 254, and 256 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant in which the Fc region comprises at least one non-natural amino acid selected from the group consisting of 239D, 330L, and 332E in the EU index numbering described in Kabat, and at least one non-natural amino acid at one or more positions selected from the group consisting of 252Y, 254T, and 256E in the EU index numbering described in Kabat.

[0150] In another embodiment, the present invention provides an antibody having a variant Fc domain, wherein the Fc region contains at least one non-natural amino acid at one or more positions selected from the group consisting of 234, 235, and 331 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant, wherein the Fc region contains at least one non-natural amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S in the EU index numbering described in Kabat. In a further specific embodiment, the Fc variant of the present invention contains the non-natural amino acid residues 234F, 235F, and 331S in the EU index numbering described in Kabat. In another specific embodiment, the Fc domain of the present invention contains the non-natural amino acid residues 234F, 235Y, and 331S in the EU index numbering described in Kabat. Optionally, the Fc region may further contain additional non-natural amino acids at one or more positions selected from the group consisting of 252, 254, and 256 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant in which the Fc region contains at least one non-natural amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S in the EU index numbering described in Kabat, and at least one non-natural amino acid at one or more positions selected from the group consisting of 252Y, 254T, and 256E in the EU index numbering described in Kabat.

[0151] In another embodiment, the present invention provides an antibody having a variant Fc domain, wherein the Fc region contains at least one non-natural amino acid at one or more positions selected from the group consisting of 239, 330, and 332 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region contains at least one non-natural amino acid selected from the group consisting of 239D, 330L, and 332E in the EU index numbering described in Kabat. Optionally, the Fc region may further contain additional non-natural amino acids at one or more positions selected from the group consisting of 252, 254, and 256 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant protein formulation in which the Fc region comprises at least one non-natural amino acid selected from the group consisting of 239D, 330L, and 332E in the EU index numbering described in Kabat, and at least one non-natural amino acid at one or more positions selected from the group consisting of 252Y, 254T, and 256E in the EU index numbering described in Kabat.

[0152] In another embodiment, the present invention provides an antibody having a variant Fc domain, wherein the Fc region contains at least one non-natural amino acid at one or more positions selected from the group consisting of 234, 235, and 331 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant protein formulation, wherein the Fc region contains at least one non-natural amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S in the EU index numbering described in Kabat. Optionally, the Fc region may further contain additional non-natural amino acids at one or more positions selected from the group consisting of 252, 254, and 256 in the EU index numbering described in Kabat. In a specific embodiment, the present invention provides an Fc variant protein formulation in which the Fc region comprises at least one non-natural amino acid selected from the group consisting of 234F, 235F, 235Y, and 331S in the EU index numbering described in Kabat, and at least one non-natural amino acid at one or more positions selected from the group consisting of 252Y, 254T, and 256E in the EU index numbering described in Kabat.

[0153] In one embodiment, the Fc variant of the present invention is a known Fc variant (e.g., Ghetie et al., 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al., 1991, J. Immunol 147:2657-2662; Lund et al, 1992, Mol Immunol 29:53-59; Alegre et al, 1994, Transplantation 57:1537-1543; Hutchins et al., 1995, Proc Natl. Acad Sci USA 92:11980-11984; Jefferis et al, 1995, Immunol Lett. 44:111-117; Lund et al., 1995, Faceb J 9:115-119;Jefferis et al,1996,Immunol Lett 54:101-104;Lund et al,1996,J Immunol 157:4963-4969;Armour et al.,1999,Eur J Immunol 29:2613-2624;Idusogie et al,2000,J Immunol 164:4178-4184;Reddy et al,2000,J Immunol 164:1925-1933;Xu et al.,2000,Cell Immunol 200:16-26;Idusogie et al,2001,J Immunol 166:2571-2575;Shields et al. al.,2001,J Biol Chem 276:6591-6604;Jefferis et al,2002,Immunol Lett 82:57-65;Presta et al.,2002,Biochem Soc Trans 30:487-490; US Patent No. 5,624,821; US ​​Patent No. 5,885,573; US Patent No. 5,677,425; US Patent No. 6,165,745; US Patent No. 6,277,375; No. 046; No. 6,121,022; No. 5,624,821; No. 5,648,260; No. 6,528,624; No. 6,194,551; No. 6,737,056; No. 6, This invention can be combined with variants disclosed in U.S. Patent Publication No. 821,505; No. 6,277,375; U.S. Patent Publication No. 2004 / 0002587 and PCT Publication Nos. WO94 / 29351; WO99 / 58572; WO00 / 42072; WO02 / 060919; WO04 / 029207; WO04 / 099249; and WO04 / 063351. Furthermore, Fc regions including deletions, additions, and / or modifications are also encompassed by this invention. Further modifications / substitutions / additions / deletions of the Fc domain will be readily apparent to those skilled in the art.

[0154] Methods for generating non-natural Fc regions are known in the art. For example, amino acid substitutions and / or deletions can be generated by mutagenesis, including, but not limited to, site-directed mutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCR mutagenesis (Higuchi, in “PCR Protocols: A Guide to Methods and Applications”, Academic Press, San Diego, pp. 177-183 (1990)), and cassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably, site-directed mutagenesis is performed by overlap-extension PCR (Higuchi, in “PCR Technology: Principles and Applications for DNA Amplification”, Stockton Press, New York, pp. 61-70 (1989)). Furthermore, the overlap-extension PCR technique (Higuchi, ibid.) can be used to introduce any desired mutation(s) into the target sequence (starting DNA). For example, the first round of PCR in the overlap-extension method involves amplifying the target sequence with an outer primer (primer 1) and an internal mutagenesis primer (primer 3), followed by further amplification with a second outer primer (primer 4) and an internal primer (primer 2), yielding two PCR segments (segments A and B). The internal mutagenesis primer (primer 3) is designed to contain a mismatch to the target sequence that specifies the desired mutation(s). In the second round of PCR, the products of the first round of PCR (segments A and B) are amplified by PCR using two outer primers (primers 1 and 4). The resulting full-length PCR segment (segment C) is digested with restriction enzymes, and the resulting restriction fragment is cloned into a suitable vector. As the first step in mutagenesis, the starting DNA (e.g., an Fc fusion protein, antibody, or simply encoding an Fc region) is cloning manipulably into a mutagenesis vector.The primers are designed to reflect the desired amino acid substitutions. Other methods useful for generating variant Fc regions are known in the art (e.g., U.S. Patent Nos. 5,624,821; 5,885,573; 5,677,425; 6,165,745; 6,277,375; 5,869,046; 6,121,022; 5,624,821; 5,648,260; 6,528,624; 6,111 See Patent No. 94,551; Patent No. 6,737,056; Patent No. 6,821,505; Patent No. 6,277,375; U.S. Patent Publication No. 2004 / 0002587 and PCT Publications WO94 / 29351; WO99 / 58572; WO00 / 42072; WO02 / 060919; WO04 / 029207; WO04 / 099249; and WO04 / 063351).

[0155] In some embodiments, antibodies having a variant Fc domain comprise one or more engineered glycoforms, i.e., carbohydrate compositions covalently bound to a molecule containing an Fc region. Engineered glycoforms may be useful for a variety of purposes, including, but not limited to, enhancing or reducing effector function. Engineered glycoforms can be produced by any method known to those skilled in the art, e.g., by using engineered or variant-expressing strains, by co-expression with one or more enzymes (e.g., DI N-acetylglucosaminyltransferase III (GnTI11)), by expression of molecules containing an Fc region in various organisms or cell lines of various organisms, or by modification of carbohydrates after expression of molecules containing an Fc region. Methods for purifying manipulated glycoforms are known in the art, and such methods are not limited to the following: Umana et al, 1999, Nat. Biotechnol 17:176-180; Davies et al., 20017 Biotechnol Bioeng 74:288-294; Shields et al, 2002, J Biol Chem 277:26733-26740; Shinkawa et al., 2003, J Biol Chem Examples include methods described in 278:3466-3473)USPat. No. 6,602,684; US Patent Application No. 10 / 277,370; US Patent Application No. 10 / 113,929; PCT No. WO00 / 61739A1; PCT No. WO01 / 292246A1; PCT No. WO02 / 311140A1; PCT No. WO02 / 30954A1; and methods described in Potelligent™ technology (Biowa, Inc., Princeton, NJ) and GlycoMAb™ glycosylation technology (GLYCART biotechnology AG, Zurich, Switzerland). For example, see WO00061739;EA01229125;US20030115614;Okazaki et al.,2004,JMB,336:1239-49.

[0156] Antibody glycosylation In yet another embodiment, the glycosylation of the antibody used according to the present invention is modified. For example, an aglycosylated antibody (i.e., the antibody does not contain glycosylation) can be produced. Glycosylation can be modified, for example, to increase the affinity of the antibody to a target antigen. Such carbohydrate modification can be achieved, for example, by modifying one or more glycosylation sites in the antibody sequence. For example, one or more variable region framework glycosylation sites can be removed, and one or more amino acid substitutions can be made to eliminate glycosylation at that site. Such aglycosylation can increase the affinity of the antibody to the antigen. Such approaches are described in more detail in U.S. Patents 5,714,350 and 6,350,861. Alternatively, one or more amino acid substitutions can be made to remove glycosylation sites present in the Fc region (e.g., asparagine 297 in IgG). Furthermore, aglycosylated antibodies can also be produced in bacterial cells that do not contain the required glycosylation mechanism.

[0157] Furthermore, antibodies with modified glycosylation types (for example, low-fucosylated antibodies with reduced fucosyl residues, or antibodies with increased bipartite GlcNAc structures) can also be produced. Such modified glycosylation patterns have been shown to enhance the ADCC (Adverse Drug Reaction Capability) activity of antibodies. Such carbohydrate modifications can be achieved, for example, by expressing antibodies in host cells with modified glycosylation mechanisms. Cells with modified glycosylation mechanisms are described in the Art and can be used as host cells to produce antibodies with modified glycosylation by expressing the recombinant antibodies of the present invention. See, for example, Shields, R. Let al. (2002) J. Biol. Chem. 277:26733-26740; Umana et al. (1999) Nat. Biotech. 17:176-1; and U.S. Patent No. 6,946,292; European Patent No. EP1,176,195; PCT Publication No. WO03 / 035835; and PCT Publication No. WO99 / 54342 (each of which is incorporated herein by reference in its entirety).

[0158] Operating the effects pedal function It may be desirable to modify the anti-CoV S glycoprotein antibody of the present invention in terms of effector function. For example, cysteine ​​residues can be introduced into the Fc region to enable the formation of interchain disulfide bonds within this region. Homodimerated antibodies thus produced may have improved internal migration ability and / or increased complement-mediated cell killing and / or antibody-dependent cell-mediated cytotoxicity (ADCC). See Caron et al., J. Exp Med., 176:1191-1195 (1992) and Shopes, B., J. Immunol., 148:2918-2922 (1992). Homodimerated antibodies with enhanced antitumor activity can also be prepared using heterobifunctional crosslinking agents as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Furthermore, antibodies can be engineered to have a dual Fc region, thereby enhancing complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0159] Other methods for manipulating the Fc region of an antibody to alter its effector function are known in the art (for example, U.S. Patent Publication 20040185045 and PCT Publication WO2004 / 016750 (both to Koenig et al., describing modifications of the Fc region to enhance the binding affinity to FcγRIIB compared to the binding affinity to FcγRIIA); also see PCT Publication WO99 / 58572 to Armour et al., WO99 / 51642 to ldusogie et al., and US6,395,272 to Deo et al. (the entirety of these disclosures is incorporated herein by reference)). Furthermore, methods for modifying the Fc region to reduce its binding affinity to FcγRIIB are also known in the art (e.g., U.S. Patent Publication No. 20010036459 and PCT Publication No. WO01 / 79299 (both to Ravetch et al., the entirety of which is incorporated herein by reference)). Modified antibodies having variant Fc regions with enhanced binding affinity to FcγRIIIA and / or FcγRIIA compared to the wild-type Fc region have also been described (e.g., PCT Publication No. WO2004 / 063351 to Stavenhagen et al. (the entirety of which is incorporated herein by reference)).

[0160] Using in vitro assays known in the art, it is possible to determine whether the anti-CoV S glycoprotein antibody used in the compositions and methods of the present invention can mediate ADCC (e.g., as described herein).

