Neutralizing Anti-SARS-cov-2 antibodies

Abstract

This disclosure provides novel neutralizing and potent anti-SARS-CoV-2 antibodies, related nucleic acids, related cells, related kits, related compositions, and related methods or uses.

Description

[0001] NEUTRALIZING ANTI-SARS-COV-2 ANTIBODIES

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119(e) of the earlier filing date of U.S. Provisional Patent No. 63 / 509,948, filed June 23, 2023, which is hereby incorporated by reference in its entirety.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] This application contains a Sequence Listing which has been submitted electronically in xml format and is hereby incorporated by reference in its entirety. Said xml copy, created on June 20, 2024, is named SeqList-070413-20802. xml and is 11,002 bytes in size.

[0006] FIELD OF THE INVENTION

[0007] The present invention relates to antibodies directed to epitopes of SARS-CoV-2 Coronavirus 2 (“SARS-CoV-2”). The present invention further relates to the preparation and use of neutralizing antibodies directed to the SARS-CoV-2 spike (S) glycoproteins for the prevention and treatment of SARS-CoV-2 infection.

[0008] BACKGROUND OF THE INVENTION

[0009] Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV-2) produced a worldwide pandemic, infecting over 456 million people and responsible for over 6 million deaths. In the United States, the FDA authorized the use of three vaccines encoding prefusion-stabilized SARS- CoV-2 spike: BNT162b2 from Pfizer-BioNtech, mRNA-1273 from Moderna, and an adenovirusbased vaccine, Ad26.COV2.S from Janssen.

[0010] The COVID-19 pandemic disproportionately affected the elderly population, which was among the most prone to hospitalization and death (de Lusignan et al., 2020; Hewitt et al., 2020; Mueller et al., 2020; Williamson et al., 2020). Fortunately, mRNA vaccination resulted in a substantial decline in COVID-19-related hospitalizations and deaths in the elderly (Britton et al., 2022; Havers et al., 2022b; Moghadas et al., 2021). However, the most pronounced increase in hospitalization and mortality associated with emerging variants in vaccinated individuals was among the elderly (Havers et al., 2022a). Cohorts of younger individuals that receive a third vaccine dose are believed to be protected from hospitalization after infection with SARS-CoV-2 variants in part because the booster dose increases diversity and the number of neutralizing antibody-producing memory B cells that can rapidly be recalled upon challenge (Andrews et al., 2022; Barda et al., 2021; Lustig et al., 2022; Muecksch et al., 2022; Tang et al., 2022; Tartof et al., 2022; Thompson et al., 2022). However, little is known about the memory B cell response in the elderly.

[0011] SUMMARY OF THE INVENTION

[0012] In one aspect, this disclosure provides a novel isolated and recombinantly modified anti- SARS-CoV-2 neutralizing antibody or antigen-binding fragment thereof that binds specifically to a SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen comprises a spike (S) polypeptide, such as a S polypeptide of a human or an animal SARS-CoV-2.

[0013] In some embodiments, the SARS-CoV-2 antigen comprises the receptor binding domain (RBD) of the S polypeptide.

[0014] In some embodiments, the antibody or antigen-binding fragment thereof is capable of neutralizing a plurality of SARS-CoV-2 strains.

[0015] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2; and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of a light chain variable region having the amino acid sequence of SEQ ID NO: 4.

[0016] In some embodiments, the antibody or antigen-binding fragment thereof comprises HCDR1, HCDR2, and HCDR3 of a heavy chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 2 and LCDR1 , LCDR2, and LCDR3 of a light chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 4.

[0017] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 2; or comprising the amino acid sequence of SEQ ID NO: 2; and / or a light chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 4 or comprising the amino acid sequence of SEQ ID NO: 4. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) that comprise a HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 and 4.

[0018] In some embodiments, the antibody or antigen-binding fragment thereof is a multivalent antibody that comprises (a) a first target binding site that specifically binds to an epitope within the S polypeptide, and (b) a second target binding site that binds to a different epitope on the S polypeptide or a different molecule. In some embodiments, the multivalent antibody is a bivalent or bispecific antibody.

[0019] In some embodiments, the antibody or the antigen-binding fragment thereof further comprises a variant Fc constant region. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a humanized monoclonal antibody. In some embodiments, the antibody is a single-chain antibody, Fab or Fab2 fragment.

[0020] In some embodiments, the antibody or antigen-binding fragment thereof is detectably labeled or conjugated to a toxin, a therapeutic agent, a polymer, a receptor, an enzyme, or a receptor ligand. In some embodiments, the polymer is polyethylene glycol (PEG).

[0021] For example, an antibody of the invention may be coupled to a toxin. Such antibodies may be used to treat animals, including humans, that are infected with the virus that is etiologically linked to SARS-CoV-2. For example, an antibody that binds to the spike protein of the coronavirus that is etiologically linked to SARS-CoV-2 may be coupled to a tetanus toxin and administered to an animal suffering from infection by the aforementioned virus. The toxin-coupled antibody is thought to bind to a portion of a spike protein presented on an infected cell, and then kill the infected cell.

[0022] An antibody of the invention may be coupled to a detectable tag (or detectable label). Such antibodies may be used within diagnostic assays to determine if an animal, such as a human, is infected with SARS-CoV-2. Examples of detectable tags include fluorescent proteins (i.e., green fluorescent protein, red fluorescent protein, yellow fluorescent protein), fluorescent markers (i.e., fluorescein isothiocyanate, rhodamine, texas red), radiolabels (i.e., 3H, 32P, 1251), enzymes (i.e., P-galactosidase, horseradish peroxidase, P-glucuronidase, alkaline phosphatase), or an affinity tag (i.e., avidin, biotin, streptavidin).

[0023] In another aspect, this disclosure provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof as described herein and optionally a pharmaceutically acceptable carrier or excipient.

[0024] In some embodiments, the pharmaceutical comprises two or more of the antibody or antigen-binding fragment thereof described herein, such as any combinations of the antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain that comprise the respective amino acid sequences of one selected from those in Tables 2-4.

[0025] In some embodiments, the two or more of the antibody or antigen-binding fragment thereof comprise a first antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising the respective amino acid sequences of a first antibody selected from those in Tables 2-4; and a second antibody or antigen-binding fragment thereof comprising a heavy chain variable region and a light chain variable region comprising the respective amino acid sequences of a second antibody selected from those in Tables 2-4, wherein the first antibody or antigen-binding fragment thereof is different from the second antibody or antigen-binding fragment thereof.

[0026] In some embodiments, the two or more of the antibody or antigen-binding fragment thereof comprise: a first antibody or antigen-binding fragment thereof comprising a HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 and 4 and a second antibody or antigen-binding fragment thereof comprising a HCVR / LCVR amino acid sequence pair, wherein the first antibody or antigen-binding fragment thereof is different from the second antibody or antigen-binding fragment thereof.

[0027] In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiments, the second therapeutic agent comprises an antiinflammatory drug or an antiviral compound. In some embodiments, the antiviral compound comprises: a nucleoside analog, a peptide, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA dependent RNA polymerase. In some embodiments, the antiviral compound may include: acyclovir, gancyclovir, vidarabine, foscarnet, cidofovir, amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, or an interferon. In some embodiments, the interferon is an interferon-a or an interferon-p.

[0028] Also within the scope of this disclosure is use of the pharmaceutical composition, as described herein, in the preparation of a medicament for the diagnosis, prophylaxis, treatment, or combination thereof of a condition resulting from a SARS-CoV-2.

[0029] In another aspect, this disclosure also provides (i) a nucleic acid molecule encoding a polypeptide chain of the antibody or antigen-binding fragment thereof described herein; (ii) a vector comprising the nucleic acid molecule as described; and (iii) a cultured host cell comprising the vector as described.

[0030] Also provided is a method for producing a polypeptide, comprising: (a) obtaining the cultured host cell as described; (b) culturing the cultured host cell in a medium under conditions permitting expression of a polypeptide encoded by the vector and assembling of an antibody or fragment thereof; and (c) purifying the antibody or fragment from the cultured cell or the medium of the cell.

[0031] In another aspect, this disclosure provides a kit comprising a pharmaceutically acceptable dose unit of the antibody or antigen-binding fragment thereof or the pharmaceutical composition described herein. Also within the scope of this disclosure is a kit for the diagnosis, prognosis, or monitoring of the treatment of SARS-CoV-2 in a subject, comprising: one or more of the antibody or antigen-binding fragment thereof as described herein; and a least one detection reagent that binds specifically to the antibody or antigen-binding fragment thereof.

[0032] In yet another aspect, this disclosure further provides a method of neutralizing SARS-CoV- 2 in a subject. The method comprises administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof or a therapeutically effective amount of the pharmaceutical composition, as described herein.

[0033] In yet another aspect, this disclosure additionally provides a method of preventing or treating a SARS-CoV-2 infection. The method comprises administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof or a therapeutically effective amount of the pharmaceutical composition, as described herein. In some embodiments, the method of neutralizing SARS-CoV-2 in a subject comprises administering to a subject in need thereof a therapeutically effective amount of a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof of the antibody or antigen-binding fragment, as described herein, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen binding fragment thereof exhibit synergistic activity or a therapeutically effective amount of the pharmaceutical composition described herein.

[0034] In some embodiments, the method of preventing or treating a SARS-CoV-2 infection, comprising administering to a subject in need thereof a therapeutically effective amount of a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof of the antibody or antigen-binding fragment, as described herein, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen binding fragment thereof exhibit synergistic activity or a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiments, the first antibody or antigen-binding fragment thereof is administered before, after, or concurrently with the second antibody or antigen-binding fragment thereof.

[0035] In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof can be any combinations of the antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain that comprise the respective amino acid sequences of an antibody selected from those in Table 3.

[0036] In some embodiments, the second therapeutic agent comprises an anti-inflammatory drug or an antiviral compound. In some embodiments, the antiviral compound comprises a nucleoside analog, a peptide, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA dependent RNA polymerase. In some embodiments, the antiviral compound may include: acyclovir, gancyclovir, vidarabine, foscamet, cidofovir, amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, or an interferon. In some embodiments, the interferon is an interferon-a or an interferon-p.

[0037] In some embodiments, the antibody or antigen-binding fragment thereof is administered before, after, or concurrently with the second therapeutic agent or therapy. In some embodiments, the antibody or antigen-binding fragment thereof is administered to the subject intravenously, subcutaneously, or intraperitoneally. In some embodiments, the antibody or antigen-binding fragment thereof is administered prophylactically or therapeutically.

[0038] In another aspect, this disclosure further provides a method for detecting the presence of SARS CoV-2 in a sample comprising the steps of: (i) contacting a sample with the antibody or antigen-binding fragment thereof described herein; and (ii) determining binding of the antibody or antigen-binding fragment to one or more SARS CoV-2 antigens, wherein binding of the antibody to the one or more SARS CoV-2 antigens is indicative of the presence of SARS CoV-2 in the sample. In some embodiments, the sample is a blood sample.

[0039] In some embodiments, the SARS-CoV-2 antigen comprises a S polypeptide, such as a S polypeptide of a human or an animal SARS-CoV-2. In some embodiments, the receptor binding domain (RBD) of the S polypeptide.

[0040] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the step of detecting comprises contacting a secondary antibody with the antibody or antigen-binding fragment thereof and wherein the secondary antibody comprises a label. In some embodiments, the label includes a fluorescent label, a chemiluminescent label, a radiolabel, and an enzyme.

[0041] In some embodiments, the step of detecting comprises detecting fluorescence or chemiluminescence. In some embodiments, the step of detecting comprises a competitive binding assay or ELISA.

[0042] In some embodiments, the method further comprises binding the sample to a solid support. In some embodiments, the solid support includes microparticles, microbeads, magnetic beads, and an affinity purification column.