[0161] Manufacturing / production of anti-CoV S glycoprotein antibodies Once the desired anti-CoV S glycoprotein antibody has been manipulated, it can be commercially produced using methods well known in the art for the large-scale production of antibodies. For example, this can be achieved using recombinant expression systems (e.g., but not limited to, systems described later).

[0162] Recombinant expression system Recombinant expression of antibodies or variants generally requires the construction of an expression vector containing a polynucleotide encoding the antibody. Once an antibody molecule, or a polynucleotide encoding the heavy or light chain of an antibody, or a portion thereof, is obtained, a vector for antibody molecule production can be generated by recombinant DNA techniques using techniques well known in the art. See, for example, U.S. Patent No. 6,331,415 (which is incorporated herein by reference in its entirety). Accordingly, a method for preparing a protein by expressing a polynucleotide containing an antibody-coding nucleotide sequence is described herein. An expression vector containing an antibody-coding sequence and appropriate transcription and translational regulatory signals can be constructed using methods well known to those skilled in the art. Such methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Accordingly, the present invention provides a replicable vector comprising a nucleotide sequence encoding an antibody molecule, a heavy or light chain of an antibody, a heavy or light chain variable domain of an antibody or a portion thereof, or a heavy or light chain CDR, and configured to act on a promoter. Such vectors contain a nucleotide sequence encoding the constant region of an antibody molecule (see, for example, International Publication Nos. WO86 / 05807 and WO89 / 01036; and U.S. Patent No. 5,122,464), and the variable domain of the antibody can be cloned into such vectors for the expression of the entire heavy chain, the entire light chain, or both the heavy and light chains.

[0163] In another embodiment, anti-CoV S glycoprotein antibodies can be produced using targeted homologous recombination to generate all or part of the anti-CoV S glycoprotein antibodies (see U.S. Patents 6,063,630, 6,187,305, and 6,692,737). In a particular embodiment, anti-CoV S glycoprotein antibodies can be produced using random recombination techniques to generate all or part of the anti-CoV S glycoprotein antibodies (see U.S. Patents 6,361,972, 6,524,818, 6,541,221, and 6,623,958). Anti-CoV S glycoprotein antibodies can also be produced in cells expressing antibodies from the genomic sequence of cells containing modified immunoglobulin loci using Cre-mediated site-directed homologous recombination (see U.S. Patent 6,091,001). The host cell line may be derived from humans or from non-human species (including, but not limited to, mice and Chinese hamsters). If the production of human or humanized antibodies is desired, the host cell line should be a human cell line. These methods can be advantageously used to manipulate stable cell lines that permanently express antibody molecules.

[0164] After transferring the expression vector to host cells using conventional techniques, the translocation cells are cultured using conventional techniques to obtain antibodies. Therefore, the present invention comprises host cells containing the antibody or a fragment thereof, its heavy chain or light chain or a part thereof, or a polynucleotide encoding the single-chain antibody of the present invention, which is receptively bound to a heterologous promoter. In certain embodiments for the expression of double-chain antibodies, as detailed below, vectors encoding both the heavy chain and light chain can be co-expressed in host cells to express the entire immunoglobulin molecule.

[0165] Various host expression vector systems can be used to express anti-CoV S glycoprotein antibodies or parts thereof, which can be used for the manipulation and production of anti-CoV S glycoprotein antibodies (see, for example, U.S. Patent No. 5,807,715). For example, combinations of mammalian cells such as Chinese hamster ovary cells (CHO) and vectors such as the major intermediate early gene promoter element derived from human cytomegalovirus are effective expression systems for antibodies (Foecking et al., Gene 45:101 (1986); and Cockett et al., Bio / Technology 8:2 (1990)). Furthermore, host cell lines can be selected that regulate the expression of the inserted sequence or that modify and process the gene product in a desired specific manner. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for protein function. Different host cells have characteristic and specific mechanisms for post-translational processing and modification of proteins and gene products. By selecting an appropriate cell line or host system, the correct modification and processing of the expressed antibody or a portion thereof can be ensured. For this purpose, eukaryotic host cells with cellular mechanisms for proper primary transcript processing, glycosylation, and gene product phosphorylation may be used. Such mammalian host cells include, but are not limited to, CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT2O, and T47D, NSO (a mouse myeloma cell line that does not endogenously produce functional immunoglobulin chains), CRL7030, and HsS78Bst cells.

[0166] In bacterial systems, multiple expression vectors can be advantageously selected depending on the intended use of the antibody molecule to be expressed. For example, when attempting to produce large quantities of such antibodies to create a pharmaceutical composition containing anti-CoV S glycoprotein antibodies, a vector that induces high levels of expression of a readily purified fusion protein product is desirable. Examples of such vectors, though not limited to them, include the E. coli expression vector pUR278 (Ruther et al., EMBO, 12:1791 (1983)) (which allows for the generation of a fusion protein by individually ligating the antibody coding sequence with the lac Z coding region in-frame into the vector); and the pIN vector (Inouye & Inouye, 1985, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, 1989, J. Biol. Chem., 24:5503-5509 (1989)). Alternatively, a pGEX vector may be used to express an exogenous polypeptide as a fusion protein with glutathione-S-transferase (GST). Generally, such fusion proteins are soluble, and purification from lysed cells can be easily performed by adsorption and binding to a glutathione-agarose affinity matrix, followed by elution in the presence of free glutathione. The pGEX vector is designed to introduce an athrombin and / or factor Xa protease cleavage site into the expression polypeptide, allowing the cloned target gene product to be released from the GST portion.

[0167] In insects, the Autographa californica nuclear polyhedron disease virus (AcNPV) is used as a vector for expressing extragenes. This virus grows in Spodoptera frugiperda cells. Antibody coding sequences can be individually cloned into non-essential regions of the virus (e.g., polyhedrin genes) and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

[0168] In mammalian host cells, multiple virus-based expression systems can be utilized. When using adenovirus as an expression vector, the coding sequence of the antibody of interest can be ligated to the adenovirus transcription / translation regulatory complex, such as the late promoter and tripartite reader sequence. This chimeric gene can then be inserted into the adenovirus genome by recombination in vitro or in vivo. By inserting the viral genome into a non-essential region (e.g., region E1 or E3), a recombinant virus viable in the infected host and capable of expressing the antibody molecule can be obtained (see, e.g., Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:355-359 (1984)). Specific start signals may also be required for efficient translation of the inserted antibody coding sequence. This signal includes the ATG start codon and adjacent sequences. Furthermore, the start codon should generally be in-frame with respect to the reading frame of the desired coding sequence to ensure translation of the entire insertion. These exogenous translational regulatory signals and start codons can have various origins, both natural and synthetic. Expression efficiency can be enhanced by including appropriate transcriptional enhancer elements, transcriptional terminators, etc. (see, for example, Bittner et al., Methods in Enzymol., 153:51-544 (1987)).

[0169] Stable expression can be used to generate recombinant proteins in high yield over the long term. For example, it is possible to generate cell lines that stably express antibody molecules. Host cells can be transformed with appropriately engineered vectors containing expression regulatory elements (e.g., promoters, enhancers, transcriptional terminators, polyadenylation sites, etc.) and selectable marker genes. After introducing the foreign DNA, the cells can be grown in concentrated medium for 1-2 days, then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection, allowing cells to grow that stably integrate the plasmid into the chromosome and form proliferation foci that can be cloned into cell lines and amplified. Using plasmids encoding anti-CoV S glycoprotein antibodies, the gene / DNA can be introduced into any cell line suitable for generation under culture.

[0170] Multiple selection systems can be used, and these include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler et al., Cell, 11:223 (1977)), hypoxanthine guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell, 22:8-17 (1980)) genes, each of which is tk - hgprt -It can be used in aprcell. Furthermore, antimetabolite resistance can be used as a basis for selecting the following genes: dhfr (confers resistance to methotrexate) (Wigler et al., Natl. Acad. Sci. USA, 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981)); gpt (confers resistance to mycophenolic acid) (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)); neo (confers resistance to aminoglycoside G-418) (Wu and Wu, Biotherapy 3:87-95 (1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem. 62:191-217 (1993); May, TIB TECH 11(5):155-2 15 (1993)); and hygro (conferring resistance to hygromycin) (Santerre et al., Gene, 30:147 (1984)). Methods commonly known in the field of recombinant DNA technology can typically be applied to the selection of desired recombinant clones, such methods are described, for example, in Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, NY (1993); Kricgler, Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY (1990); and Chapters 12 and 13, Dracopoli et al. (eds.), Current Protocols in Human Genetics, John Wiley & Sons, NY (1994); and Colberre-Garapin et al., 1981, J.Mol.Biol., 150:1 (the entire text of which is incorporated herein by reference).

[0171] The expression level of antibody molecules can be increased by vector amplification (see Bebbington and Hentschel, The use of vectors based on gene amplification for expression of cloned genes in mammalian cells in DNA cloning, Vol. 3, Academic Press, New York (1987) for an overview). If a marker within an antibody-expressing vector system is amplified, increasing the level of the inhibitor present in the host cell culture increases the copy number of the marker gene. Since the amplified region is associated with the antibody gene, antibody production also increases (Crouse et al., Mol. Cell. Biol. 3:257 (1983)). Antibody expression levels can be amplified by the use of recombinant methods and tools known to those skilled in the field of recombinant protein synthesis (including techniques for remodeling the surrounding chromatin and enhancing transgene expression in the form of an active artificial transcription domain).

[0172] Host cells may be simultaneously transfused with two expression vectors: a first vector encoding a heavy chain polypeptide and a second vector encoding a light chain polypeptide. The two vectors may contain the same or different selection markers. A single vector encoding and expressing both heavy and light chain polypeptides can also be used. In such situations, the light chain should be positioned at 5' of the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot Nature 322:562-65(1986); and Kohler, 1980, Proc. Natl. Acad. Sci. USA, 77:2197(1980)). The heavy and light chain coding sequences may include cDNA or genomic DNA.

[0173] Once antibody molecules are generated by recombinant expression, they can be purified by any method known in the art for purifying immunoglobulin molecules, for example, by chromatography (e.g., ion exchange, affinity (particularly affinity for specific antigen protein A or protein G), and sizing), centrifugation, solubility difference, or any other standard technique for protein purification. Furthermore, the antibodies or fragments of the present invention can be fused with heterologous polypeptide sequences described herein or otherwise known in the art to facilitate purification.

[0174] Antibody purification and isolation When using recombinant techniques, antibodies can be generated in the intracellular periplasmic space or secreted directly into the culture medium. When generating antibodies intracellularly, the first step is to remove particulate debris, such as host cells or lysed fragments, by, for example, centrifugation or ultrafiltration. Carter et al., Bio / Technology 10:163-167 (1992) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli. Briefly, the paste is thawed for approximately 30 minutes in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF). Cellular debris can be removed by centrifugation. When secreting antibody variants into the culture medium, generally, the supernatant from such an expression system is first concentrated using a commercially available protein concentration filter (e.g., Amicon or Millipore Pellicon ultrafiltration unit). One of the steps described above may include a protease inhibitor such as PMSF to inhibit PMSF protein degradation, and an antibiotic may be included to prevent the growth of exogenous contaminants.

[0175] Antibody compositions prepared from cells can be purified, for example, by hydroxyl apatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and / or affinity chromatography, either alone or in combination with other purification steps. The suitability of protein A as an affinity ligand depends on the species and isotype of the immunoglobulin Fc domain present in the antibody variant. Protein A can be used to purify antibodies based on human γ1, γ2, or γ4 heavy chains (Lindmark et al., J.Immunol.Methods, 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and human γ3 (Guss et al., EMBO J., 5:15671575 (1986)). The matrix to which affinity ligands bind is most often agarose, but other matrices are also available. Mechanically stable matrices (e.g., controlled porous glass or poly(styrenedivinyl)benzene) allow for faster flow rates and shorter processing times than those achievable with agarose. Bakerbond ABX resin (JTBaker, Phillipsburg, NJ) is useful for purification when the antibody contains a CH3 domain. Other protein purification techniques, such as fractional distillation on ion-exchange columns, ethanol precipitation, reverse-phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on anion or cation exchange resins (e.g., polyaspartate columns), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation, are also available depending on the antibody to be recovered.