[0043] The foregoing summary is not intended to define every aspect of the disclosure, and additional aspects are described in other sections, such as the following detailed description. The entire document is intended to be related as a unified disclosure, and it should be understood that all combinations of features described herein are contemplated, even if the combination of features are not found together in the same sentence, or paragraph, or section of this document. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, because various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

[0044] BRIEF DESCRIPTION OF THE DRAWINGS

[0045] FIGs. 1A-I shows plasma ELISAs and neutralizing activity. A, Diagram shows blood donation schedules for the younger participants 6.5 months after the third dose (top, n=14) and for the elderly participants 8.5 month after the third dose (Vax3, bottom, n=7 and 3 months after the fourth dose (Vax4, bottom, n=24). B and C, Graph shows half-maximal binding titer (BT50) for plasma IgG antibody binding to wild-type SARS-CoV-2 (WT) RBD (B), and Omicron BA.4 / 5 RBD (C). D-I, Plasma neutralizing activity against indicated SARS-CoV-2 variants. D: Wuhan- hu-1 (WT), E : Omicron BA.1, F: Omicron BA.2, G: Omicron BA.4 / 5, H: Omicron BA.2.75.2 and I: Omicron XBB.1.5. The deletions / substitutions corresponding to viral variants used in D-I were incorporated into a spike protein that also includes the R683G substitution, which disrupts the furin cleavage site and increases particle infectivity. Neutralizing activity against mutant pseudoviruses was compared to a wildtype (WT) SARS-CoV-2 spike sequence (NC_045512), carrying R683G. All experiments were performed at least in duplicate. Bars and values in B-I represent geometric mean values. Statistical significance in B-I was determined by two-tailed Kruskal -Wallis test with subsequent Dunn’s multiple comparisons.

[0046] FIGs. 2A-2F shos anti-SARS-CoV-2 RBD memory B cells after breakthrough infection. A and B, the frequency (A) and the number (B) of WT RBD-specific B cells is indicated for young participants after Vax3, (n=24), and elderly participants 8.5 month after Vax3 (n=7) or 3 months after Vax4 (n=24). C, Pie charts show the distribution of IgG antibody sequences obtained from WT-specific memory B cells from: 5 younger individuals assayed after the third mRNA dose (Vax3); 2 elderly individuals after Vax3 and 3 elderly individuals after Vax4 (See also in FIG. 7C). The number inside the circle indicates the number of sequences analyzed for the individual denoted above the circle. Pie slice size is proportional to the number of clonally related sequences. The black outline and associated numbers indicate the percentage of clonal sequences detected at each time point. Slices indicate persisting clones (same IGHV and IGLV genes, with highly similar CDR3s) found at more than one timepoint within the same individual. Grey slices indicate clones unique to the timepoint. White slices indicate sequences isolated only once per time point. D, Circus plot depicts the relationship between antibodies that share V and J gene segment sequences at both IGH and IGL. The grey and black grey lines connect related clones, clones and singles, and singles to each other, respectively. E, F, The Shannon-Weiner index for diversity analysis (E) and clonality analysis (F) of the sequences from C. All experiments were performed at least in duplicate and repeated twice. Bars and numbers in A, B, E and F represent mean. Statistics in A, and B, were determined by two-tailed Kruskal-Wallis test with subsequent Dunn’s multiple-comparisons test and in E, and F, by two-tailed Mann-Whitney test.

[0047] FIGs. 3A-3E show anti-SARS-CoV-2 RBD monoclonal antibodies. A and B, Graphs show half-maximal effective concentration (EC50) of n=255 monoclonal antibodies measured by ELISA against WT-(A), Omicron BA.4 / 5-, and Omicron XBB- and Omicron XBB.1.5 RBD protein (B). Antibodies were obtained from memory B cells from young participants after Vax3 (Young Vax3), and elderly participants after Vax3 (Elderly Vax3) and Vax4 (Elderly Vax4). C, Results of epitope mapping performed by competition BLI, comparing mAbs cloned from vaccinated younger and elderly individuals. D, Graph showing affinity measurements (ADS) for WT RBD measured by BLI for antibodies cloned from young and elderly individuals. E, same as C, but for clones and singlets, separately. All experiments were performed at least in duplicate and repeated twice. Each dot represents one antibody. Bars and numbers in A and B represent geometries mean, in D and E represent mean. Statistics in A(dotplot), B and E, were determined by two-tailed Kruskal -Wallis test with subsequent Dunn’s multiple-comparisons test and in D by two-tailed Mann-Whitney test, and two-sided Fisher’ s exact test with subsequent Bonferroni-Dunn correction for A (ring plots).

[0048] FIGs. 4A-4D illustrate epitopes and neutralizing breadth. A and B, Graphs show anti- SARS-CoV-2 neutralizing activity of monoclonal antibodies, IC50S value against WT, Omicron BA.l and Omicron BA. 4 / 5 SARS-CoV-2 pseudoviruses, for all antibodies (n=253). Ring plots show fraction of neutralizing (ICsoMOOOng / ml, grey) and non-neutralizing (ICsoMOOO ng / ml, black) antibodies against WT, Omicron BA. l, and Omicron BA.4 / 5 SARS-CoV-2 pseudoviruses. Number in inner circles indicates number of antibodies tested. C, Results of epitope mapping performed by competition BLI, comparing mAbs cloned from vaccinated younger and elderly individuals. Pie charts show the distribution of the antibody classes among WT-neutralizing antibodies (upper panel), WT+Omicron BA.1 -neutralizing antibodies (middle panel), or WT+Omicron BA.4 / 5-neutralizing antibodies (lower panel). D, Graphs showing IC50 neutralization activity of Class 1 / 2 antibodies among all antibodies in A. The deletions / substitutions corresponding to viral variants used in A, B and D were incorporated into a spike protein that also includes the R683G substitution, which disrupts the furin cleavage site and increases particle infectivity. Neutralizing activity against mutant pseudoviruses was compared to a wildtype (WT) SARS-CoV-2 spike sequence (NC_045512), carrying R683G. All experiments were performed at least in duplicate and repeated twice. Each dot represents one antibody. Bars and values in A, B, and D, represent geometric mean values. Statistics were determined by two-tailed Kruskal Wallis test with subsequent Dunn’s multiple comparisons (A, dotplot), two-sided Fisher’s exact test with sub sequent Bonferroni -Dunn correction (A, ring plots), Mann-Whitney test (B, D) and two-tailed Chi-square test (C).

[0049] FIGs. 5A-5E show the correlation between plasma anti-RBD binding activity and vaccine dosing interval, or age. A, BT50S for anti-Nucleocapsid(N) IgG. B and C, BT50S for anti- RBD IgG plotted against intervals between vaccine doses and blood collection. D and E, Age (x axis) plotted against BT50S for anti-RBD IgG (y axis)(D) or NT50S for WT (y axis)(E). All experiments were performed at least in duplicate and repeated twice. Statistical significance was determined by two-tailed Spearman’s correlation.

[0050] FIGs. 6A-6J show flow cytometry gating strategy to phenotype or sort RBD-binding memory B cells after booster vaccination in the elderly and younger individuals. A, Gating strategy for phenotyping. Gating was on lymphocytes singlets that were CD 19+ or CD20+ and CD3-CD8-CD16-Ova-. Anti-IgG, IgM, IgA, IgD, CD21, CD11c, CD38 and CD27 antibodies were used for B cell phenotype analysis. Antigen-specific cells were detected based on binding to Wuhan-Hu- 1 RBD-PE+ and RBD-AF647+. Gating was on lymphocytes singlets that were CD20+and CD3-CD8-CD16-Ova-. Anti-IgG, IgM, IgA antibodies were used for B cell phenotype analysis. Antigen-specific cells were detected based on binding to WT RBD-PE+and RBD- AF647+. B, Graphs show the absolute number of CD 19+ B cells. C, Graphs show the frequency and absolute number of age-associated B cells (ABCs). D, Graphs show the frequency and absolute number of ABCs in WT RBD+ MBCs. E and F, % of ABCs (y axis) was plotted against intervals between vaccine doses and blood collection (x axis). G, Gating strategy for single-cell sorting for CD20+ B cells for WT RBD-PE and RBD-AF647. H, Representative flow cytometry plots indicating PE-WT-RBD and AlexaFluor-647-WT-RBD binding memory B cells from 10 younger individuals after Vax3, 5 elderly individuals after Vax3, and 5 elderly individuals after Vax4. I, Pie charts show the distribution of IgG antibody sequences obtained 2 elderly individuals after Vax4 (in additional to the 3 vaccinees shown in Fig. 2D). J, Number of nucleotide somatic hypermutations (SHM) in IGHV + IGLV in WT-RBD-specific sequences from elderly or younger vaccinees. All experiments were performed at least in duplicate and repeated twice. Statistical significance in B and J, was determined by two-tailed Kruskal -Wallis test with subsequent Dunn’ s multiple comparisons, in C, D, by two-tailed Mann-Whitney test, E and F were determined by two-tailed Spearman’s correlation.

[0051] FIGs.7A-7H show frequency of V gene usage of RBD- binding memory B cells after vaccination or peripheral B cells in the elderly and younger individuals. A-C, Comparison of the frequency distribution of human V genes for heavy chain and light chains of anti-RBD antibodies. Graph shows relative abundance of human IGHV (A), IGKV (B) and IGLV (C) genes in antibodies obtained from elderly, and younger vaccinees. Statistical significance was determined by two-sided binomial test. * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001. D-H, Distribution of BCR repertoire and frequency distribution of V genes in circulating B cells. D, Pie charts show the relative size of BCR clones as slices. The areas indicated in white represent unique BCR sequences. The number above the pie chart is the donor ID for each individual. The number in the center of the pie chart represents the number of cells assayed for each individual. E, The Shannon-Weiner index for diversity analysis (left panel) and clonality analysis (right panel) of the sequences from D. F-H, Graph shows relative abundance of human IGHV (F), IGKV (G) and IGLV (H) genes in antibodies genes obtained from elderly, and younger vaccinees. Statistical significance was determined by Mann-Whitney test for E and two-sided binomial test for A-C, F- H * = p<0.05, ** = p<0.01, *** = p<0.001, **** = p<0.0001.

[0052] FIGs. 8A-8I show additional characterization of antibodies’ binding activity, epitopes and somatic hypermutations. A-D, Graphs show anti-SARS-CoV-2 binding activity ELISA half- maximal concentration (ECso) values for all antibodies, clones and singlets of n=255 monoclonal antibodies against WT-RBD (A), Omicron BA.4 / 5-RBD (B), Omicron XBB-RBD (C) and Omicron XBB.1.5-RBD (D). E, Schematic representation of the BLI experiment for antibodies isolated from the elderly or younger vaccinees. Graphs represent the binding of the second antibody (2nd Ab) to preformed first antibody (1st Ab)-RBD complexes. Dotted line denotes when 1st Ab and 2nd Ab are the same. F, Results of epitope mapping performed by competition BLI, comparing mAbs cloned from each participant, related to Fig. 3C. G, Results of epitope mapping performed by competition BLI, comparing mAbs belonged to Class 1 / 2 or Class 2 / 3 / 5, cloned from vaccinated younger individuals compared to elderly individuals. Each dot represents one antibody. H, Number of nucleotide somatic hypermutations (SHM) in IGHV + IGLV in WT-RBD- specific sequences from clone and singlets. I, Number of nucleotide somatic hypermutations (SHM) in IGHV + IGLV in WT-RBD-specific sequences from classl / 2 and non-class 1 / 2 antibodies. Horizontal bars and numbers indicate geometric mean values. Statistical significance was determined by two-tailed Kruskal -Wallis test with subsequent Dunn’s multiple comparisons (A-D, H, I) or by two-tailed Chi-square test (G). All experiments were performed at least twice.

[0053] FIGs. 9A-9D show additional characterization of antibodies’ neutralizing breadth.. A, Graphs show anti-SARS-CoV-2 neutralizing activity (IC50S) of monoclonal antibodies (n=253) measured by a SARS-CoV-2 pseudotype virus neutralization assay using WT spike, for all tested antibodies, clones and singlets. B, Graphs show anti-SARS-CoV-2 neutralizing activity (ICsos) of monoclonal antibodies isolated from the elderly vaccinees after Vax3 or Vax4, against WT, Omicron BA.1 and Omicron BA.4 / 5 SARS-CoV-2 pseudoviruses, Pie charts illustrate the fraction of neutralizing (IC50 < 1,000 ng / ml) antibodies (grey slices) and non-neutralizing (IC50 > 1,000 ng / ml) antibodies (dark slices), inner circle shows the number of antibodies tested per group. C and D, Graphs showing IC50 neutralization activity of Class 4, 1 / 4(C) and Class 5, 3 / 5, 2 / 3 / 5 antibodies among all antibodies in FIG. 4A. The deletions / substitutions corresponding to viral variants were incorporated into a spike protein that also includes the R683G substitution, which disrupts the furin cleavage site and increases particle infectivity. Neutralizing activity against mutant pseudoviruses was compared to a wildtype (WT) SARS-CoV-2 spike sequence (NC 045512), carrying R683G. All experiments were performed at least in duplicate and repeated twice. Each dot represents one antibody. Bars and values represent geometric mean values. Horizontal bars and numbers indicate geometric mean values. Statistical significance was determined by Mann-Whitney test, and for the ring plots in B by two-sided Fisher’s exact test.