[0176] After any optional pre-purification step(s), the mixture containing the antibody of interest and impurities can be subjected to low-pH hydrophobic interaction chromatography using an elution buffer with a pH of approximately 2.5–4.5, and can be carried out at a low salt concentration (e.g., approximately 0–0.25 M salt).

[0177] Therapeutic anti-CoV S glycoprotein antibody The anti-CoV S glycoprotein antibody used in the compositions and methods of the present invention may be a human antibody or a humanized antibody that can treat COVID-19 or neutralize the SARS-CoV-2 virus or its variants. In certain embodiments, the anti-CoV S glycoprotein antibody may be a chimeric antibody or a mouse antibody. In certain embodiments, the anti-CoV S glycoprotein antibody may be a monoclonal human antibody, a humanized antibody, or a chimeric antibody. The anti-CoV S glycoprotein antibody used in the compositions and methods of the present invention may be a human antibody or a humanized antibody of the IgG1 or IgG3 human isotype, or any IgG1 or IgG3 allele found in the human population. In other embodiments, the anti-CoV S glycoprotein antibody used in the compositions and methods of the present invention may be a human antibody or a humanized antibody of the IgG2 or IgG4 human isotype, or any IgG2 or IgG4 allele found in the human population.

[0178] In a particular embodiment, the antibody is an isotype switch variant of a known antibody (for example, against IgG1 or IgG3 human isotypes) as described above.

[0179] The anti-CoV S glycoprotein antibody used in the compositions and methods of this disclosure may be a naked antibody, an immunoconjugate, or a fusion protein.

[0180] Screening of SARS-CoV-2 S-glycoprotein-binding antibodies Binding assays can be used to identify antibodies that bind to the SARS-CoV-2 S glycoprotein. Binding assays can be performed as direct binding assays or competitive binding assays. Binding can be detected using standard ELISA or standard flow cytometry assays. Direct binding assays test the binding of a candidate antibody to the SARS-CoV-2 S glycoprotein. Competitive binding assays, on the other hand, evaluate the ability of a candidate antibody to compete with known anti-CoV S glycoprotein antibodies or other compounds that bind to the SARS-CoV-2 S glycoprotein.

[0181] In a direct binding assay, the SARS-CoV-2 S glycoprotein is brought into contact with the candidate antibody under conditions that allow the candidate antibody to bind to the SARS-CoV-2 S glycoprotein. Binding can be performed in solution or on a solid surface. The candidate antibody may be pre-labeled for detection. Any detectable compound, such as, but not limited to, luminescent, fluorescent, radioisotopes or groups containing them, or non-isotope labeling (e.g., enzymes or dyes), can be used for labeling. After a sufficient incubation period for binding to occur, the reactants are subjected to conditions and procedures to remove excess or nonspecifically bound antibody. Typically, this involves washing with a suitable buffer. Finally, the presence of a complex of the candidate antibody and the SARS-CoV-2 S glycoprotein is detected.

[0182] In competitive binding assays, the ability of a candidate antibody to inhibit or substitute the binding of a known anti-CoV S glycoprotein antibody (or other compound) to the SARS-CoV-2 S glycoprotein is evaluated. A labeled known SARS-CoV-2 S glycoprotein binder can be mixed with the candidate antibody and placed under conditions in which the interaction between them would normally occur, with or without the candidate antibody. The amount of labeled known binder that binds to the SARS-CoV-2 glycoprotein can be compared to the amount of binding in the presence or absence of the candidate antibody.

[0183] In one embodiment, the binding assay is performed using one or more components immobilized on a solid surface to facilitate the formation and detection of antibody-antigen complexes. In various embodiments, the solid support may be, but is not limited to, polyvinylidene fluoride, polycarbonate, polystyrene, polypropylene, polyethylene, glass, nitrocellulose, dextran, nylon, polyacrylamide, and agarose. Support configurations may include beads, membranes, microparticles, and the inner surface of a reaction vessel (e.g., a microtiter plate, test tube, or other reaction vessel). Immobilization of the SARS-CoV-2 S glycoprotein or its fragments, or other components, can be achieved by covalent or non-covalent bonding. In one embodiment, attachment can be performed indirectly, i.e., via an attached antibody. In another embodiment, the SARS-CoV-2 S glycoprotein and negative control are tagged with an epitope (e.g., glutathione S-transferase (GST)) so that a commercially available antibody (e.g., anti-GST (Santa Cruz Biotechnology)) can mediate attachment to the solid surface.

[0184] For example, such affinity binding assays can be performed using SARS-CoV-2 S glycoprotein immobilized on a solid support. Typically, the unimmobilized component of the binding reaction (in this case, a candidate anti-CoV S glycoprotein antibody) is labeled for detection. Various labeling methods are available and can be used (e.g., luminescence, color development, fluorescence, or radioisotope labeling, or groups containing these, and non-isotope labeling (e.g., enzymes or dyes)). In one embodiment, the candidate anti-CoV S glycoprotein antibody is labeled with a fluorophore such as fluorescein isothiocyanate (FITC, available from Sigma Chemicals, St. Louis). Such affinity binding assays can be performed using SARS-CoV-2 S glycoprotein immobilized on a solid surface. The anti-CoV S glycoprotein antibody is then incubated with the antigen, and the specific binding of the antibody is detected by methods known in the art (including, but not limited to, BiaCore analysis, ELISA, FMET, and R1A).

[0185] Finally, any labels remaining on the solid surface can be detected by any detection method known in the art. For example, if a candidate anti-CoV S glycoprotein antibody is labeled with a fluorophore, the complex can be detected using a fluorometer.

[0186] SARS-CoV-2 S glycoprotein can be added to binding assays in the form of intact cells expressing SARS-CoV-2 S glycoprotein, or in the form of an isolated membrane containing human SARS-CoV-2 S glycoprotein. Therefore, direct binding to SARS-CoV-2 glycoprotein can be assayed in intact cells under culture or in animal models, both in the presence and absence of candidate anti-CoV S glycoprotein antibodies. Labeled candidate anti-CoV S glycoprotein antibodies can be mixed with cells expressing SARS-CoV-2 S glycoprotein, and the candidate anti-CoV S glycoprotein antibodies can be added. Using isolated membranes, candidate anti-CoV S glycoprotein antibodies that interact with SARS-CoV-2 S glycoprotein can be identified. For example, in a typical experiment using isolated membranes, cells can be genetically engineered to express SARS-CoV-2 S glycoprotein. The membrane can be collected using standard techniques and used in in vitro binding assays. Labeled candidate anti-CoV S glycoprotein antibodies (e.g., fluorescently labeled antibodies) are conjugated to the membrane, and specific activity is assayed. Specific binding is quantified by comparison with a binding assay performed in the presence of an excess of unlabeled (low-temperature) candidate anti-CoV S glycoprotein antibody. Alternatively, antibodies that bind to a portion of the SARS-CoV-2 S glycoprotein can be identified using a non-cell-based assay system with polypeptides corresponding to one or more regions of the SARS-CoV-2 S glycoprotein (e.g., RBD), or fusion proteins containing one or more regions of the SARS-CoV-2 S glycoprotein. In the non-cell-based assay, recombinant human SARS-CoV-2 S glycoprotein is attached to a solid substrate such as a test tube, microliter well, or column by means well known to those skilled in the art (see Ausubel et al. above). The ability of the test antibody to bind to the SARS-CoV-2 S glycoprotein is then assayed.

[0187] The binding reaction can also be carried out in solution. In this assay, the labeled component can interact with its binding partner(s) in solution. If such separation is possible due to a size difference between the labeled component and its binding partner(s), separation can be achieved by passing the binding reaction product through an ultrafiltration membrane with pores that allow unbound labeled components to pass through but not its binding partner(s) or the labeled component(s) bound to its partner(s). Separation can also be achieved using any reagent capable of capturing the binding partner(s) of the labeled component from solution (e.g., an antibody against the binding partner).

[0188] In one embodiment, for example, a phage library can be screened by passing phages from a serial phage display library through a column containing SARS-CoV-2 S glycoprotein or a portion thereof (e.g., RBD of SARS-CoV-2 S glycoprotein), or its derivatives, analogs, fragments, or domains, bound to a solid phase such as plastic beads. By changing the stringency of the wash buffer, phages expressing peptides with high affinity for SARS-CoV-2 S glycoprotein can be enriched. The phages isolated from the column can be cloned, and their affinity can be directly measured. Once it is known which antibody and its amino acid sequence confers the strongest binding to SARS-CoV-2 S glycoprotein, a computer model can be used to identify molecular contacts between SARS-CoV-2 S glycoprotein and candidate antibodies.

[0189] In another specific embodiment, the solid support is a membrane containing the SARS-CoV-2 S glycoprotein and is attached to a microtiter dish. Candidate antibodies can be bound to cells expressing the library antibody, for example, cultured under conditions that allow expression of the library member in a microliter dish. Library members that bind to SARS-CoV-2 are collected. Such methods are generally described as examples in Parmley and Smith, 1988, Gene, 73:305-318; Fowlkes et al., 1992, BioTechniques, 13:422-427; PCT publication number W094 / 18318; and the references cited above herein. The antibody identified as binding to the SARS-CoV-2 S glycoprotein may be any of the above types or modifications of the antibody.

[0190] Screening of antibodies against human ADCC effector function Human IgG class antibodies, which have a long half-life in serum and possess functional characteristics such as the ability to mediate various effector functions, are used in certain embodiments of the present invention (Monoclonal Antibodies: Principles and Applications, Wiley-Liss, Inc., Chapter 1 (1995)). Human IgG class antibodies are further classified into the following four subclasses: IgG1, IgG2, IgG3, and IgG4. Numerous studies have been conducted on ADCC and CDC as effector functions of IgG class antibodies, and it has been reported that the IgG1 subclass has the highest ADCC and CDC activity in humans among human IgG class antibodies (Chemical Immunology, 65, 88 (1997)).

[0191] The expression of ADCC and CDC activity by human IgG1 subclass antibodies generally involves the binding of the antibody's Fc region to the antibody receptor (hereinafter referred to as "FcγR") present on the surface of effector cells (e.g., killer cells, natural killer cells, or activated macrophages). Various complement components can be bound. Regarding binding, it has been suggested that several amino acid residues in the hinge region and the second domain of the C region of the antibody (hereinafter referred to as the "Cγ2 domain") are important (Eur.J.Immunol.,23,1098(1993),Immunology,86,319(1995),Chemical Immunology,65,88(1997)), and that glycans within the Cγ2 domain (Chemical Immunology,65,88(1997)) are also important.

[0192] Anti-CoV S glycoprotein antibodies can be modified in terms of effector function, for example, to enhance the antibody's ADCC and / or complement-dependent cell-mediated cytotoxicity (CDC). This can be achieved by introducing one or more amino acid substitutions into the antibody's Fc region. Alternatively, cysteine ​​residues may be introduced into the Fc region to allow the formation of interchain disulfide bonds within this region. In this way, homodimeric antibodies with improved internalization ability that can increase complement-mediated cell death and ADCC can be produced (Caron et al., J.Exp.Med., 176:1191-1195 (1992) and Shopes, J.Immunol., 148:2918-2922 (1992)). Heterobifunctional crosslinking agents can also be used to produce homodimeric antibodies with enhanced antitumor activity (Wolff et al., Cancer Research, 53:2560-2565 (1993)). Furthermore, antibodies can be manipulated to have two or more Fc regions to enhance their complement lysis and ADCC capabilities (Stevenson et al., Anti-Cancer Drug Design, (3)219-230(1989)).

[0193] Other methods for manipulating the Fc region of an antibody to alter its effector function are known in the art (for example, U.S. Patent Publication 20040185045 and PCT Publication WO2004 / 016750 (both to Koenig et al., describing modifications of the Fc region to enhance the binding affinity to FcγRIIB compared to the binding affinity to FCγRIIA); also see PCT Publication WO99 / 58572 to Armour et al., WO99 / 51642 to Idusogic et al., and US6,395,272 to Dco et al. (these disclosures are incorporated herein by reference in their entirety)). Furthermore, methods for modifying the Fc region to reduce its binding affinity to FcγRIIB are also known in the art (e.g., U.S. Patent Publication No. 20010036459 and PCT Publication No. WO01 / 79299 (both to Ravetch et al., the entirety of which is incorporated herein by reference)). Modified antibodies having variant Fc regions with enhanced binding affinity to FcγRIIIA and / or FcγRIIA compared to the wild-type Fc region have also been described (e.g., see PCT Publication No. WO2004 / 063351 to Stavenhagen et al. (the entirety of which is incorporated herein by reference)).

[0194] At least four different types of FcγR have been identified, called FcγRI(CD64), FcγRII(CD32), FcγRIII(CD16), and FcγRIV, respectively. In humans, FcγRII and FcγRIII are further classified into FcγRIIa and FcγR1Ib, and FcγRIIIa and FcγRIIIb, respectively. FcγR is a membrane protein belonging to the immunoglobulin superfamily. FcγRII, FcγRIII, and FcγRIV have an a-chain with an extracellular domain containing two immunoglobulin-like domains, while FcγRI has an a-chain with an extracellular domain containing three immunoglobulin-like domains, with the a-chain being involved in IgG binding activity. Furthermore, FcγRI and FcγRIII contain a γ or ζ chain as a component that has signal transduction function in relation to the α chain (Annu. Rev. Immunol., 18, 709 (2000), Annu. Rev. Immunol., 19, 275 (2001)). FcγRIV is described in Bruhns et al., Clin. Invest. Med., (Canada) 27:3D (2004).

[0195] To evaluate the ADCC activity of an anti-CoV S glycoprotein antibody of interest, an in vitro ADCC assay, such as those described in U.S. Patent No. 5,500,362 or No. 5,821,337, can be used. Alternatively, the assay may be performed using a commercially available kit (e.g., CytoTox 96® (Promega)). Useful effector cells for such assays include, but are not limited to, peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, and NK cell lines. NK cell lines expressing transgenic Fc receptors (e.g., CD16) and associated signaling polypeptides (e.g., FCεRI-γ) can also function as effector cells (see, for example, WO2006 / 023148A2 against Campbell). For example, the ability of any specific antibody to mediate complement activation and / or ADCC-mediated lysis of target cells can be assayed. Cells of interest are grown and labeled in vitro. Antibodies are added to cell cultures in combination with immune cells that can be activated by antigen-antibody complexes, i.e., effector cells involved in the ADCC response. The complement activation of these antibodies can also be tested. In either case, cell lysis of target cells is detected by the release of a label from the lysed cells. The degree of target cell lysis can also be quantified by detecting the release of cytoplasmic proteins (e.g., LDH) into the supernatant. In practice, antibodies can be screened using the patient's own serum as a source of complement and / or immune cells. Antibodies capable of mediating human ADCC in vitro can be used therapeutically for that particular patient. Furthermore, the ADCC activity of molecules of interest can also be evaluated in vivo in animal models, such as those disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA), 95:652-656 (1998). In addition, techniques for modulating (i.e., increasing or decreasing) the ADCC level of antibodies, and optionally their CDC activity, are well known in the art. See, for example, U.S. Patent No. 6,194,551. The antibodies of the present invention may be capable of inducing ADCC and / or CDC, or may be modified to have such ability.Assays for quantifying ADCC function can be performed using human effector cells to evaluate human ADCC function. Such assays may also include those intended for screening antibodies that induce, mediate, enhance, or block cell death via necrotic and / or apoptotic mechanisms. Such methods (including assays utilizing cell viability dyes, methods for detecting and analyzing caspases, and assays for measuring DNA cleavage) can be used to assess the apoptotic activity of cells cultured in vitro with an anti-CoV S glycoprotein antibody of interest.

[0196] For example, the Annexin V or TdT-mediated dUTP nick end labeling (TUNEL) assay can be performed to detect apoptotic activity, as described in Decker et al., Blood (USA) 103:2718-2725 (2004). The TUNEL assay involves culturing cells of interest with fluorescein-labeled dUTP for incorporation into DNA strand breaks. The cells are then processed for analysis by flow cytometry. The Annexin V assay detects the appearance of phosphatidylserine (PS) on the outer cell membrane of apoptotic cells using fluorescein-conjugated Annexin V that specifically recognizes exposed PS molecules. At the same time, a cell viability dye such as propidium iodide can be used to exclude late-stage apoptotic cells. Cells are stained with labeled Annexin V and analyzed by flow cytometry.

[0197] Neutralizing Antibody In various embodiments, the anti-CoV S glycoprotein antibodies described herein are neutralizing antibodies. In various embodiments, the anti-CoV S glycoprotein antibodies neutralize SARS-CoV-2 virus or variants thereof.

[0198] Anti-CoV S Glycoprotein Antibody Conjugate According to certain specific aspects of the present invention, compounds can be conjugated to anti-CoV S glycoprotein antibodies for use in the compositions and methods of the present invention. In certain specific embodiments, these conjugates can be produced as fusion proteins.

[0199] Covalent modifications of anti-CoV S glycoprotein antibodies are encompassed within the scope of the present invention. This can be carried out by chemical synthesis, or where applicable, by enzymatic or chemical cleavage of the antibody. Other types of covalent modifications of anti-CoV S glycoprotein antibodies are introduced into the molecule by reacting targeted amino acid residues of the antibody with an organic derivatizing agent that is capable of reacting with selected side chains or N-terminal or C-terminal residues.

[0200] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Similarly, iodo reagents can also be used. Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidazolyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimide, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0201] Histidyl residues are derivatized by reaction with diethyl pyrocarbonate at pH 5.5 to 7.0. This is because this agent is relatively specific for the histidyl side chain. Parabromophenacyl bromide is also useful. The reaction can be carried out in 0.1 M sodium cacodylate at pH 6.0.

[0202] Lysyl and amino-terminal residues are reacted with succinic acid or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residue. Other suitable reagents for derivatizing α-amino-containing residues and / or ε-amino-containing residues include transaminase-catalyzed reactions with imide esters, such as methyl picoline imidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, 0-methyl isourea, 2,4-pentanedione, and glyoxylate.

[0203] Arginyl residues can be modified by reaction with one or more conventional reagents, such as phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginyl residues generally requires reaction under alkaline conditions due to the high pKa of the guanidine functional group. Furthermore, these reagents can react with the arginine epsilon-amino group in addition to the ε-amino group of lysine.

[0204] Specific modification of tyrosyl residues, particularly the introduction of spectral labels to tyrosyl residues, can be carried out by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidisole and tetranitromethane are used to form O-acetyltyrosyl species and 3-nitro derivatives, respectively. 125 I or 131 Iodine is used to prepare labeled proteins for use in radioimmunoassays.

[0205] The carboxyl side chain (aspartyl or glutamyl) is selectively modified by reaction with a carbodiimide (RN=C=N-R'), where R and R' are different alkyl groups, for example, 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, the aspartyl and glutamyl residues are converted to asparaginyl and glutamyl residues by reaction with ammonium ions.

[0206] Glutaminyl and asparaginyl residues are often deamidated to the corresponding glutamyl and aspartyl residues, respectively. These residues are deamidated under neutral or basic conditions. The deamidation forms of these residues are within the scope of the present invention.

[0207] Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of ceryl or threonyl residues, methylation of α-amino groups of lysine, arginine, and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, WH Freeman & Co., San Francisco, pp. 79-86 (1983)), acetylation of N-terminal amines, and amidation of any C-terminal carboxyl group.

[0208] Another type of covalent modification involves chemically or enzymatically attaching a glycoside to an antibody. This procedure is advantageous in that it does not require the production of antibodies in host cells capable of glycosylation for N- or O-linked glycosylation. Depending on the coupling mechanism used, the sugar(s) can be attached to (a) arginine and histidine, (b) a free carboxyl group, (c) a free sulfhydryl group (e.g., of cysteine), (d) a free hydroxyl group (e.g., of serine, threonine, or hydroxyproline), (e) an aromatic residue (e.g., of phenylalanine, tyrosine, or tryptophan), or (f) the amide group of glutamine. These methods are described in WO87 / 05330 (published September 11, 1987) and Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0209] Pharmaceutical composition The present invention also relates to a composition comprising an anti-CoV S glycoprotein antibody, and to a method of using the composition for the treatment of COVID-19 in human subjects.

[0210] The present invention relates to a pharmaceutical composition comprising an anti-CoV S glycoprotein antibody of the IgG1 or IgG3 human isotype. The present invention also relates to a pharmaceutical composition comprising an anti-CoV S glycoprotein antibody of the IgG2 or IgG4 human isotype that can mediate human ADCC. In certain embodiments, the present invention also relates to a pharmaceutical composition comprising a monoclonal human antibody, a humanized antibody, or a chimeric anti-CoV S glycoprotein antibody that can be produced by means known in the art.

[0211] In other specific embodiments, anti-CoV S glycoprotein antibodies can mediate ADCC, complement-dependent cell-mediated cytotoxicity, or apoptosis.

[0212] Antibody half-life In various embodiments, the half-life of the anti-CoV S glycoprotein antibody described herein is approximately 1 hour to approximately 60 days. For example, anti-CoV The half-life of S glycoprotein antibodies is approximately 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, and 17 days. 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days Maximum of approximately 29 days, maximum of approximately 30 days, maximum of approximately 31 days, maximum of approximately 32 days, maximum of approximately 33 days, maximum of approximately 34 days, maximum of approximately 35 days, maximum of approximately 36 days, maximum of approximately 37 days, maximum of approximately 38 days, maximum of approximately 39 days, The maximum half-life is approximately 40 days, 41 days, 42 days, 43 days, 44 days, 45 days, 46 days, 47 days, 48 ​​days, 49 days, 50 days, 51 days, 52 days, 53 days, 54 days, 55 days, 56 days, 57 days, 58 days, 59 days, or 60 days. In certain embodiments, the half-life of the antibody of the composition and method of the present invention can be extended by methods known in the art. Such extension can reduce the amount and / or frequency of administration of the antibody composition. Antibodies with improved in vivo half-lives and methods for preparing them are disclosed in U.S. Patent No. 6,277,375 and International Publication Nos. WO98 / 23289 and WO97 / 3461.

[0213] Furthermore, the in vivo serum circulation of anti-CoV S glycoprotein antibodies can also be extended by site-specific conjugation of inert polymer molecules such as high molecular weight polyethylene glycol (PEG) to the N or C terminus of the antibody, or by attaching them to the antibody with or without a multifunctional linker via epsilon-amino groups present on lysyl residues. Linear or branched polymer derivatization is used to minimize the loss of biological activity. The degree of conjugation can be carefully monitored by SDS-PAGE and mass spectrometry to ensure proper binding between the PEG molecule and the antibody. Unreacted PEG can be separated from the antibody-PEG conjugate by size exclusion or ion-exchange chromatography. In addition to testing for binding activity, PEG-derivative antibodies can also be tested for in vivo efficacy using methods known to those skilled in the art (e.g., by the immunoassays described herein).

[0214] Furthermore, the antibodies of the compositions and methods of the present invention can be conjugated with albumin to make the antibodies more stable in vivo or to lengthen their half-life in vivo. Such techniques are well known in the art. See, for example, International Publications WO93 / 15199, WO93 / 15200, and WO01 / 77137, and European Patent EP413,622 (all of which are incorporated herein by reference).

[0215] Pharmaceutical preparations, administration, and drug dispensing The pharmaceutical formulation of the present invention contains an anti-CoV S glycoprotein antibody as an active ingredient. The formulation contains a naked antibody, an immunoconjugate, or a fusion protein in a unit of weight or volume suitable for administration to a human patient, in an amount effective to produce a desired response, and is preferably sterile.

[0216] Anti-CoV S glycoprotein antibody compositions can be formulated with a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means one or more non-toxic materials that do not interfere with the efficacy of the biological activity of the active ingredient. Such preparations may typically include salts, buffers, preservatives, a suitable carrier, and optionally other therapeutic agents. Such pharmaceutically acceptable preparations may also typically include a suitable solid or liquid filler, diluent, or encapsulating material suitable for administration to humans. When used pharmaceutically, salts should be pharmaceutically acceptable, but pharmaceutically unacceptable salts may also be conveniently used to prepare such pharmaceutically acceptable salts and are not excluded from the scope of this invention. Such pharmacokinetic and pharmaceutically acceptable salts include, but are not limited to, salts prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, malonic acid, acetic acid, salicylic acid, citric acid, boric acid, formic acid, malonic acid, succinic acid, etc. Furthermore, pharmaceutically acceptable salts may be prepared as alkali metals or alkaline earth metals such as sodium, potassium, or calcium salts. The term "carrier" refers to natural or synthetic organic or inorganic components that are combined with the active ingredient to facilitate application. In addition, the components of the pharmaceutical composition may be mixed with the antibody of the present invention, and with each other, in such a manner that there are no interactions that substantially impair the desired pharmaceutical efficacy.