[0054] DETAILED DESCRIPTION OF THE INVENTION

[0055] SARS-CoV-2 represents a serious public health concern. Methods to diagnose and treat persons who are infected with SARS-CoV-2 provide the opportunity to either prevent or control further spread of infection by SARS-CoV-2. These methods are especially important due to the ability of SARS-CoV-2 to infect persons through an airborne route. This invention is based, at least in part, on unexpected neutralizing activities of the disclosed anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof. These antibodies and antigen-binding fragments constitute a novel therapeutic strategy in protection from SARS-CoV-2 infections.

[0056] NEUTRALIZING ANTI-SARS-COV-2 ANTIBODIES

[0057] Antibodies

[0058] In one aspect, this disclosure provides novel isolated and recombinantly modified anti- SARS-CoV-2 neutralizing antibodies or antigen-binding fragments thereof. These antibodies refer to a class of broadly neutralizing antibodies that neutralize multiple SARS-CoV-2 virus strains. The antibodies are able to protect a subject prophylactically and therapeutically against a lethal challenge with a SARS-CoV-2 virus.

[0059] In one aspect, this disclosure provides an isolated anti-SARS-CoV-2 antibody or antigenbinding fragment thereof that binds specifically to a SARS-CoV-2 antigen. In some embodiments, the SARS-CoV-2 antigen comprises a portion of a spike (S) polypeptide, such as a S polypeptide of a human or an animal SARS-CoV-2.

[0060] In some embodiments, the SARS-CoV-2 antigen comprises a Spike (S) polypeptide, such as a S polypeptide of a human or an animal SARS-CoV-2. In some embodiments, the SARS-CoV- 2 antigen comprises the receptor-binding domain (RBD) of the S polypeptide.

[0061] In some embodiments, the antibody or antigen-binding fragment thereof is capable of neutralizing a SARS-CoV-2 virus at an IC50 concentration of less than 50 (e. ., 1, 5, 10, 20, 30, 40, 50) pg / ml.

[0062] The spike protein is important because it is present on the outside of intact SARS-CoV-2. Thus, it presents a target that can be used to inhibit or eliminate an intact virus before the virus has an opportunity to infect a cell. A representative amino acid sequence is provided below:

[0063] (Accession ID: NC_045512.2) (SEQ ID NO: 9)

[0064] MFVFLVLLPLVSSQCVNLTTRTQLPPAYTNSFTRGVYYPDKVFRSSVLHSTQDLFLPFFS NVTWFHAIHVSGTNGTKRFDNPVLPFNDGVYFASTEKSNIIRGWIFGTTLDSKTQSLLIV NNATNVVIKVCEFQFCNDPFLGVYYHKNNKSWMESEFRVYSSANNCTFEYVSQPFLMD LEGKQGNFKNLREFVFKNIDGYFKIYSKHTPINLVRDLPQGF S ALEPL VDLPIGINITRFQT LLALHRS YLTPGD S S SGWT AGAAA YYVGYLQPRTFLLKYNENGTITD AVDC ALDPL SET KCTLKSFTVEKGIYQTSNFRVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISN CVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIA DYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGST PCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKN KCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVS VITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPTWRVYSTGSNVFQTRAGCLIGAEH VNNSYECDIPIGAGICASYQTQTNSPRRARSVASQSIIAYTMSLGAENSVAYSNNSIAIPT NFTISVTTEILPVSMTKTSVDCTMYICGDSTECSNLLLQYGSFCTQLNRALTGIAVEQDK NTQEVFAQVKQIYKTPPIKDFGGFNFSQILPDPSKPSKRSFIEDLLFNKVTLADAGFIKQY GDCLGDIAARDLICAQKFNGLTVLPPLLTDEMIAQYTSALLAGTITSGWTFGAGAALQIP FAMQMAYRFNGIGVTQNVLYENQKLIANQFNSAIGKIQDSLSSTASALGKLQDVVNQN AQALNTLVKQLSSNFGAISSVLNDILSRLDKVEAEVQIDRLITGRLQSLQTYVTQQLIRAA EIRASANLAATKMSECVLGQSKRVDFCGKGYHLMSFPQSAPHGVVFLHVTYVPAQEKN FTTAPAICHDGKAHFPREGVFVSNGTHWFVTQRNFYEPQIITTDNTFVSGNCDVVIGIVN NTVYDPLQPELDSFKEELDKYFKNHTSPDVDLGDISGINASVVNIQKEIDRLNEVAKNLN ESLIDLQELGKYEQYIKWPWYIWLGFIAGLIAIVMVTIMLCCMTSCCSCLKGCCSCGSCC KFDEDD SEP VLKGVKLHYT

[0065] The total length of SARS-CoV-2 S is 1273 amino acids and consists of a signal peptide (amino acids 1-13) located attheN-terminus, the SI subunit (14-685 residues), and the S2 subunit (686-1273 residues); the last two regions are responsible for receptor binding and membrane fusion, respectively. In the SI subunit, there is an N-terminal domain (14-305 residues) and a receptor-binding domain (RBD, 319-541 residues); the fusion peptide (FP) (788-806 residues), heptapeptide repeat sequence 1 (HR1) (912-984 residues), HR2 (1163-1213 residues), TM domain (1213-1237 residues), and cytoplasm domain (1237-1273 residues) comprise the S2 subunit. S protein trimers visually form a characteristic bulbous, crown-like halo surrounding the viral particle. Based on the structure of coronavirus S protein monomers, the SI and S2 subunits form the bulbous head and stalk region.

[0066] Listed below in Tables 2-4 are amino acid sequences of the heavy chain (HC) variable regions (HCVR) and light chain (LC) variable regions (LCVR) of exemplary antibodies.

[0067] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region having an amino acid sequence with at least 75% (e. , 75%, 78%, 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%) identity to one selected from those in Tables 2-4; and / or (ii) a light chain variable region having an amino acid sequence with at least 75% (e.g., 75%, 78%, 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%) identity to one selected from those in Tables 2-4.

[0068] In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a heavy chain variable region having an amino acid sequence selected from those in Tables 2-4; and / or (ii) a light chain variable region having an amino acid sequence selected from those in Tables 2-4.

[0069] In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region that comprises an amino acid sequence pair selected from those in Tables 2-4.

[0070] In some embodiments, the antibody or antigen-binding fragment thereof comprises three heavy chain complementarity determining regions (HCDRs) (HCDR1, HCDR2, and HCDR3) of a heavy chain variable region having the amino acid sequence of SEQ ID NO: 2 and three light chain CDRs (LCDR1, LCDR2, and LCDR3) of a light chain variable region having the amino acid sequence of SEQ ID NO: 4.

[0071] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a heavy chain variable region comprising an amino acid sequence with at least 75% (e.g., 75%, 78%, 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%) identity to the amino acid sequence of SEQ ID NO: 2; or comprising the amino acid sequence of SEQ ID NO: 2, and / or a light chain variable region comprising an amino acid sequence with at least 75% (e.g., 75%, 78%, 80%, 82%, 84%, 85%, 86%, 88%, 90%, 92%, 94%, 95%, 96%, 97%, 98%, 99%) identity to the amino acid sequence of SEQ ID NO: 4 or comprising the amino acid sequence of SEQ ID NO: 4.

[0072] A person of ordinary skill in the art will understand that various CDR numbering schemes (such as the Kabat, Chothia, Enhanced Chothia, IMGT, AHoAbM, Contact numbering schemes) can be used to determine CDR positions. In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) and a light chain variable region (LCVR) that comprise a HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 and 4.

[0073] In some embodiments, the antibody or antigen-binding fragment thereof comprises (a) a first target binding site that specifically binds to an epitope within the S polypeptide, and (b) a second target binding site that binds to a different epitope on the S polypeptide or on a different molecule. In some embodiments, the multivalent antibody is a bivalent or bispecific antibody.

[0074] In some embodiments, the antibody or the antigen-binding fragment thereof further comprises a variant Fc constant region. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric antibody, a humanized antibody, or a humanized monoclonal antibody. In some embodiments, the antibody is a single-chain antibody, Fab or Fab2 fragment.

[0075] In some embodiments, the antibody or the antigen-binding fragment thereof further comprises a variant Fc constant region. The antibody can be a monoclonal antibody. In some embodiments, the antibody can be a chimeric antibody, a humanized antibody, or a humanized monoclonal antibody. In some embodiments, the antibody can be a single-chain antibody, Fab or Fab2 fragment.

[0076] In some embodiments, the antibody or antigen-binding fragment thereof can be detectably labeled or conjugated to a toxin, a therapeutic agent, a polymer (e.g, polyethylene glycol (PEG)), a receptor, an enzyme, or a receptor ligand. For example, an antibody of the present invention may be coupled to a toxin (e.g, a tetanus toxin). Such antibodies may be used to treat animals, including humans, that are infected with the virus that is etiologically linked to SARS-CoV-2. The toxin-coupled antibody is thought to bind to a portion of a spike protein presented on an infected cell, and then kill the infected cell.

[0077] In another example, an antibody of the present invention may be coupled to a detectable tag. Such antibodies may be used within diagnostic assays to determine if an animal, such as a human, is infected with SARS-CoV-2. Examples of detectable tags include fluorescent proteins (z.e., green fluorescent protein, red fluorescent protein, yellow fluorescent protein), fluorescent markers (z.c., fluorescein isothiocyanate, rhodamine, texas red), radiolabels (z'.c., 3H, 32P, 1251), enzymes (z.e., P-galactosidase, horseradish peroxidase, P-glucuronidase, alkaline phosphatase), or an affinity tag (z.e., avidin, biotin, streptavidin). Methods to couple antibodies to a detectable tag are known in the art. Harlow et al., Antibodies: A Laboratory Manual, page 319 (Cold Spring Harbor Pub. 1988).

[0078] Fragment

[0079] In some embodiments, an antibody provided herein is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and single-chain Fv (scFv) fragments, and other fragments described below, e.g., diabodies, triabodies tetrabodies, and single-domain antibodies. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185; and U.S. Pat. Nos. 5,571,894 and 5,587,458. For discussion of Fab and F(ab')2 fragments comprising salvage receptor binding epitope residues and having increased in vivo half-life, see U.S. Pat. No. 5,869,046.

[0080] Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993 / 01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0081] Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In some embodiments, a single-domain antibody is a human single-domain antibody (DOMANTIS, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516).

[0082] Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0083] Chimeric and Humanized Antibodies

[0084] In some embodiments, an antibody provided herein is a chimeric antibody. Certain chimeric antibodies are described, e.g., in U.S. Pat. No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81 :6851-6855 (1984)). In one example, a chimeric antibody comprises a nonhuman variable region e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non- human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a “class switched” antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0085] In some embodiments, a chimeric antibody is a humanized antibody. Typically, a nonhuman antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, e.g., CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0086] Humanized antibodies and methods of making them are reviewed, e.g., in Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008), and are further described, e.g., in Riechmann et al., Nature 332:323-329 (1988); Queen et al., Proc. Nat'l Acad. Sci. USA 86: 10029-10033 (1989); U.S. Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri et al., Methods 36:25- 34 (2005) (describing specificity determining region (SDR) grafting); Padlan, Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acqua et al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbourn et al., Methods 36:61 -68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing the “guided selection” approach to FR shuffling).

[0087] Human framework regions that may be used for humanization include but are not limited to: framework regions selected using the “best-fit” method (see, e.g., Sims et al. J. Immunol. 151 :2296 (1993)); framework regions derived from the consensus sequence of human antibodies of a particular subgroup of light or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta et al. J. Immunol., 151 :2623 (1993)); human mature (somatically mutated) framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13: 1619-1633 (2008)); and framework regions derived from screening FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272: 10678-10684 (1997) and Rosok etal., J. Biol. Chem. 271 :22611-22618 (1996)).

[0088] Human Antibodies In some embodiments, an antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art or using techniques described herein. Human antibodies are described generally in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin. Immunol. 20:450-459 (2008).

[0089] Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. For review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23: 1117-1125 (2005). See also, e.g, U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE® technology; U.S. Pat. No. 5,770,429 describing HUMAB technology; U.S. Pat. No. 7,041,870 describing K-M MOUSE technology, and U.S. Patent Application Publication No. US 2007 / 0061900, describing VELOCIMOUSE® technology). Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region.

[0090] Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. (See, e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987); and Boemer et al., J. Immunol., 147: 86 (1991).) Human antibodies generated via human B-cell hybridoma technology are also described in Li etal., Proc. Natl. Acad. Sci. USA, 103:3557- 3562 (2006). Additional methods include those described, for example, in U.S. Pat. No. 7,189,826 (describing production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing human-human hybridomas). Human hybridoma technology (Trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3): 185-91 (2005). Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain. Techniques for selecting human antibodies from antibody libraries are described below.

[0091] Antibodies of the invention may be isolated by screening combinatorial libraries for antibodies with the desired activity or activities. For example, a variety of methods are known in the art for generating phage display libraries and screening such libraries for antibodies possessing the desired binding characteristics. Such methods are reviewed, e.g., in Hoogenboom et aL, in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001) and further described, e.g., in the McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et aL, J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods in Molecular Biology 248: 161-175 (Lo, ed., Human Press, Totowa, N.J., 2003); Sidhu et aL, J. Mol. Biol. 338(2): 299-310 (2004); Lee et aL, J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004).

[0092] In certain phage display methods, repertoires of VH and VL genes are separately cloned by polymerase chain reaction (PCR) and recombined randomly in phage libraries, which can then be screened for antigen-binding phage as described in Winter etaL, Ann. Rev. Immunol., 12: 433- 455 (1994). Phage typically displays antibody fragments, either as scFv fragments or as Fab fragments. Libraries from immunized sources provide high-affinity antibodies to the immunogen without the requirement of constructing hybridomas. Alternatively, the naive repertoire can be cloned (e.g., from human) to provide a single source of antibodies to a wide range of non-self and also self-antigens without any immunization as described by Griffiths et aL, EMBO J, 12: 725- 734 (1993). Finally, naive libraries can also be made synthetically by cloning unrearranged V- gene segments from stem cells and using PCR primers containing random sequences to encode the highly variable CDR3 regions and to accomplish rearrangement in vitro, as described by Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Pat. No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360. Antibodies or antibody fragments isolated from human antibody libraries are considered human antibodies or human antibody fragments herein.

[0093] Variants

[0094] In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into and / or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g, antigen binding.

[0095] Substitution, Insertion, and Deletion Variants

[0096] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Sites of interest for substitutional mutagenesis include the HVRs and FRs. Conservative substitutions are defined herein. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, e.g., retained / improved antigen binding, decreased immunogenicity, or improved antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0097] Accordingly, an antibody of the invention can comprise one or more conservative modifications of the CDRs, heavy chain variable region, or light variable regions described herein. A conservative modification or functional equivalent of a peptide, polypeptide, or protein disclosed in this invention refers to a polypeptide derivative of the peptide, polypeptide, or protein, e.g., a protein having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof. It substantially retains the activity to of the parent peptide, polypeptide, or protein (such as those disclosed in this invention). In general, a conservative modification or functional equivalent is at least 60% (e.g., any number between 60% and 100%, inclusive, e.g, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%) identical to a parent. Accordingly, within the scope of this invention are heavy chain variable region or light variable regions having one or more point mutations, insertions, deletions, truncations, a fusion protein, or a combination thereof, as well as antibodies having the variant regions. As used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (z.e., % homology=# of identical positions / total # of positions x 100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below.

[0098] The percent identity between two amino acid sequences can be determined using the algorithm of E. Meyers and W. Miller (Comput. Appl. Biosci., 4: 11-17 (1988)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch (J. Mol. Biol. 48:444-453 (1970)) algorithm, which has been incorporated into the GAP program in the GCG software package (available at www. cg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.

[0099] Additionally or alternatively, the protein sequences of the present invention can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the XBLAST program (version 2.0) of Altschul, et al. (1990) J. Mol. Biol. 215:403-10. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the antibody molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g, XBLAST and NBLAST) can be used. (See ncbi.nlm.nih.gov).

[0100] As used herein, the term “conservative modifications” refers to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include: (i) amino acids with basic side chains (e.g, lysine, arginine, histidine), (ii) acidic side chains (e.g, aspartic acid, glutamic acid), (iii) uncharged polar side chains (e.g, glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), (iv) nonpolar side chains (c’.g, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), (v) beta-branched side chains (e.g, threonine, valine, isoleucine), and (vi) aromatic side chains (e.g, tyrosine, phenylalanine, tryptophan, histidine).

[0101] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0102] An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, e.g, using phage display -based affinity maturation techniques such as those described in, e.g, Hoogenboom et al., in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001). Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C- terminus of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0103] Glycosylation Variants

[0104] In some embodiments, an antibody provided herein is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0105] For example, an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation). Glycosylation can be altered to, for example, increase the affinity of the antibody for antigen. Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence. For example, one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site. Such aglycosylation may increase the affinity of the antibody for antigen. Such an approach is described in further detail in U.S. Patent Nos.

[0106] 5,714,350 and 6,350,861 by Co et al.

[0107] Glycosylation of the constant region on N297 may be prevented by mutating the N297 residue to another residue, e.g., N297A, and / or by mutating an adjacent amino acid, e.g., 298 to thereby reduce glycosylation on N297.

[0108] Additionally or alternatively, an antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GlcNac structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies. Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies described herein to thereby produce an antibody with altered glycosylation. For example, EP 1,176,195 by Hanai et al. describes a cell line with a functionally disrupted FUT8 gene, which encodes a fucosyltransferase, such that antibodies expressed in such a cell line exhibit hypofucosylation. PCT Publication WO 03 / 035835 by Presta describes a variant Chinese Hamster Ovary cell line, Led 3 cells, with reduced ability to attach fucose to Asn(297)-linked carbohydrates, also resulting in hypofucosylation of antibodies expressed in that host cell (see also Shields, R.L. et al. (2002) J. Biol. Chem. 277:26733-26740). PCT Publication WO 99 / 54342 by Umana et al. describes cell lines engineered to express glycoprotein-modifying glycosyltransferases (e.g., beta(l,4)-N- acetylglucosaminyltransferase III (GnTIII)) such that antibodies expressed in the engineered cell lines exhibit increased bisecting GlcNac structures which result in increased ADCC activity of the antibodies (see also Umana et al. (1999) Nat. Biotech. 17: 176-180).

[0109] Fc Region Variants

[0110] The variable regions of the antibody described herein can be linked (e.g., covalently linked or fused) to an Fc, e.g., an IgGl, IgG2, IgG3 or IgG4 Fc, which may be of any allotype or isoallotype, e.g., for IgGl: Glm, Glml(a), Glm2(x), Glm3(f), Glml7(z); for IgG2: G2m, G2m23(n); for IgG3: G3m, G3m21(gl), G3m28(g5), G3ml l(b0), G3m5(bl), G3ml3(b3), G3ml4(b4), G3ml0(b5), G3ml5(s), G3ml6(t), G3m6(c3), G3m24(c5), G3m26(u), G3m27(v); and for K: Km, Kml, Km2, Km3 (see, e.g. , Jefferies et al. (2009) mAbs 1 : 1). In some embodiments, the antibodies variable regions described herein are linked to an Fc that binds to one or more activating Fc receptors (Fcyl, Fcylla, or Fcyllla), and thereby stimulate ADCC and may cause T cell depletion. In some embodiments, the antibody variable regions described herein are linked to an Fc that causes depletion.

[0111] In some embodiments, the antibody variable regions described herein may be linked to an Fc comprising one or more modifications, typically to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and / or antigendependent cellular cytotoxicity. Furthermore, an antibody described herein may be chemically modified (e.g. one or more chemical moi eties can be attached to the antibody) or be modified to alter its glycosylation, to alter one or more functional properties of the antibody. The numbering of residues in the Fc region is that of the EU index of Kabat.

[0112] The Fc region encompasses domains derived from the constant region of an immunoglobulin, preferably a human immunoglobulin, including a fragment, analog, variant, mutant, or derivative of the constant region. Suitable immunoglobulins include IgGl, IgG2, IgG3, IgG4, and other classes such as IgA, IgD, IgE, and IgM. The constant region of an immunoglobulin is defined as a naturally- occurring or synthetically-produced polypeptide homologous to the immunoglobulin C-terminal region, and can include a CHI domain, a hinge, a CH2 domain, a CH3 domain, or a CH4 domain, separately or in combination. In some embodiments, an antibody of this invention has an Fc region other than that of a wild type IgAl . The antibody can have an Fc region from that of IgG (e.g., IgGl, IgG2, IgG3, and IgG4) or other classes such as IgA2, IgD, IgE, and IgM. The Fc can be a mutant form of IgAl .

[0113] The constant region of an immunoglobulin is responsible for many important antibody functions, including Fc receptor (FcR) binding and complement fixation. There are five major classes of heavy chain constant region, classified as IgA, IgG, IgD, IgE, IgM, each with characteristic effector functions designated by isotype. For example, IgG is separated into four subclasses known as IgGl, IgG2, IgG3, and IgG4.

[0114] Ig molecules interact with multiple classes of cellular receptors. For example, IgG molecules interact with three classes of Fey receptors (FcyR) specific for the IgG class of antibody, namely FcyRI, FcyRII, and FcyRIIL. The important sequences for the binding of IgG to the FcyR receptors have been reported to be located in the CH2 and CH3 domains. The serum half-life of an antibody is influenced by the ability of that antibody to bind to an FcR.

[0115] In some embodiments, the Fc region is a variant Fc region, e.g., an Fc sequence that has been modified (e.g., by amino acid substitution, deletion and / or insertion) relative to a parent Fc sequence (e.g., an unmodified Fc polypeptide that is subsequently modified to generate a variant), to provide desirable structural features and / or biological activity. For example, one may make modifications in the Fc region in order to generate an Fc variant that (a) has increased or decreased ADCC, (b) increased or decreased CDC, (c) has increased or decreased affinity for Clq and / or (d) has increased or decreased affinity for an Fc receptor relative to the parent Fc. Such Fc region variants will generally comprise at least one amino acid modification in the Fc region. Combining amino acid modifications is thought to be particularly desirable. For example, the variant Fc region may include two, three, four, five, etc., substitutions therein, e.g., of the specific Fc region positions identified herein.

[0116] A variant Fc region may also comprise a sequence alteration wherein amino acids involved in disulfide bond formation are removed or replaced with other amino acids. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the antibodies described herein. Even when cysteine residues are removed, single chain Fc domains can still form a dimeric Fc domain that is held together non-covalently. In other embodiments, the Fc region may be modified to make it more compatible with a selected host cell. For example, one may remove the PA sequence near the N-terminus of a typical native Fc region, which may be recognized by a digestive enzyme in E. coli, such as proline iminopeptidase. In other embodiments, one or more glycosylation sites within the Fc domain may be removed. Residues that are typically glycosylated (e.g. , asparagine) may confer cytolytic response. Such residues may be deleted or substituted with unglycosylated residues (e.g., alanine). In other embodiments, sites involved in interaction with complement, such as the Clq binding site, may be removed from the Fc region. For example, one may delete or substitute the EKK sequence of human IgGl. In some embodiments, sites that affect binding to Fc receptors may be removed, preferably sites other than salvage receptor binding sites. In other embodiments, an Fc region may be modified to remove an ADCC site. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992) with regard to ADCC sites in IgGl. Specific examples of variant Fc domains are disclosed, for example, i In one embodiment, the hinge region of Fc is modified such that the number of cysteine residues in the hinge region is altered, e.g., increased or decreased. This approach is described further in U.S. Patent No. 5,677,425 by Bodmer et al. The number of cysteine residues in the hinge region of Fc is altered to, for example, facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody. In one embodiment, the Fc hinge region of an antibody is mutated to decrease the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the CH2-CH3 domain interface region of the Fc- hinge fragment such that the antibody has impaired Staphylococcal protein A (SpA) binding relative to native Fc-hinge domain SpA binding. This approach is described in further detail in U.S. Patent No. 6,165,745 by Ward et al.