[0217] According to certain specific embodiments of the present invention, the anti-CoV S glycoprotein antibody composition can be prepared for storage in the form of a lyophilized formulation or an aqueous solution by mixing the antibody or immunoconjugate having a desired purity with an optional physiologically acceptable carrier, excipient, or stabilizer (Remington's Pharmaceutical Sciences, 16th edition, Osol, A. Ed. (1999)). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and such carriers, excipients, or stabilizers include buffers, for example, phosphate, citrate, and other organic acids; antioxidants (including ascorbic acid and methionine); preservatives (for example, octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates (including glucose, mannose, or dextrins); chelating agents such as EDTA; sugars (for example, sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions such as sodium; metal complexes (for example, Zn-protein complexes); and / or nonionic surfactants such as TWEEN(registered trademark), PLURONICS(registered trademark), or polyethylene glycol (PEG).

[0218] In addition, the anti-CoV S glycoprotein antibody composition can optionally contain suitable preservatives (for example, benzalkonium chloride, chlorobutanol, parabens, and thimerosal).

[0219] Anti-CoV S glycoprotein antibody compositions can conveniently be presented in unit dose form and can be prepared by any method well known in the pharmaceutical field. All methods involve the step of conjugating the active agent to a carrier constituting one or more auxiliary components. Generally, anti-CoV S glycoprotein antibody compositions are prepared by uniformly and closely associating the active compound with a liquid carrier, a fine powder solid carrier, or both, and then shaping the product if necessary.

[0220] Compositions suitable for parenteral administration conveniently include a sterile aqueous or non-aqueous preparation of an anti-CoV S glycoprotein antibody, which is preferably isotonic with the recipient's blood. This preparation can be formulated using suitable dispersants or wetting agents and suspending agents according to known methods. Alternatively, sterile injection preparations may be sterile injection solutions or suspensions in a non-toxic, parenterally acceptable diluent or solvent (e.g., as a solution in 1,3-butanediol). Acceptable vehicles and solvents that can be used include water, Ringer's solution, and isotonic sodium chloride solution. Furthermore, sterile fixatives have conventionally been used as solvents or suspension media. For this purpose, any non-irritating fixative, including synthetic monoglycerides or diglycerides, can be used. Additionally, fatty acids such as oleic acid can also be used in the preparation of injection solutions. Carrier formulations suitable for oral, subcutaneous, intravenous, and intramuscular administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0221] Furthermore, the active ingredient can be encapsulated in microcapsules prepared by coacervation techniques or interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively), colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0222] Formulations used for in vivo administration are typically sterile. This is easily achieved by filtering through a sterile filtration membrane.

[0223] Sustained-release preparations may be prepared. A preferred example of a sustained-release preparation is a semipermeable matrix of a solid hydrophobic polymer containing an anti-CoV S glycoprotein antibody, which takes the form of a molded article (e.g., a film or microcapsule). Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactide (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactate-glycolic acid copolymers, e.g., LUPRON DEPOT® (injectable microspheres composed of lactate-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid. Polymers such as ethylene vinyl acetate and lactate glycolic acid allow molecules to be released for 100 days, while certain hydrogels release proteins in a shorter period. If encapsulated antibodies remain in the body for extended periods, exposure to moisture at 37°C can result in denaturation or aggregation, potentially leading to loss of biological activity and altered immunogenicity. Depending on the mechanism involved, reasonable strategies for stabilization can be devised. For example, if the aggregation mechanism is found to be intermolecular SS bond formation via thio-disulfide exchange, stabilization can be achieved through modification of sultidyl residues, freeze-drying from acidic solutions, control of water content, use of appropriate additives, and development of specific polymer matrix compositions. In certain embodiments, the pharmaceutically acceptable carriers used in the compositions of the present invention do not affect human ADCC or CDC.

[0224] The anti-CoV S glycoprotein antibodies disclosed herein can also be formulated as immunoliposomes. “Liposomes” are vesicles composed of various types of lipids, phospholipids, and / or surfactants, which are useful for the delivery of drugs (e.g., the anti-CoV S glycoprotein antibodies disclosed herein) to humans. The components of liposomes are generally arranged in a bilayer configuration, similar to the lipid arrangement of biological membranes. Liposomes containing the antibodies of the present invention are prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA, 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA, 77:4030 (1980); and U.S. Patents No. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be produced by reverse-phase evaporation using a lipid composition containing phosphatidylcholine, cholesterol, and PEG-derivativeized phosphatidylethanolamine (PEG-PE). The liposomes are extruded through a filter of a specified pore size to obtain liposomes with the desired diameter. The antibodies of the present invention can be conjugated with liposomes via a disulfide exchange reaction, as described in Martin et al., J. Biol. Chem., 257:286-288 (1982). Therapeutic agents can also be contained within the liposomes. See Gabizon et al. J. National Cancer Inst. (19) 1484 (1989).

[0225] In certain embodiments, the pharmaceutical composition of the present invention is stable at 4°C. In certain embodiments, the pharmaceutical composition of the present invention is stable at room temperature.

[0226] The compositions of the present invention can be administered to human patients by any route, including, but not limited to, intravenous, intradermal, transdermal, subcutaneous, intramuscular, inhalation (e.g., via aerosol), buccal (e.g., sublingual), topical (e.g., both skin and mucous membrane surfaces, including the airway surface), intrathecal, intra-articular, intrapleural, intracerebral, intra-arterial, intraperitoneal, oral, intralymphatic, intranasal, intrarectal, or vaginal administration, perfusion via a local catheter, or direct intra-focal injection. In one embodiment, the compositions of the present invention are administered by intravenous push or intravenous infusion over a specified period (e.g., 0.5 to 2 hours). The compositions of the present invention can be delivered by peristaltic means or in depot form, but the most suitable route in any given case depends on factors such as the species, age, sex, and general condition of the subject, the nature and severity of the condition being treated, and / or the nature of the particular composition to be administered (i.e., dosage, formulation), as is well known in the art.

[0227] In some embodiments, the dose of the composition containing anti-CoV S glycoprotein antibodies is measured in mg / kg of the patient's body weight. In other embodiments, the dose of the composition containing anti-CoV S glycoprotein antibodies is measured in mg / kg of the patient's lean body mass (i.e., body weight minus body fat content). In yet another embodiment, the dose of the composition containing anti-CoV S glycoprotein antibodies is measured in mg / m² of the patient's body surface area. 2 The dose is measured in units of mg. In other embodiments, the dose of a composition containing an anti-CoV S glycoprotein antibody is measured in mg per dose administered to a patient. Any measured dose can be used with the compositions and methods of the present invention, and the dose units can be converted by means of the art.

[0228] Those skilled in the art will understand that the dosage can be selected based on many factors, including the age, sex, species, and condition of the subject. For example, the effective amount of the composition of the present invention can be estimated from dose-response curves derived from in vitro test systems or animal model (e.g., Cotton rats or monkeys) test systems. Models and methods for evaluating the effects of antibodies are known in the art (Wooldridge et al., Blood, 89(8):2994-2998 (1997) (the whole thereof is incorporated herein by reference)).

[0229] Examples of administration regimens that can be used in the method of the present invention include, but are not limited to, daily, three times a week (intermittently), weekly, every 14 days, every month, every 6 to 8 weeks, every 2 months, every 6 months, or every year.

[0230] In various embodiments, the dose of anti-CoV S glycoprotein antibody ranges from 10 mg to approximately 2 g. For example, doses of anti-CoV S glycoprotein antibody range from approximately 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, and 850 mg. mg, approximately 900 mg, approximately 950 mg, approximately 1 g, approximately 1.05 g, approximately 1.1 g, approximately 1.15 g, approximately 1.2 g, approximately 1.25 g, approximately 1.3 g, approximately 1.35 g, approximately 1.4 g, approximately 1.45 g, approximately 1.5 g, approximately 1.55 g, approximately 1.6 g, approximately 1.65 g, approximately 1.7 g, approximately 1.75 g, approximately 1.8 g, approximately 1.85 g, approximately 1.9 g, approximately 1.95 g, approximately 2 g, or any range or partial range between these.

[0231] In some embodiments, the Disclosure provides a method for treating a subject infected with the SARS-CoV-2 virus or a variant thereof, comprising administering a composition comprising an anti-CoV S glycoprotein antibody as described herein. In some embodiments, the SARS-CoV-2 variant has a PANGO lineage selected from the group consisting of B.1.1.529, BA.1, BA.1.1, BA.2, BA.3, BA.4, BA.5, B.1.1.7, B.1.351, P.1, BA.1.617.2, AY, B.1.427, B.1.429, B.1.525, B.1.526, B.1.617.1, B.1.617.3, P.2, B.1.621, or B.1.621.1.

[0232] Toxicity testing The tolerability, toxicity, and / or efficacy of the compositions and / or therapeutic regimens of the present invention can be quantified, for example, by standard pharmaceutical procedures in cell cultures or experimental animals for quantifying the LD50 (a dose that is lethal to 50% of the population), ED50 (a dose that is therapeutically effective to 50% of the population), and IC50 (a dose that is effective in achieving 50% inhibition).

[0233] Data obtained from cell culture assays and animal studies can be used to formulate dosage ranges and / or therapeutic regimens for human use. Doses of such agents are within the circulating concentration range, including the ED50, and are minimal to no toxicity. Doses may vary within this range depending on the form of administration used and the route of administration utilized. For any therapeutic method used in the methods of the present invention, the effective therapeutic dose can be estimated using a suitable animal model. Depending on the species of the animal model, the dose can be scaled for human use according to a formulation recognized in the art, such as, for example, Freireich et al., Quantitative comparison of toxicity of anticancer agents in mouse, rat, monkey, dog, and human, Cancer Chemotherapy Reports, NCI 1966 40:219-244. Data obtained from cell culture assays may be useful in predicting potential toxicity. Animal studies can be used to formulate specific doses to achieve the circulating plasma concentration range, including the IC50 (i.e., the concentration of the test compound that achieves maximum half-dose inhibition of symptoms) quantified in cell culture. Such information can be used to more accurately quantify useful doses in humans. Plasma drug levels can be measured, for example, by high-performance liquid chromatography, ELISA, or cell-based assays.

[0234] Diagnostic assays, product identity assays, lot release assays In various embodiments, the antibodies and their fragments described herein are used for the manufacture of diagnostic assays, product identity assays, and lot release assays. In various embodiments, the antibodies or their fragments are used in ELISA assays. In various embodiments, the antibodies or their fragments are used in flow cytometry experiments. In various embodiments, the antibodies or their fragments are used to determine whether a sample contains SARS-CoV-2 S glycoprotein. [Examples]

[0235] Example 1: Production of SARS-CoV-2 S glycoprotein and its subunits SARS-CoV-2 S glycoprotein nanoparticles were generated in Spodoptera frugiperda (Sf9) cells using the procedure for the production of SARS-CoV-2 S glycoprotein nanoparticles described in International Publication No. 2021 / 154812 (which is incorporated herein by reference in its entirety for all purposes). Compared to native SARS-CoV-2 S glycoprotein, the SARS-CoV-2 S glycoprotein described herein contains the amino acid sequence QQAQ (SEQ ID NO: 76) and inactive fulin cleavage sites with proline at amino acid positions 973 and 974 (SARS-CoV-2 glycoproteins are numbered according to SARS-CoV-2 S glycoprotein SEQ ID NO: 73). The amino acid sequences of each generated SARS-CoV-2 S glycoprotein are shown in Table A. [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Table A-6] [Table A-7] [Table A-8]

[0236] The recombinant SARS-CoV-2 S receptor-binding domain and the N-terminal domain containing the 8X-His tag were subcloned, expressed, and purified. Briefly, the N-terminal domain (NTD, residues 1-294) of the SARS-CoV-2 XBB.1.5 spike was codon-optimized for recombinant expression and synthesized into a pcDNA3.1(+) mammalian expression plasmid (Genscript, Piscataway, NJ, USA). The XBB.1.5 NTD construct was transiently translocated into Expi293F(registered trademark) cell suspension cultures in low-serum medium using ExpiFectamine(registered trademark) 293 translocation reagent. Five days after translocation, the secreted protein was purified using Ni-NTA Sepharose 6 Fast Flow IMAC resin (CYTIVA(registered trademark)). The fractions containing XBB.1.5 NTD were combined, buffered with 25 mM NaPi, 150 mM NaCl, and 0.03% PS80, and concentrated to approximately 1.0 mg / mL.