[0117] In yet other embodiments, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function(s) of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320, and 322 can be replaced with a different amino acid residue such that the antibody has an altered affinity for an effector ligand but retains the antigen-binding ability of the parent antibody. The effector ligand to which affinity is altered can be, for example, an Fc receptor or the CI component of complement. This approach is described in further detail in U.S. Patent Nos. 5,624,821 and 5,648,260, both by Winter etal.

[0118] In another example, one or more amino acids selected from amino acid residues 329, 331 , and 322 can be replaced with a different amino acid residue such that the antibody has altered Clq binding and / or reduced or abolished CDC. This approach is described in further detail in U.S. Patent Nos. 6,194,551 by Idusogie et al.

[0119] In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered to thereby alter the ability of the antibody to fix complement. This approach is described further in PCT Publication WO 94 / 29351 by Bodmer et al.

[0120] In yet another example, the Fc region may be modified to increase ADCC and / or to increase the affinity for an Fey receptor by modifying one or more amino acids at the following positions:

[0121] 234, 235, 236, 238, 239, 240, 241 , 243, 244, 245, 247, 248, 249, 252, 254, 255, 256, 258, 262,

[0122] 263, 264, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,

[0123] 295, 296, 298, 299, 301, 303, 305, 307, 309, 312, 313, 315, 320, 322, 324, 325, 326, 327, 329, 1 330, 331, 332, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 433, 434, 435, 436, 437, 438 or 439. Exemplary substitutions include 236A, 239D, 239E, 268D, 267E, 268E, 268F, 324T, 332D, and 332E. Exemplary variants include 239D / 332E, 236A / 332E, 236A / 239D / 332E, 268F / 324T, 267E / 268F, 267E / 324T, and 267E / 268F7324T. Other modifications for enhancing FcyR and complement interactions include but are not limited to substitutions 298A, 333A, 334A, 326A, 2471, 339D, 339Q, 280H, 290S, 298D, 298V, 243L, 292P, 300L, 396L, 3051, and 396L. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0124] Fc modifications that increase binding to an Fey receptor include amino acid modifications at any one or more of amino acid positions 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 279, 280, 283, 285, 298, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 312, 315, 324, 327, 329, 330, 335, 337, 3338, 340, 360, 373, 376, 379, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index as in abat (WO00 / 42072).

[0125] Other Fc modifications that can be made to Fes are those for reducing or ablating binding to FcyR and / or complement proteins, thereby reducing or ablating Fc-mediated effector functions such as ADCC, antibody-dependent cellular phagocytosis (ADCP), and CDC. Exemplary modifications include but are not limited to substitutions, insertions, and deletions at positions 234, 235, 236, 237, 267, 269, 325, and 328, wherein numbering is according to the EU index. Exemplary substitutions include but are not limited to 234G, 235G, 236R, 237K, 267R, 269R, 325L, and 328R, wherein numbering is according to the EU index. An Fc variant may comprise 236R / 328R. Other modifications for reducing FcyR and complement interactions include substitutions 297A, 234A, 235A, 237A, 318A, 228P, 236E, 268Q, 309L, 330S, 33 IS, 220S, 226S, 229S, 238S, 233P, and 234V, as well as removal of the glycosylation at position 297 by mutational or enzymatic means or by production in organisms such as bacteria that do not glycosylate proteins. These and other modifications are reviewed in Strohl, 2009, Current Opinion in Biotechnology 20:685-691.

[0126] Optionally, the Fc region may comprise a non-naturally occurring amino acid residue at additional and / or alternative positions known to one skilled in the art (see, e.g., U.S. Pat. Nos. 5,624,821; 6,277,375; 6,737,056; 6,194,551; 7,317,091; 8,101,720; WO00 / 42072; WOOl / 58957; W002 / 06919; W004 / 016750; W004 / 029207; WO04 / 035752; WO04 / 074455; WO04 / 099249; W004 / 063351; W005 / 070963; W005 / 040217, WO05 / 092925 and W006 / 020114).

[0127] Fc variants that enhance affinity for an inhibitory receptor FcyRIIb may also be used. Such variants may provide an Fc fusion protein with immune-modulatory activities related to FcyRIIb cells, including, for example, B cells and monocytes. In one embodiment, the Fc variants provide selectively enhanced affinity to FcyRIIb relative to one or more activating receptors. Modifications for altering binding to FcyRIIb include one or more modifications at a position selected from the group consisting of 234, 235, 236, 237, 239, 266, 267, 268, 325, 326, 327, 328, and 332, according to the EU index. Exemplary substitutions for enhancing FcyRIIb affinity include but are not limited to 234D, 234E, 234F, 234W, 235D, 235F, 235R, 235Y, 236D, 236N, 237D, 237N, 239D, 239E, 266M, 267D, 267E, 268D, 268E, 327D, 327E, 328F, 328W, 328Y, and 332E. Exemplary substitutions include 235Y, 236D, 239D, 266M, 267E, 268D, 268E, 328F, 328W, and 328Y. Other Fc variants for enhancing binding to FcyRIIb include 235Y / 267E, 236D / 267E, 239D / 268D, 239D / 267E, 267E / 268D, 267E / 268E, and 267E / 328F.

[0128] The affinities and binding properties of an Fc region for its ligand may be determined by a variety of in vitro assay methods (biochemical or immunological based assays) known in the art, including but not limited to equilibrium methods (e.g., ELISA, or radioimmunoassay), or kinetics (e.g., BIACORE analysis), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence resonance energy transfer (FRET), gel electrophoresis and chromatography (c'.g, gel filtration). These and other methods may utilize a label on one or more of the components being examined and / or employ a variety of detection methods, including but not limited to chromogenic, fluorescent, luminescent, or isotopic labels. A detailed description of binding affinities and kinetics can be found in Paul, W. E., ed., Fundamental Immunology, 4th Ed., Lippincott-Raven, Philadelphia (1999), which focuses on antibody-immunogen interactions.

[0129] In some embodiments, the antibody is modified to increase its biological half-life. Various approaches are possible. For example, this may be done by increasing the binding affinity of the Fc region for FcRn. For example, one or more of the following residues can be mutated: 252, 254, 256, 433, 435, 436, as described in U.S. Pat. No. 6,277,375. Specific exemplary substitutions include one or more of the following: T252L, T254S, and / or T256F. Alternatively, to increase the biological half-life, the antibody can be altered within the CHI or CL region to contain a salvage receptor binding epitope taken from two loops of a CH2 domain of an Fc region of an IgG, as described in U.S. Patent Nos. 5,869,046 and 6, 121,022 by Presta et al. Other exemplary variants that increase binding to FcRn and / or improve pharmacokinetic properties include substitutions at positions 259, 308, 428, and 434, including for example 2591, 308F, 428L, 428M, 434S, 434H, 434F, 434Y, and 434M. Other variants that increase Fc binding to FcRn include: 250E, 250Q, 428L, 428F, 250Q / 428L (Hinton et al„ 2004, J. Biol. Chem. 279(8): 6213-6216, Hinton etal. 2006 Journal of Immunology 176:346-356), 256A, 272A, 286A, 305A, 307A, 307Q, 311A, 312A, 376A, 378Q, 380A, 382A, 434A (Shields et al, Journal of Biological Chemistry, 2001, 276(9):6591-6604), 252F, 252T, 252Y, 252W, 254T, 256S, 256R, 256Q, 256E, 256D, 256T, 309P, 31 IS, 433R, 433 S, 4331, 433P, 433Q, 434H, 434F, 434Y, 252Y / 254T / 256E, 433K / 434F / 436H, 308T / 309P / 311S (Dall Acqua e / c / / . Journal of Immunology, 2002, 169:5171-5180, Dall'Acqua et al., 2006, Journal of Biological Chemistry 281 :23514-23524). Other modifications for modulating FcRn binding are described in Yeung et al., 2010, J Immunol, 182:7663-7671. In some embodiments, hybrid IgG isotypes with particular biological characteristics may be used. For example, an IgGl / IgG3 hybrid variant may be constructed by substituting IgG 1 positions in the CH2 and / or CH3 region with the amino acids from IgG3 at positions where the two isotypes differ. Thus a hybrid variant IgG antibody may be constructed that comprises one or more substitutions, e.g, 274Q, 276K, 300F, 339T, 356E, 358M, 384S, 392N, 397M, 4221, 435R, and 436F. In other embodiments described herein, an IgGl / IgG2 hybrid variant may be constructed by substituting IgG2 positions in the CH2 and / or CH3 region with amino acids from IgGl at positions where the two isotypes differ. Thus, a hybrid variant IgG antibody may be constructed chat comprises one or more substitutions, e.g., one or more of the following amino acid substitutions: 233E, 234L, 235L, 236G (referring to an insertion of a glycine at position 236), and 321 h.

[0130] Moreover, the binding sites on human IgGl for FcyRl, FcyRII, FcyRIII, and FcRn have been mapped, and variants with improved binding have been described (see Shields, R.L. et al. (2001) J. Biol. Chem. 276:6591-6604). Specific mutations at positions 256, 290, 298, 333, 334, and 339 were shown to improve binding to FcyRIII. Additionally, the following combination mutants were shown to improve FcyRIII binding: T256A / S298A, S298A / E333A, S298A / K224A, and S298A / E333A / K334A, which has been shown to exhibit enhanced FcyRIIIa binding and ADCC activity (Shields et al., 2001). Other IgGl variants with strongly enhanced binding to FcyRIIIa have been identified, including variants with S239D / I332E and S239D / I332E / A330L mutations which showed the greatest increase in affinity for FcyRIIIa, a decrease in FcyRIIb binding, and strong cytotoxic activity in cynomolgus monkeys (Lazar et al., 2006). Introduction of the triple mutations into antibodies such as alemtuzumab (CD52- specific), trastuzumab (HER2 / neu- specific), rituximab (CD20- specific), and cetuximab (EGFR- specific) translated into greatly enhanced ADCC activity in vitro, and the S239D / I332E variant showed an enhanced capacity to deplete B cells in monkeys (Lazar et al., 2006). In addition, IgGl mutants containing L235V, F243L, R292P, Y300L and P396L mutations which exhibited enhanced binding to FcyRIIIa and concomitantly enhanced ADCC activity in transgenic mice expressing human FcyRIIIa in models of B cell malignancies and breast cancer have been identified (Stavenhagen et al., 2007; Nordstrom et al., 2011). Other Fc mutants that may be used include S298A / E333A / L334A, S239D / I332E, S239D / I332E / A330L, L235V / F243L / R292P / Y300L / P396L, and M428L / N434S.

[0131] In some embodiments, an Fc is chosen that has reduced binding to FcyRs. An exemplary Fc, e.g., IgGl Fc, with reduced FcyR binding, comprises the following three amino acid substitutions: L234A, L235E, and G237A.

[0132] In some embodiments, an Fc is chosen that has reduced complement fixation. An exemplary Fc, e.g., IgGl Fc, with reduced complement fixation, has the following two amino acid substitutions: A330S and P331S.

[0133] In some embodiments, an Fc is chosen that has essentially no effector function, i.e., it has reduced binding to FcyRs and reduced complement fixation. An exemplary Fc, e.g., IgGl Fc, that is effectorless, comprises the following five mutations: L234A, L235E, G237A, A330S, and P331S.

[0134] When using an IgG4 constant domain, it is usually preferable to include the substitution S228P, which mimics the hinge sequence in IgGl and thereby stabilizes IgG4 molecules.

[0135] Multivalent Antibodies

[0136] In one embodiment, the antibodies of the invention may be monovalent or multivalent (e.g., bivalent, trivalent, etc.). As used herein, the term “valency” refers to the number of potential target binding sites associated with an antibody. Each target binding site specifically binds one target molecule or specific position or locus on a target molecule. When an antibody is monovalent, each binding site of the molecule will specifically bind to a single antigen position or epitope. When an antibody comprises more than one target binding site (multivalent), each target binding site may specifically bind the same or different molecules (e.g., may bind to different ligands or different antigens, or different epitopes or positions on the same antigen). See, for example, U.S.P.N. 2009 / 0130105. In each case, at least one of the binding sites will comprise an epitope, motif or domain associated with a DLL3 isoform.

[0137] In one embodiment, the antibodies are bispecific antibodies in which the two chains have different specificities, as described in Millstein et al., 1983, Nature, 305:537-539. Other embodiments include antibodies with additional specificities, such as trispecific antibodies. Other more sophisticated compatible multispecific constructs and methods of their fabrication are set forth in U.S.P.N. 2009 / 0155255, as well as WO 94 / 04690; Suresh et al., 1986, Methods in Enzymology, 121 :210; and WO96 / 27011.

[0138] As stated above, multivalent antibodies may immunospecifically bind to different epitopes of the desired target molecule or may immunospecifically bind to both the target molecule as well as a heterologous epitope, such as a heterologous polypeptide or solid support material. In some embodiments, the multivalent antibodies may include bispecific antibodies or trispecific antibodies. Bispecific antibodies also include cross-linked or “heteroconjugate” antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.

[0139] In some embodiments, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences, such as an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2, and / or CH3 regions, using methods well known to those of ordinary skill in the art.

[0140] Antibody Derivatives An antibody provided herein may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water-soluble polymers.

[0141] Non-limiting examples of water-soluble polymers include, but are not limited to, PEG, copolymers of ethylene glycol / propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly- 1,3 -di oxolane, poly-1, 3, 6-trioxane, ethylene / maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, polypropylene oxide / ethylene oxide co-polymers, polyoxyethylated polyols (c.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

[0142] In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube (Kam etal., Proc. Natl. Acad. Sci. USA 102: 11600- 11605 (2005)). The radiation may be of any wavelength and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody-nonproteinaceous moiety are killed.

[0143] Another modification of the antibodies described herein is pegylation. An antibody can be pegylated to, for example, increase the biological (e.g, serum) half-life of the antibody. To pegylate an antibody, the antibody, or fragment thereof, typically is reacted with PEG, such as a reactive ester or aldehyde derivative of PEG, under conditions in which one or more PEG groups become attached to the antibody or antibody fragment. Preferably, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive PEG molecule (or an analogous reactive water-soluble polymer). As used herein, the term “polyethylene glycol” is intended to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (CI -CIO) alkoxy- or aryloxy-polyethylene glycol or polyethylene glycol-maleimide. In some embodiments, the antibody to be pegylated is an aglycosylated antibody. Methods for pegylating proteins are known in the art and can be applied to the antibodies described herein. See, for example, EP0154 316 by Nishimura et al. and EP0401384 by Ishikawa et al.

[0144] The present invention also encompasses a human monoclonal antibody described herein conjugated to a therapeutic agent, a polymer, a detectable label, or enzyme. In one embodiment, the therapeutic agent is a cytotoxic agent. In one embodiment, the polymer is PEG.

[0145] Nucleic Acids, Expression Cassettes, and Vectors

[0146] The present invention provides isolated nucleic acid segments that encode the polypeptides, peptide fragments, and coupled proteins of the invention. The nucleic acid segments of the invention also include segments that encode for the same amino acids due to the degeneracy of the genetic code. For example, the amino acid threonine is encoded by ACU, ACC, ACA, and ACG and is therefore degenerate. It is intended that the invention includes all variations of the polynucleotide segments that encode for the same amino acids. Such mutations are known in the art (Watson et al., Molecular Biology of the Gene, Benjamin Cummings 1987). Mutations also include alteration of a nucleic acid segment to encode for conservative amino acid changes, for example, the substitution of leucine for isoleucine and so forth. Such mutations are also known in the art. Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.

[0147] The nucleic acid segments of the invention may be contained within a vector. A vector may include, but is not limited to, any plasmid, phagemid, F-factor, virus, cosmid, or phage in a double- or single-stranded linear or circular form which may or may not be self transmissible or mobilizable. The vector can also transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extra-chromosomally (e.g., autonomous replicating plasmid with an origin of replication).

[0148] The nucleic acid segment in the vector can be under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in vitro or in a host cell, such as a eukaryotic cell, or a microbe, e.g., bacteria. The vector may be a shuttle vector that functions in multiple hosts. The vector may also be a cloning vector that typically contains one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion. Such insertion can occur without loss of essential biological function of the cloning vector. A cloning vector may also contain a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector. Examples of marker genes are tetracycline resistance or ampicillin resistance. Many cloning vectors are commercially available (Stratagene, New England Biolabs, Clonetech).

[0149] The nucleic acid segments of the invention may also be inserted into an expression vector. Typically, an expression vector contains prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance gene to provide for the amplification and selection of the expression vector in a bacterial host; regulatory elements that control initiation of transcription such as a promoter; and DNA elements that control the processing of transcripts such as introns, or a transcription termination / polyadenylation sequence.

[0150] Methods to introduce nucleic acid segment into a vector are available in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). Briefly, a vector into which a nucleic acid segment is to be inserted is treated with one or more restriction enzymes (restriction endonuclease) to produce a linearized vector having a blunt end, a “sticky” end with a 5' or a 3' overhang, or any combination of the above. The vector may also be treated with a restriction enzyme and subsequently treated with another modifying enzyme, such as a polymerase, an exonuclease, a phosphatase or a kinase, to create a linearized vector that has characteristics useful for ligation of a nucleic acid segment into the vector. The nucleic acid segment that is to be inserted into the vector is treated with one or more restriction enzymes to create a linearized segment having a blunt end, a “sticky” end with a 5' or a 3' overhang, or any combination of the above. The nucleic acid segment may also be treated with a restriction enzyme and subsequently treated with another DNA modifying enzyme. Such DNA modifying enzymes include, but are not limited to, polymerase, exonuclease, phosphatase or a kinase, to create a nucleic acid segment that has characteristics useful for ligation of a nucleic acid segment into the vector.

[0151] The treated vector and nucleic acid segment are then ligated together to form a construct containing a nucleic acid segment according to methods available in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). Briefly, the treated nucleic acid fragment, and the treated vector are combined in the presence of a suitable buffer and ligase. The mixture is then incubated under appropriate conditions to allow the ligase to ligate the nucleic acid fragment into the vector.

[0152] The invention also provides an expression cassette that contains a nucleic acid sequence capable of directing expression of a particular nucleic acid segment of the invention, either in vitro or in a host cell. Also, a nucleic acid segment of the invention may be inserted into the expression cassette such that an anti-sense message is produced. The expression cassette is an isolatable unit such that the expression cassette may be in linear form and functional for in vitro transcription and translation assays. The materials and procedures to conduct these assays are commercially available from Promega Corp. (Madison, Wis ). For example, an in vitro transcript may be produced by placing a nucleic acid sequence under the control of a T7 promoter and then using T7 RNA polymerase to produce an in vitro transcript. This transcript may then be translated in vitro through use of a rabbit reticulocyte lysate. Alternatively, the expression cassette can be incorporated into a vector allowing for replication and amplification of the expression cassette within a host cell or also in vitro transcription and translation of a nucleic acid segment.

[0153] Such an expression cassette may contain one or a plurality of restriction sites allowing for placement of the nucleic acid segment under the regulation of a regulatory sequence. The expression cassette can also contain a termination signal operably linked to the nucleic acid segment as well as regulatory sequences required for proper translation of the nucleic acid segment. The expression cassette containing the nucleic acid segment may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression cassette may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Expression of the nucleic acid segment in the expression cassette may be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus.

[0154] The expression cassette may include in the 5 '-3' direction of transcription, a transcriptional and translational initiation region, a nucleic acid segment and a transcriptional and translational termination region functional in vivo and / or in vitro. The termination region may be native with the transcriptional initiation region, may be native with the nucleic acid segment, or may be derived from another source.

[0155] The regulatory sequence can be a polynucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include, but are not limited to, enhancers, promoters, repressor binding sites, translation leader sequences, introns, and polyadenylation signal sequences. They may include natural and synthetic sequences as well as sequences, which may be a combination of synthetic and natural sequences. While regulatory sequences are not limited to promoters, some useful regulatory sequences include constitutive promoters, inducible promoters, regulated promoters, tissue-specific promoters, viral promoters, and synthetic promoters.

[0156] A promoter is a nucleotide sequence that controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. A promoter includes a minimal promoter, consisting only of all basal elements needed for transcription initiation, such as a TATA-box and / or initiator that is a short DNA sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. A promoter may be derived entirely from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0157] The invention also provides a construct containing a vector and an expression cassette. The vector may be selected from, but not limited to, any vector previously described. Into this vector may be inserted an expression cassette through methods known in the art and previously described (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001)). In one embodiment, the regulatory sequences of the expression cassette may be derived from a source other than the vector into which the expression cassette is inserted. In another embodiment, a construct containing a vector and an expression cassette is formed upon insertion of a nucleic acid segment of the invention into a vector that itself contains regulatory sequences. Thus, an expression cassette is formed upon insertion of the nucleic acid segment into the vector. Vectors containing regulatory sequences are available commercially, and methods for their use are known in the art (Clonetech, Promega, Stratagene).

[0158] In another aspect, this disclosure also provides (i) a nucleic acid molecule encoding a polypeptide chain of the antibody or antigen-binding fragment thereof described herein; (ii) a vector comprising the nucleic acid molecule as described; and (iii) a cultured host cell comprising the vector as described. Also provided is a method for producing a polypeptide, comprising: (a) obtaining the cultured host cell as described; (b) culturing the cultured host cell in a medium under conditions permitting expression of a polypeptide encoded by the vector and assembling of an antibody or fragment thereof; and (c) purifying the antibody or fragment from the cultured cell or the medium of the cell.

[0159] Methods of Production

[0160] Antibodies may be produced using recombinant methods and compositions, e.g., as described in U.S. Pat. No. 4,816,567. In one embodiment, an isolated nucleic acid encoding an antibody described herein is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and / or an amino acid sequence comprising the VH of the antibody (e.g., the light and / or heavy chains of the antibody). In a further embodiment, one or more vectors (e.g., expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g., has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, e.g., a Chinese Hamster Ovary (CHO) cell or lymphoid cell (e.g., YO, NSO, Sp20 cell). In one embodiment, a method of making an antibody is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). For recombinant production of an antibody, a nucleic acid encoding an antibody, e.g., as described herein, is isolated and inserted into one or more vectors for further cloning and / or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody).

[0161] Suitable host cells for cloning or expression of antibody-encoding vectors include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression of antibody fragments in E. coli.) After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

[0162] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been “humanized,” resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gemgross, Nat. Biotech. 22: 1409-1414 (2004), and Li etal., Nat. Biotech. 24:210-215 (2006).

[0163] Suitable host cells for the expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified, which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

[0164] Plant cell cultures can also be utilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES technology for producing antibodies in transgenic plants).

[0165] Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells as described, e.g., in Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK); mouse sertoli cells (TM4 cells as described, e.g., in Mather, Biol. Reprod. 23:243- 251 (1980)); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells, as described, e.g., in Mather et al., Annals N.Y. Acad. Sci. 383:44-68 (1982); MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include CHO cells, including DHFR- CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255-268 (2003).

[0166] COMPOSITIONS AND KITS

[0167] The antibodies of this invention represent an excellent way for the development of antiviral therapies either alone or in antibody cocktails with additional anti-SARS-CoV-2 virus antibodies for the treatment of human SARS-CoV-2 infections in humans.

[0168] In another aspect, the present invention provides a pharmaceutical composition comprising the antibodies of the present invention described herein formulated together with a pharmaceutically acceptable carrier. The composition may optionally contain one or more additional pharmaceutically active ingredients, such as another antibody or a therapeutic agent.