[0237] Example 2: Production of anti-SARS-CoV-2 S glycoprotein antibody To generate monoclonal antibodies against XBB.1.5 rS, female BALB / c mice (M. musculus; n=6) were immunized intramuscularly with 5 μg of XBB.1.5 rS (SEQ ID NO: 65) and 5 μg of saponin adjuvant on day 0 and day 14.

[0238] A schematic diagram of the XBB 1.5 rS glycoprotein is shown in Figure 1. The XBB 1.5 rS glycoprotein has 42 mutations compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 74, of which 12 are in the NTD and 22 are in the RBD. The table below shows the mutations in the SARS-CoV-2 S glycoprotein of the SARS-CoV-2 variant compared to the SARS-CoV-2 S glycoprotein of SEQ ID NO: 74. [Table 6]

[0239] The table above highlights the amino acid changes, deletions, and insertions of the XBB lineages shown for XBB.1.5, XBB.2.3, XBB.1.16.6, and EG.1.5 spikes, compared to the SARS-CoV-2 S glycoprotein ("Wuhan-Hu-1") of SEQ ID NO: 74. Common mutations are represented by X, while mutational drift is indicated as O, +, or R for each respective variant.

[0240] The saponin adjuvant contained approximately 85% by weight of a fraction A ISCOM matrix derived from Quillaja Saponaria Molina and approximately 15% by weight of a fraction C ISCOM matrix derived from Quillaja Saponaria Molina, relative to the total weight of the saponin adjuvant in the composition. Further information regarding the fraction A ISCOM matrix and the fraction C ISCOM matrix derived from Quillaja Saponaria Molina is provided in International Publication 2021 / 154812 (which is incorporated herein by reference in its entirety for all purposes). Two mice with the highest anti-rS IgG and hACE2 receptor inhibitory activity were selected and boosted with 5 μg of XBB.1.5 rS intraperitoneally on day 29 (without adjuvant). Splenectomy was performed on day 33, followed by collection of spleen cells, isolation of IgG-expressing cells by negative selection, and subsequent fusion, expansion, and screening of hybridomas. Positive clones were enlarged, and hybridoma isotypes were determined.

[0241] The following antibodies were identified: NVX.205.10, NVX.172.10, NVX.324.6, and NVX.62.12. NVX.205.10 contains the VL domain of SEQ ID NO: 3 and the VH domain of SEQ ID NO: 7. NVX.172.10 contains the VL domain of SEQ ID NO: 2 and the VH domain of SEQ ID NO: 6. NVX.324.6 contains the VL domain of SEQ ID NO: 4 and the VH domain of SEQ ID NO: 8. NVX.62.12 contains the VL domain of SEQ ID NO: 1 and the VH domain of SEQ ID NO: 5. The respective CDRs for NVX.205.10, NVX.172.10, NVX324.6, and NVX.62.12 are shown in Table 2 of this application.

[0242] Example 3: Characterization of the binding of antibodies NVX.205.10, NVX.172.10, NVX.324.6, and NVX.62.12 to SARS-CoV-2 S glycoprotein Objective: We investigated the ability of antibodies ("mAbs") NVX.205.10, NVX.172.10, NVX.324.6, and NVX.62.12 to bind to the CoV S glycoprotein derived from parental SARS-CoV-2 virus (SARS-CoV-2 virus possessing the SARS-CoV-2 S glycoprotein of SEQ ID NO: 73) and multiple SARS-CoV-2 variants. We also investigated the ability of these antibodies to inhibit the interaction between hACE2 and the SARS-CoV-2 S glycoprotein. Finally, we examined the ability of these antibodies to neutralize viable SARS-CoV-2 virus and pseudoviruses.

[0243] Results: None of the antibodies bound to the ancestral SARS-CoV-2 S glycoprotein (SEQ ID NO: 74). ELISA quantification revealed that all four mAbs bound to XBB.1.5, XBB.1.16, XBB.2.3, and EG.5.1, while three bound to XBB.1.16.6 and FL.1.5.1. Only one of these four mAbs showed hACE2 binding inhibitory activity against all tested XBB subseries variants (mAb NVX.205.10), another mAb showed hACE2 binding inhibitory activity against XBB.1.5, XBB.2.3, and EG.5.1 (mAb NVX.172.10). The remaining two mAbs (NVX.324.6 and NVX.62.12) showed no hACE2 binding inhibitory activity against any of the tested variants. Despite these differences in hACE2 binding inhibitory activity, these mAbs were found to potently neutralize SARS-CoV-2 omicron XBB.1.5, XBB.2.3, XBB.1.16, XBB.1.16.6, and EG.5.1 in pseudoviral neutralization assays, and all mAbs except NVX.172.10 were found to neutralize FL.1.5.1 (Figures 2A-2D). [Table 7]

[0244] To further investigate the binding activity of these mAbs, binding kinetics were performed on NVX.205.10 and NVX.324.6 using biolayer interferometry (BLI) and biolayer inferometry. First, binding to the XBB.1.5, XBB.2.3, XBB.1.16, XBB.1.16.6, EG.5.1, and FL.1.5.1 spike proteins was evaluated (Figures 3 and 4). The binding constants (ka values; a / Ms) for NVX.205.10 to these target variant ("VoC") spike glycoproteins ranged from 2.0 × 10⁴ to 5.6 × 10⁴, while the ka values ​​for binding to NVX.324.6 ranged from 1.6 × 10⁴ to 4.5 × 10⁴. On the other hand, the dissociation constants (kd; 1 / dis) of both mAbs were less than 1.0 × 10⁻⁷ in all VoC spikes (see table below). [Table 8]

[0245] In contrast, both mAbs exhibited undetectable binding and dissociation to the prototype spike (ancestral SARS-CoV-2; Wuhan-Hu-1), indicating a lack of binding. Octet's BLI also showed that NVX.205.10 bound to the XBB.1.5 receptor-binding domain (RBD), while NVX.324.6 bound to the XBB.1.5 NTD (Figure 5 and the table below). [Table 9]

[0246] These binding domains were further confirmed by SDS-PAGE and Western blotting (Figures 6 and 7).

[0247] Next, using negative staining (NS) transmission electron microscopy (TEM), epitopes of Fab isolated from hybridoma mAbs were mapped to SARS-CoV-2 XBB.1.5 rS (Figures 8A-8D). Monoclonal Fab was incubated with XBB.1.5 antigen prior to imaging, and the dominant epitope was identified using 2D / 3D classification. Purified SARS-CoV-2 variant XBB.1.5 trimers showed binding to their respective Fabs and exhibited NTD or RBD-specific binding in various conformations (Figures 8A-8D). Furthermore, 3D reconstruction from NS-TEM revealed that SARS-CoV-2 XBB.1.5 trimers can simultaneously bind two or three NTD-binding Fabs and one or two RBD-binding Fabs in "down or up" conformations (Figure 9). 3D reconstruction also showed that Fabs can bind to RBDs in conformations that minimize arbitrary steric hindrance with NTDs (Figure 9).

[0248] Furthermore, Fab binding to each subdomain (NTD or RBD) was found to be independent of any collisions, suggesting specificity and a broad neutralization mechanism (Figures 8A-8D, Figure 9). It was also shown that NTD-specific NVX.324.6 and RBD-specific NVX.205.10 form stable complexes with XBB.1.5 rS in their pre-fusion three-dimensional structures.

[0249] To map NTD-specific antibody binding, a homology model of the XBB.1.5 NTD was compared with docking NVX.324.6 to the structures of four known neutralizing antibodies (PDB 2L2F, 7L2E, 7SWW, 7L2E) (most of which are supersite-specific). Using the NTD-oriented PDB structure, a structural superposition for NTD-Cα was obtained to mimic the mAb binding site. The model shown in Figure 10 indicates that this NTD-specific antibody may bind to the XBB.1.5 spike via the supersite N-3 and N-5 loops. This supersite is described in Lok et al. 2021, 29(5):744-746 (the entire article is incorporated herein by reference for all purposes).

[0250] To verify the EM density of NS-TEM 3D reconstitutions corresponding to RBD-specific Fabs (multiple Fabs), a Fab-spike complex homology model was created and fitted to the spike trimer-bound Fab (multiple Fabs) of the NVX.205.10 antibody at the "up" position of the RBD, illustrating the overall characteristics of antibody recognition. The 3D reconstitution revealed the main three-dimensional structure of XBB.1.5, with two RBDs adopting the "up" position, each bound to one Fab, and a third RBD existing in a "down" three-dimensional structure without a Fab (Figure 11). Mapping using known structures of RBD-Fab complexes (PDBs-6XCN, 6XCM, 7WDO, 3HC3, 8HCA) revealed that the neutralizing epitope on the RBD of NVX.205.10 is located on the outer surface of the receptor-binding motif (RBM) region, indicating that this antibody is a class 1-2 antibody.

[0251] Method (Recombinant CoV S Glycoprotein Production): SARS-CoV-2 S glycoprotein nanoparticles (also called CoV S glycoprotein) were produced according to the method of Example 1.

[0252] Method (Antibody Generation): Antibodies were generated according to the method described in Example 2.

[0253] Method (ELISA): 96-well microtiters were coated with 1.0 μg / mL of SARS-CoV-2 S protein. After blocking nonspecific binding, serial dilutions of monoclonal antibodies were added, and antibody binding was measured using horseradish peroxidase (HRP)-conjugated anti-mice. Substrate turnover was measured at OD 450 nm. EC50 values ​​were calculated by 4-parameter curve fitting.

[0254] Methods (hACE2 receptor inhibition): The ability of antibodies to block the interaction between the human angiotensin-converting enzyme 2 (hACE2) receptor and CoV S glycoprotein was evaluated by ELISA. Briefly, 1.0 μg / mL of CoV S glycoprotein was coated onto a 96-well plate overnight at 4°C. The plate was washed with PBS-T and nonspecific binding was blocked with TBS Startblock blocking buffer. Serum or mAb solutions were serially diluted from 1:20 to 2-fold and added to the coated wells at room temperature over 1 hour. After washing, 30 ng / mL of histidine-tagged hACE2 was added to the wells at room temperature over 1 hour. HRP-conjugated anti-histidine IgG was added and incubated for 1 hour, followed by TMB substrate. The plates were read at OD 450 nm using a SpectraMax Plus plate reader, and the data were analyzed with SoftMax Pro software. The inhibition percentage for each dilution in each sample was calculated using the following formula in the SoftMax Pro program: 100 - [(MeanResults / ControlValue@PositiveControl)*100].

[0255] A plot of MAb concentration versus inhibition % was created, and curve fitting was performed using 4-parameter logistic curve fitting. The MAb concentration (IC50) at which hACE2 inhibits 50% binding to the SARS-CoV-2 rS protein was quantified.

[0256] Methods (Pseudoviral Neutralization Assay): Pseudoviruses encoding the SARS-CoV-2 S glycoprotein were generated using a lentiviral platform. All spike protein sequences included a deletion of the cytoplasmic tail. HEK293T cells were seeded one day before translocation and incubated overnight at 37°C. Translocation was performed when the cell monolayer reached 60–75% confluence. This translocation used a cationic lipid delivery system with a set of plasmids encoding a lentiviral backbone, a dual reporter plasmid expressing luciferase and Zs green together, a plasmid expressing the SARS-CoV-2 glycoprotein, and plasmids expressing other HIV proteins for pseudovirion formation. 48 hours after translocation, the supernatant was collected, centrifuged, and filtered through a 0.45 μm filter to obtain the pseudoviral stock. Commercially available pseudoviruses for Omicron XBB.1.5, XBB.1.16, XBB.1.16.6, XBB.2.3, and EG.5.1 were obtained from eEnzyme®, and only the luciferase reporter gene for detecting pseudovirus entry was incorporated. Aliquots of the pseudovirus stock were stored at -80°C.