[0169] In some embodiments, the pharmaceutical comprises two or more of the antibody or antigen-binding fragment thereof described herein, such as any combinations of the antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain that comprise the respective amino acid sequences described herein.

[0170] The pharmaceutical compositions of the invention also can be administered in a combination therapy with, for example, another immune-stimulatory agent, an antiviral agent, a vaccine, etc. In some embodiments, a composition comprises an antibody of this invention at a concentration of at least 1 mg / ml, 5 mg / ml, 10 mg / ml, 50 mg / ml, 100 mg / ml, 150 mg / ml, 200 mg / ml, 1-300 mg / ml, or 100-300 mg / ml.

[0171] In some embodiments, the second therapeutic agent comprises an anti-inflammatory drug or an antiviral compound. In some embodiments, the antiviral compound comprises: a nucleoside analog, a peptide, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA dependent RNA polymerase. In some embodiments, the antiviral compound may include: acyclovir, gancyclovir, vidarabine, foscarnet, cidofovir, amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, or an interferon. In some embodiments, the interferon is an interferon-a or an interferon-p.

[0172] Also within the scope of this disclosure is use of the pharmaceutical composition in the preparation of a medicament for the diagnosis, prophylaxis, treatment, or combination thereof of a condition resulting from a SARS-CoV-2.

[0173] The pharmaceutical composition can comprise any number of excipients. Excipients that can be used include carriers, surface-active agents, thickening or emulsifying agents, solid binders, dispersion or suspension aids, solubilizers, colorants, flavoring agents, coatings, disintegrating agents, lubricants, sweeteners, preservatives, isotonic agents, and combinations thereof. The selection and use of suitable excipients is taught in Gennaro, ed., Remington: The Science and Practice of Pharmacy, 20th Ed. (Lippincott Williams & Wilkins 2003), the disclosure of which is incorporated herein by reference.

[0174] Preferably, a pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (c'.g, by injection or infusion). Depending on the route of administration, the active compound can be coated in a material to protect it from the action of acids and other natural conditions that may inactivate it. The phrase “parenteral administration” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, an antibody of the present invention described herein can be administered via a non-parenteral route, such as a topical, epidermal, or mucosal route of administration, e.g., intranasally, orally, vaginally, rectally, sublingually, or topically.

[0175] The pharmaceutical compositions of the invention may be prepared in many forms that include tablets, hard or soft gelatin capsules, aqueous solutions, suspensions, liposomes, and other slow-release formulations, such as shaped polymeric gels. An oral dosage form may be formulated such that the antibody is released into the intestine after passing through the stomach. Such formulations are described in U.S. Pat. No. 6,306,434 and in the references contained therein.

[0176] Oral liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups, or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

[0177] An antibody can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampules, prefilled syringes, small volume infusion containers, or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Pharmaceutical compositions suitable for rectal administration can be prepared as unit dose suppositories. Suitable carriers include saline solution and other materials commonly used in the art.

[0178] For administration by inhalation, an antibody can be conveniently delivered from an insufflator, nebulizer, a pressurized pack, or other convenient means of delivering an aerosol spray. Pressurized packs may comprise a suitable propellant such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount.

[0179] Alternatively, for administration by inhalation or insufflation, an antibody may take the form of a dry powder composition, for example, a powder mix of a modulator and a suitable powder base such as lactose or starch. The powder composition may be presented in a unit dosage form in, for example, capsules or cartridges or, e.g. , gelatin or blister packs from which the powder may be administered with the aid of an inhalator or insufflator. For intra-nasal administration, an antibody may be administered via a liquid spray, such as via a plastic bottle atomizer.

[0180] Pharmaceutical compositions of the invention may also contain other ingredients such as flavorings, colorings, anti -microbial agents, or preservatives. It will be appreciated that the amount of an antibody required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated, and the age and condition of the patient. Ultimately the attendant health care provider may determine a proper dosage. In addition, a pharmaceutical composition may be formulated as a single unit dosage form.

[0181] The pharmaceutical composition of the present invention can be in the form of sterile aqueous solutions or dispersions. It can also be formulated in a microemulsion, liposome, or other ordered structure suitable to high drug concentration.

[0182] An antibody of the present invention described herein can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the antibody in the patient. In general, human antibodies show the longest half-life, followed by humanized antibodies, chimeric antibodies, and nonhuman antibodies. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patient can be administered a prophylactic regime.

[0183] The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will vary depending upon the subject being treated and the particular mode of administration and will generally be that amount of the composition, which produces a therapeutic effect. Generally, out of one hundred percent, this amount will range from about 0.01% to about 99% of active ingredient, preferably from about 0.1% to about 70%, most preferably from about 1% to about 30% of active ingredient in combination with a pharmaceutically acceptable carrier.

[0184] Dosage regimens can be adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time, or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Alternatively, the antibody can be administered as a sustained release formulation, in which case less frequent administration is required. For administration of the antibody, the dosage ranges from about 0.0001 to 800 mg / kg, and more usually 0.01 to 5 mg / kg, of the host body weight. For example, dosages can be 0.3 mg / kg body weight, 1 mg / kg body weight, 3 mg / kg body weight, 5 mg / kg body weight or 10 mg / kg body weight or within the range of 1-10 mg / kg. An exemplary treatment regime entails administration once per week, once every two weeks, once every three weeks, once every four weeks, once a month, once every 3 months or once every three to 6 months. Preferred dosage regimens for an antibody of the invention include 1 mg / kg body weight or 3 mg / kg body weight via intravenous administration, with the antibody being given using one of the following dosing schedules: (i) every four weeks for six dosages, then every three months; (ii) every three weeks; (iii) 3 mg / kg body weight once followed by 1 mg / kg body weight every three weeks. In some methods, dosage is adjusted to achieve a plasma antibody concentration of about 1-1000 pg / ml, and in some methods, about 25-300 pg / ml. A “therapeutically effective dosage” of an antibody of the invention preferably results in a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction. For example, for the treatment of SARS- CoV-2 infection in a subject, a “therapeutically effective dosage” preferably inhibits SARS-CoV- 2 virus replication or uptake by host cells by at least about 20%, more preferably by at least about 40%, even more preferably by at least about 60%, and still more preferably by at least about 80% relative to untreated subjects. A therapeutically effective amount of a therapeutic compound can neutralize SARS-CoV-2 virus, or otherwise ameliorate symptoms in a subject, which is typically a human or can be another mammal.

[0185] The pharmaceutical composition can be a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene-vinyl acetate, poly anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. See, e.g., Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0186] Therapeutic compositions can be administered via medical devices such as (1) needleless hypodermic injection devices (e.g, US 5,399,163; 5,383,851; 5,312,335; 5,064,413; 4,941,880; 4,790,824; and 4,596,556); (2) micro-infusion pumps (US 4,487,603); (3) transdermal devices (US 4,486,194); (4) infusion apparati (US 4,447,233 and 4,447,224); and (5) osmotic devices (US 4,439,196 and 4,475,196); the disclosures of which are incorporated herein by reference.

[0187] In some embodiments, the human monoclonal antibodies of the invention described herein can be formulated to ensure proper distribution in vivo. For example, to ensure that the therapeutic compounds of the invention cross the blood-brain barrier, they can be formulated in liposomes, which may additionally comprise targeting moieties to enhance selective transport to specific cells or organs. See, e.g, US 4,522,811; 5,374,548; 5,416,016; and 5,399,331; V.V. Ranade (1989) Clin. Pharmacol. 29:685; Umezawa et al., (1988) Biochem. Biophys. Res. Commun. 153:1038; Bloeman etal. (1995) FEBS Lett. 357: 140; M. Owais etal. (1995) Antimicrob. Agents Chemother. 39: 180; Briscoe etal. (1995) Am. Physiol. 1233: 134; Schreier etal. (1994). Biol. Chem. 269:9090; Keinanen and Laukkanen (1994) FEBS Lett. 346:123; and Killion and Fidler (1994) Immunomethods 4:273.

[0188] In some embodiments, the initial dose may be followed by administration of a second or a plurality of subsequent doses of the antibody or antigen-binding fragment thereof in an amount that can be approximately the same or less than that of the initial dose, wherein the subsequent doses are separated by at least 1 day to 3 days; at least one week, at least 2 weeks; at least 3 weeks; at least 4 weeks; at least 5 weeks; at least 6 weeks; at least 7 weeks; at least 8 weeks; at least 9 weeks; at least 10 weeks; at least 12 weeks; or at least 14 weeks.

[0189] Various delivery systems are known and can be used to administer the pharmaceutical composition of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the mutant viruses, receptor-mediated endocytosis (see, e.g., Wu etal. (1987) J. Biol. Chem. 262:4429-4432). Methods of introduction include, but are not limited to, intradermal, transdermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The composition may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. The pharmaceutical composition can also be delivered in a vesicle, in particular, a liposome (see, for example, Langer (1990) Science 249: 1527-1533). The use of nanoparticles to deliver the antibodies of the present invention is also contemplated herein. Antibody-conjugated nanoparticles may be used both for therapeutic and diagnostic applications. Antibody-conjugated nanoparticles and methods of preparation and use are described in detail by Arruebo, M., et al. 2009 (“Antibody-conjugated nanoparticles for biomedical applications” in J. Nanomat. Volume 2009, Article ID 439389), incorporated herein by reference. Nanoparticles may be developed and conjugated to antibodies contained in pharmaceutical compositions to target cells. Nanoparticles for drug delivery have also been described in, for example, US 8257740, or US 8246995, each incorporated herein in its entirety.

[0190] In certain situations, the pharmaceutical composition can be delivered in a controlled release system. In one embodiment, a pump may be used. In another embodiment, polymeric materials can be used. In yet another embodiment, a controlled release system can be placed in proximity of the composition's target, thus requiring only a fraction of the systemic dose.

[0191] The injectable preparations may include dosage forms for intravenous, subcutaneous, intracutaneous, intracranial, intraperitoneal, intramuscular injections, drip infusions, etc. These injectable preparations may be prepared by methods publicly known. For example, the injectable preparations may be prepared, e.g., by dissolving, suspending, or emulsifying the antibody or its salt described herein in a sterile aqueous medium or an oily medium conventionally used for injections. As the aqueous medium for injections, there are, for example, physiological saline, an isotonic solution containing glucose and other auxiliary agents, etc., which may be used in combination with an appropriate solubilizing agent such as an alcohol (e.g., ethanol), a polyalcohol (e.g., propylene glycol, polyethylene glycol), a nonionic surfactant [e.g., polysorbate 80, HCO-50 (polyoxyethylene (50 mol) adduct of hydrogenated castor oil)], etc. As the oily medium, there are employed, e.g., sesame oil, soybean oil, etc., which may be used in combination with a solubilizing agent such as benzyl benzoate, benzyl alcohol, etc. The injection thus prepared is preferably filled in an appropriate ampoule.

[0192] A pharmaceutical composition of the present invention can be delivered subcutaneously or intravenously with a standard needle and syringe. In addition, with respect to subcutaneous delivery, a pen delivery device readily has applications in delivering a pharmaceutical composition of the present invention. Such a pen delivery device can be reusable or disposable. A reusable pen delivery device generally utilizes a replaceable cartridge that contains a pharmaceutical composition. Once all of the pharmaceutical composition within the cartridge has been administered and the cartridge is empty, the empty cartridge can readily be discarded and replaced with a new cartridge that contains the pharmaceutical composition. The pen delivery device can then be reused. In a disposable pen delivery device, there is no replaceable cartridge. Rather, the disposable pen delivery device comes prefilled with the pharmaceutical composition held in a reservoir within the device. Once the reservoir is emptied of the pharmaceutical composition, the entire device is discarded.

[0193] Numerous reusable pens and autoinjector delivery devices have applications in the subcutaneous delivery of a pharmaceutical composition of the present invention. Examples include, but certainly are not limited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Burghdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (Sanofi -Aventis, Frankfurt, Germany), to name only a few. Examples of disposable pen delivery devices having applications in subcutaneous delivery of a pharmaceutical composition of the present invention include, but certainly are not limited to, the SOLOSTAR™ pen (Sanofi- Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (Eli Lilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, CA), the PENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.) and the HUMIRA™ Pen (Abbott Labs, Abbott Park, IL), to name only a few.