[0257] For commercially available pseudoviruses from each new production lot or shipment, prior to serological testing, the pseudovirus was titrated under assay conditions to determine a working dilution targeting RLU; 100,000. Next, a pseudovirus neutralization assay was performed using a HEK293T cell line (HEK293T / ACE2) that stably expresses hACE2. Serum samples were inactivated by thermal inactivation in a 56°C water bath for 30 minutes, followed immediately by cooling to 4°C. Monoclonal antibodies were prepared at either a starting concentration of 1 or 5 μg / mL, and the serum samples were diluted 1:20 or 1:50 in low-serum medium. The prepared monoclonal antibodies or serum were added to 96-well cell culture plates and diluted 3-fold by double repeats. Next, 50 microliters of SARS-CoV-2 pseudovirus stock (corresponding to 100,000 RLU, ranging from 50,000 to 250,000) were added to each well, followed by incubation at 37°C for 1 hour. Next, 2.0 × 10⁴ HEK293T / hACE2 cells were added to wells in 100 μL of HEK293T cell culture medium (DMEM without phenol red + 5% FBS + 1% penicillin + streptomycin + glutamine) (containing 1.25 μg / ml puromycin), followed by incubation at 37°C for 72 hours. After incubation, 50 μL of luciferase substrate was added to each well. The plates were incubated at room temperature for 5 minutes without ambient light. Intracellular viral entry was quantified by measuring luminescence with a microplate reader. Pseudoviral neutralizing antibody titers in mAbs or serum were quantified by the absence or reduction of luminescence in the wells. The data were analyzed, and neutralization curves were created for each sample using GraphPad Prism. The 50% pseudoviral neutralizing titer (pVN50) was calculated using 4-parameter curve fitting. Each plate contained sample-free wells along with at least one positive and negative monoclonal antibody against each pseudovirus tested.

[0258] Methods (Evaluation of antibody binding to SARS-CoV-2 spike (S) glycoprotein by biolayer interferometry): Biolayer interferometry (BLI) kinetic assay was performed using an Octet® RH96 instrument (Sartorius® AG, Germany) to measure the binding of mAbs to rS. Antibodies NVX.205.10 and NVX.324.6 were bound to anti-mouse Fc biosensor chips at 5 μg / mL for 600 seconds, and the BLI test was performed. After immobilization, a washing step was performed, at which point baseline measurements were taken. Binding of SARS-CoV-2 rS at various concentrations (300 nM to 4.7 nM) was measured for 600 seconds, followed by a 600-second dissociation step. Binding kinetics were analyzed using Octet Software Analysis Studio 12.2. To measure the binding kinetics of MAbs to spike RBD-His, His-conjugated protein (2 μg / mL) was bound to a Ni-NTA biosensor for 600 seconds. After baseline measurements, the binding of MAb at various concentrations (20 μg / mL–0.31 μg / mL) was measured for 600 seconds, followed by the measurement of dissociation for 600 seconds.

[0259] Method (Western Blot): The binding of antibodies (MAb) to full-length XBB.1.5 rS, RBD, and NTD was evaluated by Western blotting. Protein samples were prepared in 1× NuPAGE LDS sample buffer and heated at 95°C for 10 minutes. The samples were loaded at a rate of 2 μg per lane onto NuPAGE 4-12% Bis-Tris gels and electrophoresis was performed at 200V for 35 minutes in 1× NuPAGE MOPS running buffer. For SDS-PAGE, the gels were stained according to the manufacturer's recommendations.

[0260] For Western blotting, proteins separated by SDS-PAGE were transferred to a nitrocellulose membrane at 20V for 7 minutes. The membrane was blocked in 5% skim milk powder prepared with 0.05% PBST at room temperature for 45–75 minutes. After blocking, the membrane was incubated with 1 μg / mL mAb at room temperature for 45–75 minutes. After washing, the membrane was incubated with AP-conjugated anti-mouse IgG antibody at room temperature for 45–75 minutes. After washing, the membrane was stained with BCIP / NBT substrate.

[0261] Method (Preparation and data acquisition of negative-stained transmission electron microscopy (NS-TEM) samples): To perform electron microscopy (EM), Fab was generated by digesting each hybridoma mAb. Briefly, the mAb was digested using either the Pierce Fab or F(ab')2 kit. After digestion, the cleaved Fab was buffered with 1×PBS pH 7.4 using a spin desalting column.

[0262] To image the complex of negatively stained SARS-CoV-2 rS with mouse Fab, Fab:Spike was mixed in a 10:1 molar ratio at ambient temperature for 30 minutes. Approximately 4 μL of the mixture was applied to a continuous carbon TEM grid immediately after glow discharge and incubated for 1 minute. The grid was then washed three times with deionized water, followed by floating 35 μL of water droplets on top. The grid was then floated in 35 μL of 1% uranyl formate and incubated for approximately 10 seconds twice, followed by a 30-second incubation. Excess staining was removed by gentle blotting, and the grid was air-dried overnight at room temperature.

[0263] All images were collected using an FEI 200kV Arctica transmission electron microscope operating at 200keV. Images were acquired at a magnification of approximately 73,000x, a pixel size of 1.997 Å / pixel, low-dose mode, and a defocus of 0.5 μm. The total dose of the microscope images was approximately 59 e / Ų. Image processing was performed using the cryoSPARC V.4 software package. X images were imported, CTF estimation was performed, and particles were picked using a Blob picker to obtain an initial particle stack limited to 300–600 Å in size. Particles were extracted in 512-pixel boxes and Fourier-tested to 256 pixels. 2D class averaging was performed, and the best class similar to the intact structure of the spike protein was selected from the initial 2D classification and used to create a template. Particles were re-picked using a template picker, and the resulting particle stack was subjected to several cycles of 2D classification and filtering to reconstruct the 3D model. Map segmentation and model docking to density were performed using UCSF ChimeraX. Enumerated embodiments of this disclosure The following are specific enumerated embodiments. <1> ~ <35> These are illustrative examples only and do not otherwise limit the scope of the subject matter of this disclosure as defined by the claims. These enumerated embodiments encompass all combinations, subcombinations, and multiple reference (e.g., multiple dependency) combinations described herein. 1. An antibody or fragment thereof that binds to the spike (S) glycoprotein of coronavirus 2 (CoV) for sudden acute respiratory syndrome, wherein the antibody or fragment thereof (i) A light chain variable complementarity determination region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 9, 12, 15, and 18, (ii) A light chain variable complementarity determination region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 10, 13, 16, and 19, (iii) A light chain variable complementarity determination region 3 (VL CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 11, 14, 17, and 20, (iv) A heavy chain variable complementarity determination region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 21, 24, 27, and 30, (v) A heavy chain variable complementarity determination region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 22, 25, 28, and 31, and (vi) The antibody or a fragment thereof, comprising a heavy chain variable complementarity determining region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 26, 29, and 32. 2. An antibody or fragment thereof that binds to the spike (S) protein of coronavirus 2 (SARS-CoV-2), wherein the antibody or fragment thereof (i) Heavy chain variable (VH) domains comprising amino acid sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs. 5 to 8, and (ii) The antibody or a fragment thereof comprising a light chain variable (VL) domain containing an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs: 1 to 4. 3. The antibody or its fragment is (i) Heavy chain variable (VH) domains comprising amino acid sequences having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs. 5 to 8, and (ii) The antibody or fragment thereof according to Embodiment 1, comprising a light chain variable (VL) domain containing an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs: 1 to 4. 4. The antibody or its fragment is (i) A light chain variable complementarity determination region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 9, 12, 15, and 18, (ii) A light chain variable complementarity determination region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 10, 13, 16, and 19, (iii) A light chain variable complementarity determination region 3 (VL CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 11, 14, 17, and 20, (iv) A heavy chain variable complementarity determination region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 21, 24, 27, and 30, (v) A heavy chain variable complementarity determination region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 22, 25, 28, and 31, and (vi) The antibody or fragment thereof according to Embodiment 2, comprising a heavy chain variable complementarity determining region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 26, 29, and 32. 5. The antibody or fragment thereof according to the enumerated embodiments 1 to 4, wherein the antibody or fragment thereof is selected from the group consisting of VH CDR1 described in any one of SEQ ID NOs: 21, 24, 27, and 30; VH CDR2 described in any one of SEQ ID NOs: 22, 25, 28, and 31; VH CDR3 described in any one of SEQ ID NOs: 23, 26, 29, and 32; VL CDR1 described in any one of SEQ ID NOs: 9, 12, 15, and 18; VL CDR2 described in any one of SEQ ID NOs: 10, 13, 16, and 19; and VL CDR3 described in any one of SEQ ID NOs: 11, 14, 17, and 20. 6. The antibody or fragment according to any one of the enumerated embodiments 1 to 4, wherein the antibody or fragment comprises VH CDR1 described in SEQ ID NO: 21, VH CDR2 described in SEQ ID NO: 22, and VH CDR3 described in SEQ ID NO: 23, VL CDR1 described in SEQ ID NO: 9, VL CDR2 described in SEQ ID NO: 10, and VL CDR3 described in SEQ ID NO: 11. 7. The antibody or its fragment is The antibody or fragment thereof according to any one of the above embodiments 1 to 4, including VH CDR1 described in SEQ ID NO: 24, VH CDR2 described in SEQ ID NO: 25, VH CDR3 described in SEQ ID NO: 26, VL CDR1 described in SEQ ID NO: 12, VL CDR2 described in SEQ ID NO: 13, and VL CDR3 described in SEQ ID NO: 14. 8. The antibody or its fragment is The antibody or fragment thereof according to any one of the above embodiments 1 to 4, including VH CDR1 described in SEQ ID NO: 27, VH CDR2 described in SEQ ID NO: 28, VH CDR3 described in SEQ ID NO: 29, VL CDR1 described in SEQ ID NO: 15, VL CDR2 described in SEQ ID NO: 16, and VL CDR3 described in SEQ ID NO: 17. 9. The antibody or its fragment is The antibody or fragment thereof according to any one of the above embodiments 1 to 4, including VH CDR1 described in SEQ ID NO: 30, VH CDR2 described in SEQ ID NO: 31, VH CDR3 described in SEQ ID NO: 32, VL CDR1 described in SEQ ID NO: 18, VL CDR2 described in SEQ ID NO: 19, and VL CDR3 described in SEQ ID NO: 20. 10. The antibody or its fragment is (i) VH containing any one amino acid sequence from SEQ ID NOs. 5 to 8, (ii) The antibody or fragment thereof according to Embodiments 1 to 4, selected from the group consisting of VLs containing any one of the amino acid sequences of SEQ ID NOs: 1 to 4. 11. The antibody or its fragment is (i) VH containing the amino acid sequence of SEQ ID NO: 5, and (ii) The antibody or fragment thereof according to any one of the above embodiments 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO: 1. 12. The antibody or its fragment is (i) VH containing the amino acid sequence of SEQ ID NO: 6, and (ii) The antibody or fragment thereof according to any one of the above embodiments 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO: 2. 13. The antibody or its fragment is (i) VH containing the amino acid sequence of SEQ ID NO: 7, and (ii) The antibody or fragment thereof according to any one of the above embodiments 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO: 3. 14. The antibody or its fragment is (i) VH containing the amino acid sequence of SEQ ID NO: 8, and (ii) The antibody or fragment thereof according to any one of the above embodiments 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO: 4. 15. The antibody or fragment thereof according to any one of the listed embodiments 1 to 13, wherein the antibody or fragment thereof is a monoclonal antibody, Fab, F(ab')2, Fab', scFv, or a single-domain antibody (sdAb). 16. The antibody or fragment thereof according to any one of the listed embodiments 1 to 15, wherein the antibody or fragment thereof comprises an IgG1 or IgG4 domain. 17. The antibody or its fragment has an equilibrium dissociation constant (K) of 50 nM or less, 10 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, or 0.001 nM or less relative to the CoV S glycoprotein or its variant. D An antibody or fragment thereof according to any one of the enumerated embodiments 1 to 16, having the following characteristics. 18. The antibody or its fragment contains 1.0 × 10⁶ units per liter of CoV S glycoprotein or its variant. -9 Less than 1 mole (M), 1.0 × 10 -10 Less than M, 1.0 × 10-11 Less than M, or 1.0 × 10 -12 An antibody or fragment thereof according to any one of the listed embodiments 1 to 16, which binds with an equilibrium dissociation constant (Kd) of less than M. 19. The antibody or fragment thereof according to any one of the enumerated embodiments 1 to 18, wherein the antibody or fragment thereof is bound to one or more CoV S polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptides described in SEQ ID NOs. 65 to 72. 20. The antibody or fragment thereof according to any one of the listed embodiments 1 to 19, wherein the antibody or fragment thereof binds to about 2 to about 20 CoV S glycoproteins. 21. The antibody or fragment thereof according to any one of the listed embodiments 1 to 20, wherein the antibody or fragment thereof is a broad-spectrum neutralizing antibody. 22. The antibody or fragment thereof according to the enumerated embodiments 1 to 21, wherein the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein. 23. The antibody or fragment thereof according to any one of the enumerated embodiments 1 to 22, wherein the antibody or fragment thereof is of human type. 24. An isolated nucleic acid molecule encoding an antibody or a fragment thereof as described in any one of the listed embodiments 1 to 23. 25. An expression vector comprising the nucleic acid described in the enumerated embodiment 24. 26. A host cell containing the expression vector described in the enumerated embodiment 25. 27. A pharmaceutical composition comprising an antibody or fragment thereof as described in any one of the listed embodiments 1 to 23, and a pharmaceutically acceptable carrier. 28. The pharmaceutical composition according to the enumerated embodiment 27, comprising an antibody or fragment thereof as described in any one of the enumerated embodiments 1 to 23, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10. 29. A method for treating a subject in need of treatment who is infected with the SARS-CoV-2 virus or a variant thereof, comprising administering to the subject an antibody or fragment thereof according to any one of the enumerated embodiments 1 to 23, or a pharmaceutical composition according to any one of the enumerated embodiments 27 to 28. 30. The method according to the enumerated embodiment 29, wherein the subject is 65 years of age or older. 31. The method according to any one of the enumerated embodiments 29 to 30, wherein the subject is in an immune-deficient state. 32. The method according to any one of the enumerated embodiments 29 to 31, wherein the subject is a pregnant woman. 33. A method for determining whether a sample contains SARS-CoV-2 spike (S) glycoprotein, (a) Exposing the sample to the antibody or fragment described in any one of the listed embodiments 1 to 23, (b) detecting the antibody or a fragment thereof in the biological sample, The method wherein, if the antibody or a fragment thereof is detected in the sample, the sample contains the SARS-CoV-2 S glycoprotein. 34. The method according to the enumerated embodiment 33, comprising washing the sample between steps (a) and (b). 35. Detection of the antibody or its fragment in the sample is (i) Exposing the sample to a secondary antibody, (ii) detecting the secondary antibody, The method according to any one of the enumerated embodiments 33 to 34, wherein the secondary antibody is bound to the antibody or fragment described in any one of the enumerated embodiments 1 to 23. 36. The method according to any one of the listed embodiments 33 to 35, wherein the sample is serum, plasma, blood, saliva, nasopharyngeal swab, or mucus. 37. The method according to any one of the listed embodiments 33 to 35, wherein the sample is a composition containing SARS-CoV-2 S glycoprotein.

Claims

1. An antibody or fragment thereof that binds to the spike (S) glycoprotein of coronavirus 2 (CoV) for sudden acute respiratory syndrome, wherein the antibody or fragment thereof (i) A light chain variable complementarity determination region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 9, 12, 15, and 18, (ii) A light chain variable complementarity determination region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 10, 13, 16, and 19, (iii) A light chain variable complementarity determination region 3 (VL CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 11, 14, 17, and 20, (iv) A heavy chain variable complementarity determination region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 21, 24, 27, and 30, (v) A heavy chain variable complementarity determination region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 22, 25, 28, and 31, and (vi) The antibody or a fragment thereof, comprising a heavy chain variable complementarity determining region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 26, 29, and 32.

2. An antibody or fragment thereof that binds to the spike (S) protein of the coronavirus 2 (SARS-CoV-2), wherein the antibody or fragment thereof (i) Heavy chain variable (VH) domains comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs. 5 to 8, and (ii) The antibody or fragment thereof, comprising a light chain variable (VL) domain containing an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs: 1 to 4.

3. The antibody or fragment thereof (i) Heavy chain variable (VH) domains comprising an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs. 5 to 8, and (ii) The antibody or fragment thereof according to claim 1, comprising a light chain variable (VL) domain containing an amino acid sequence having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with any one polypeptide of SEQ ID NOs: 1 to 4.

4. The antibody or fragment thereof (i) A light chain variable complementarity determination region 1 (VL CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 9, 12, 15, and 18, (ii) A light chain variable complementarity determination region 2 (VL CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 10, 13, 16, and 19, (iii) A light chain variable complementarity determination region 3 (VL CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 11, 14, 17, and 20, (iv) A heavy chain variable complementarity determination region 1 (VH CDR1) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of sequence numbers 21, 24, 27, and 30, (v) A heavy chain variable complementarity determination region 2 (VH CDR2) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 22, 25, 28, and 31, and (vi) The antibody or fragment thereof according to claim 2, comprising a heavy chain variable complementarity determining region 3 (VH CDR3) having at least 80%, at least 85%, at least 90%, at least 95%, or 100% identity with a sequence selected from the group consisting of SEQ ID NOs: 23, 26, 29, and 32.

5. The antibody or fragment thereof according to claims 1 to 4, wherein the antibody or fragment thereof is selected from the group consisting of VH CDR1 described in any one of SEQ ID NOs: 21, 24, 27, and 30; VH CDR2 described in any one of SEQ ID NOs: 22, 25, 28, and 31; VH CDR3 described in any one of SEQ ID NOs: 23, 26, 29, and 32; VL CDR1 described in any one of SEQ ID NOs: 9, 12, 15, and 18; VL CDR2 described in any one of SEQ ID NOs: 10, 13, 16, and 19; and VL CDR3 described in any one of SEQ ID NOs: 11, 14, 17, and 20.

6. The antibody or fragment according to any one of claims 1 to 4, wherein the antibody or fragment comprises VH CDR1 described in SEQ ID NO: 21, VH CDR2 described in SEQ ID NO: 22, and VH CDR3 described in SEQ ID NO: 23, VL CDR1 described in SEQ ID NO: 9, VL CDR2 described in SEQ ID NO: 10, and VL CDR3 described in SEQ ID NO:

11.

7. The antibody or fragment thereof An antibody or fragment thereof according to any one of claims 1 to 4, comprising VH CDR1 as described in SEQ ID NO: 24, VH CDR2 as described in SEQ ID NO: 25, VH CDR3 as described in SEQ ID NO: 26, VL CDR1 as described in SEQ ID NO: 12, VL CDR2 as described in SEQ ID NO: 13, and VL CDR3 as described in SEQ ID NO:

14.

8. The antibody or fragment thereof An antibody or fragment thereof according to any one of claims 1 to 4, comprising VH CDR1 as described in SEQ ID NO: 27, VH CDR2 as described in SEQ ID NO: 28, VH CDR3 as described in SEQ ID NO: 29, VL CDR1 as described in SEQ ID NO: 15, VL CDR2 as described in SEQ ID NO: 16, and VL CDR3 as described in SEQ ID NO:

17.

9. The antibody or fragment thereof An antibody or fragment thereof according to any one of claims 1 to 4, comprising VH CDR1 as described in SEQ ID NO: 30, VH CDR2 as described in SEQ ID NO: 31, VH CDR3 as described in SEQ ID NO: 32, VL CDR1 as described in SEQ ID NO: 18, VL CDR2 as described in SEQ ID NO: 19, and VL CDR3 as described in SEQ ID NO:

20.

10. The antibody or fragment thereof (i) VH containing any one amino acid sequence from SEQ ID NOs. 5 to 8, (ii) The antibody or fragment thereof according to claims 1 to 4, selected from the group consisting of VLs containing any one amino acid sequence of SEQ ID NOs: 1-4.

11. The antibody or fragment thereof (i) VH containing the amino acid sequence of SEQ ID NO: 5, (ii) The antibody or fragment thereof according to any one of claims 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO:

1.

12. The antibody or fragment thereof (i) VH containing the amino acid sequence of SEQ ID NO: 6, (ii) The antibody or fragment thereof according to any one of claims 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO:

2.

13. The antibody or fragment thereof (i) VH containing the amino acid sequence of SEQ ID NO: 7, (ii) The antibody or fragment thereof according to any one of claims 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO:

3.

14. The antibody or fragment thereof (i) VH containing the amino acid sequence of SEQ ID NO: 8, (ii) The antibody or fragment thereof according to any one of claims 1 to 4, comprising a VL containing the amino acid sequence of SEQ ID NO:

4.

15. The antibody or its fragment is a monoclonal antibody, Fab, F(ab') ２ The antibody or fragment thereof according to any one of claims 1 to 13, which is Fab', scFv, or a single-domain antibody (sdAb).

16. The antibody or fragment thereof according to any one of claims 1 to 15, wherein the antibody or fragment thereof comprises an IgG1 or IgG4 domain.

17. The antibody or its fragment has an equilibrium dissociation constant (K) of 50 nM or less, 10 nM or less, 1 nM or less, 0.5 nM or less, 0.1 nM or less, 0.05 nM or less, 0.01 nM or less, or 0.001 nM or less relative to the CoV S glycoprotein or its variant. Ｄ The antibody or fragment thereof according to any one of claims 1 to 16, having the following characteristics.

18. The antibody or its fragment contains 1.0 × 10⁶ of CoV S glycoprotein or its variant per liter. －９ Less than 1 mole (M), 1.0 × 10 －１０ Less than M, 1.0 x 10 －１１ Less than M, or 1.0 × 10 －１２ An antibody or fragment thereof according to any one of claims 1 to 16, which binds with an equilibrium dissociation constant (Kd) of less than M.

19. The antibody or fragment according to any one of claims 1 to 18, wherein the antibody or fragment is bound to one or more CoV S polypeptides having at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identity with the polypeptides described in SEQ ID NOs.

20. The antibody or fragment thereof according to any one of claims 1 to 19, wherein the antibody or fragment thereof is bound to about 2 to about 20 CoV S glycoproteins.

21. The antibody or fragment thereof according to any one of claims 1 to 20, wherein the antibody or fragment thereof is a broad-spectrum neutralizing antibody.

22. The antibody or fragment thereof according to claims 1 to 21, wherein the antibody or fragment thereof binds to an epitope on the CoV S glycoprotein.

23. The antibody or fragment thereof according to any one of claims 1 to 22, wherein the antibody or fragment thereof is of human type.

24. An isolated nucleic acid molecule encoding an antibody or a fragment thereof according to any one of claims 1 to 23.

25. An expression vector comprising the nucleic acid described in claim 24.

26. A host cell comprising the expression vector described in claim 25.

27. A pharmaceutical composition comprising an antibody or fragment thereof according to any one of claims 1 to 23 and a pharmaceutically acceptable carrier.

28. The pharmaceutical composition according to claim 27, comprising up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 antibodies or fragments thereof according to any one of claims 1 to 23.

29. A method for treating a subject in need of treatment who is infected with the SARS-CoV-2 virus or a variant thereof, comprising administering to the subject an antibody or fragment thereof according to any one of claims 1 to 23, or a pharmaceutical composition according to any one of claims 27 to 28.

30. The method according to claim 29, wherein the subject is 65 years of age or older.

31. The method according to any one of claims 29 to 30, wherein the subject is in an immune-deficient state.

32. The method according to any one of claims 29 to 31, wherein the subject is a pregnant woman.

33. A method for determining whether a sample contains SARS-CoV-2 spike (S) glycoprotein, (a) Exposing the sample to the antibody or fragment described in any one of claims 1 to 23, (b) detecting the antibody or a fragment thereof in the biological sample, The method wherein, if the antibody or a fragment thereof is detected in the sample, the sample contains the SARS-CoV-2 S glycoprotein.

34. The method according to claim 33, comprising washing the sample between steps (a) and (b).

35. To detect the antibody or its fragment in the sample, (i) Exposing the sample to a secondary antibody, (ii) detecting the secondary antibody, The method according to any one of claims 33 to 34, wherein the secondary antibody is bound to the antibody or fragment described in any one of claims 1 to 23.

36. The method according to any one of claims 33 to 35, wherein the sample is serum, plasma, blood, saliva, nasopharyngeal swab, or mucus.

37. The method according to any one of claims 33 to 35, wherein the sample is a composition containing SARS-CoV-2 S glycoprotein.

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