[0194] Advantageously, the pharmaceutical compositions for oral or parenteral use described herein are prepared into dosage forms in a unit dose suited to fit a dose of the active ingredients. Such dosage forms in a unit dose include, for example, tablets, pills, capsules, injections (ampoules), suppositories, etc. The amount of the antibody contained is generally about 5 to about 500 mg per dosage form in a unit dose; especially in the form of injection, it is preferred that the antibody is contained in about 5 to about 300 mg and in about 10 to about 300 mg for the other dosage forms.

[0195] In another aspect, this disclosure provides a kit comprising a pharmaceutically acceptable dose unit of the antibody or antigen-binding fragment thereof or the pharmaceutical composition as described herein. Also within the scope of this disclosure is a kit for the diagnosis, prognosis, or monitoring of treatment of SARS-CoV-2 in a subject, comprising: the antibody or antigenbinding fragment thereof as described; and a least one detection reagent that binds specifically to the antibody or antigen-binding fragment thereof.

[0196] In some embodiments, the kit also includes a container that contains the composition and optionally informational material. The informational material can be descriptive, instructional, marketing, or other material that relates to the methods described herein and / or the use of the agents for therapeutic benefit. In an embodiment, the kit also includes an additional therapeutic agent, as described herein. For example, the kit includes a first container that contains the composition and a second container for the additional therapeutic agent.

[0197] The informational material of the kits is not limited in its form. In some embodiments, the informational material can include information about production of the composition, concentration, date of expiration, batch or production site information, and so forth. In one embodiment, the informational material relates to methods of administering the composition, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject in need thereof. In one embodiment, the instructions provide a dosing regimen, dosing schedule, and / or route of administration of the composition or the additional therapeutic agent. The information can be provided in a variety of formats, including printed text, computer-readable material, video recording, audio recording, or information that contains a link or address to substantive material.

[0198] The kit can include one or more containers for the composition. In some embodiments, the kit contains separate containers, dividers, or compartments for the composition and informational material. For example, the composition can be contained in a bottle or vial, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle or vial that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents.

[0199] The kit optionally includes a device suitable for administration of the composition or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading. Such a kit may optionally contain a syringe to allow for injection of the antibody contained within the kit into an animal, such as a human.

[0200] METHODS OF USE

[0201] Methods of Treatment

[0202] The antibodies, compositions, and formulations described herein can be used to neutralize SARS-CoV-2 virus, thereby treating or preventing SARS-CoV-2 infections.

[0203] Accordingly, in one aspect, this disclosure further provides a method of neutralizing SARS-CoV-2 in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof or a therapeutically effective amount of the pharmaceutical composition, as described herein.

[0204] In another aspect, this disclosure additionally provides a method of preventing or treating a SARS-CoV-2 infection, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof or a therapeutically effective amount of the pharmaceutical composition, as described herein.

[0205] The neutralizing of the SARS-CoV-2 virus can be done via (i) inhibiting SARS-CoV-2 virus binding to a target cell; (ii) inhibiting SARS-CoV-2 virus uptake by a target cell; (iii) inhibiting SARS-CoV-2 virus replication; and (iv) inhibiting SARS-CoV-2 virus particles release from infected cells. One skilled in the art possesses the ability to perform any assay to assess neutralization of SARS-CoV-2 virus.

[0206] Notably, the neutralizing properties of antibodies may be assessed by a variety of tests, which all may assess the consequences of (i) inhibition of SARS-CoV-2 virus binding to a target cell; (ii) inhibition of SARS-CoV-2 virus uptake by a target cell; (iii) inhibition of SARS-CoV-2 virus replication; and (iv) inhibition of SARS-CoV-2 virus particles release from infected cells. In other words, implementing different tests may lead to the observation of the same consequence, i.e., the loss of infectivity of the SARS-CoV-2 virus. Thus, in one embodiment, the present invention provides a method of neutralizing a SARS-CoV-2 virus in a subject comprising administering to the subject a therapeutically effective amount of the antibody of the present invention described herein. Another aspect of the present invention provides a method of treating a SARS-CoV-2- related disease. Such a method includes therapeutic (following SARS-CoV-2 infection) and prophylactic (prior to SARS-CoV-2 exposure, infection, or pathology). For example, therapeutic and prophylactic methods of treating an individual for a SARS-CoV-2 infection include treatment of an individual having or at risk of having a SARS-CoV-2 infection or pathology, treating an individual with a SARS-CoV-2 infection, and methods of protecting an individual from a SARS- CoV-2 infection, to decrease or reduce the probability of a SARS-CoV-2 infection in an individual, to decrease or reduce susceptibility of an individual to a SARS-CoV-2 infection, or to inhibit or prevent a SARS-CoV-2 infection in an individual, and to decrease, reduce, inhibit or suppress transmission of a SARS-CoV-2 from an infected individual to an uninfected individual. Such methods include administering an antibody of the present invention or a composition comprising the antibody disclosed herein to therapeutically or prophylactically treat (vaccinate or immunize) an individual having or at risk of having a SARS-CoV-2 infection or pathology. Accordingly, methods can treat the SARS-CoV-2 infection or pathology, or provide the individual with protection from infection (e.g., prophylactic protection).

[0207] In one embodiment, a method of treating a SARS-CoV-2-related disease comprises administering to an individual in need thereof an antibody or therapeutic composition disclosed herein in an amount sufficient to reduce one or more physiological conditions or symptoms associated with a SARS-CoV-2 infection or pathology, thereby treating the SARS-CoV-2 -related disease.

[0208] In one embodiment, an antibody or therapeutic composition disclosed herein is used to treat a SARS-CoV-2-related disease. Use of an antibody or therapeutic composition disclosed herein treats a SARS-CoV-2-related disease by reducing one or more physiological conditions or symptoms associated with a SARS-CoV-2 infection or pathology. In aspects of this embodiment, administration of an antibody or therapeutic composition disclosed herein is in an amount sufficient to reduce one or more physiological conditions or symptoms associated with a SARS- CoV-2 infection or pathology, thereby treating the SARS-CoV-2 -based disease. In other aspects of this embodiment, administration of an antibody or therapeutic composition disclosed herein is in an amount sufficient to increase, induce, enhance, augment, promote or stimulate SARS-CoV- 2 clearance or removal; or decrease, reduce, inhibit, suppress, prevent, control, or limit transmission of SARS-CoV-2 to another individual. One or more physiological conditions or symptoms associated with a SARS-CoV-2 infection or pathology will respond to a method of treatment disclosed herein. The symptoms of SARS-CoV-2 infection or pathology vary, depending on the phase of infection.

[0209] In some embodiments, the method of neutralizing SARS-CoV-2 in a subject comprises administering to a subject in need thereof a therapeutically effective amount of a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof of the antibody or antigen-binding fragment, as described herein, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen binding fragment thereof exhibit synergistic activity or a therapeutically effective amount of the pharmaceutical composition described herein.

[0210] In some embodiments, the method of preventing or treating a SARS-CoV-2 infection, comprising administering to a subject in need thereof a therapeutically effective amount of a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof of the antibody or antigen-binding fragment, as described herein, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen binding fragment thereof exhibit synergistic activity or a therapeutically effective amount of the pharmaceutical composition described herein. In some embodiments, the first antibody or antigen-binding fragment thereof is administered before, after, or concurrently with the second antibody or antigen-binding fragment thereof.

[0211] In some embodiments, the first antibody or antigen-binding fragment thereof and the second antibody or antigen-binding fragment thereof can be any combinations of the antibody or antigen-binding fragment thereof comprising a heavy chain and a light chain that comprise the respective amino acid sequences described herein.

[0212] In some embodiments, the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising a HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 and 4.

[0213] In some embodiments, the second antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair, wherein the first antibody or antigen-binding fragment thereof is different from the second antibody or antigen-binding fragment thereof. In some embodiments, the second therapeutic agent comprises an anti-inflammatory drug or an antiviral compound. In some embodiments, the antiviral compound comprises: a nucleoside analog, a peptide, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA dependent RNA polymerase. In some embodiments, the antiviral compound may include: acyclovir, gancyclovir, vidarabine, foscarnet, cidofovir, amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, or an interferon. In some embodiments, the interferon is an interferon-a or an interferon- .

[0214] In some embodiments, the antibody or antigen-binding fragment thereof is administered before, after, or concurrently with the second therapeutic agent or therapy. In some embodiments, the antibody or antigen-binding fragment thereof is administered to the subject intravenously, subcutaneously, or intraperitoneally. In some embodiments, the antibody or antigen-binding fragment thereof is administered prophylactically or therapeutically.

[0215] The antibodies described herein can be used together with one or more of other anti- S ARS- CoV-2 virus antibodies to neutralize SARS-CoV-2 virus, thereby treating SARS-CoV-2 infections.

[0216] Combination Therapies

[0217] Combination therapies may include an anti- SARS-CoV-2 antibody of the invention and any additional therapeutic agent that may be advantageously combined with an antibody of the invention or with a biologically active fragment of an antibody of the invention. The antibodies of the present invention may be combined synergistically with one or more drugs or therapy used to treat a disease or disorder associated with a viral infection, such as a SARS-CoV-2 infection. In some embodiments, the antibodies of the invention may be combined with a second therapeutic agent to ameliorate one or more symptoms of said disease. In some embodiments, the antibodies of the invention may be combined with a second antibody to provide synergistic activity in ameliorating one or more symptoms of said disease. In some embodiments, the first antibody or antigen-binding fragment thereof is administered before, after, or concurrently with the second antibody or antigen-binding fragment thereof.

[0218] For example, the antibody described herein can be used in various detection methods for use in, e.g., monitoring the progression of a SARS-CoV-2 infection; monitoring patient response to treatment for such an infection, etc. The present disclosure provides methods of detecting a neuraminidase polypeptide in a biological sample obtained from an individual. The methods generally involve: a) contacting the biological sample with a subject anti- neuraminidase antibody; and b) detecting binding, if any, of the antibody to an epitope present in the sample. In some instances, the antibody comprises a detectable label. The level of neuraminidase polypeptide detected in the biological sample can provide an indication of the stage, degree, or severity of a SARS-CoV-2 infection. The level of the neuraminidase polypeptide detected in the biological sample can provide an indication of the individual's response to treatment for a SARS-CoV-2 infection.

[0219] In some embodiments, the second therapeutic agent is another antibody to a SARS-COV- 2 protein or a fragment thereof. It is contemplated herein to use a combination (“cocktail”) of antibodies with broad neutralization or inhibitory activity against SARS-COV-2. In some embodiments, non-competing antibodies may be combined and administered to a subject in need thereof. In some embodiments, the antibodies comprising the combination bind to distinct nonoverlapping epitopes on the protein. In some embodiments, the second antibody may possess a longer half-life in human serum.

[0220] As used herein, the term “in combination with” means that additional therapeutically active component(s) may be administered prior to, concurrent with, or after the administration of the anti- SARS-COV-2 antibody of the present invention. The term “in combination with” also includes sequential or concomitant administration of an anti-SARS-COV-2 antibody and a second therapeutic agent.

[0221] The additional therapeutically active component(s) may be administered to a subject prior to administration of an anti-SARS-COV-2 antibody of the present invention. For example, a first component may be deemed to be administered “prior to” a second component if the first component is administered 1 week before, 72 hours before, 60 hours before, 48 hours before, 36 hours before, 24 hours before, 12 hours before, 6 hours before, 5 hours before, 4 hours before, 3 hours before, 2 hours before, 1 hour before, 30 minutes before, 15 minutes before, 10 minutes before, 5 minutes before, or less than 1 minute before administration of the second component. In other embodiments, the additional therapeutically active component(s) may be administered to a subject after administration of an anti-SARS-COV-2 antibody of the present invention. For example, a first component may be deemed to be administered “after” a second component if the first component is administered 1 minute after, 5 minutes after, 10 minutes after, 15 minutes after, 30 minutes after, 1 hour after, 2 hours after, 3 hours after, 4 hours after, 5 hours after, 6 hours after, 12 hours after, 24 hours after, 36 hours after, 48 hours after, 60 hours after, 72 hours after administration of the second component. In yet other embodiments, the additional therapeutically active component(s) may be administered to a subject concurrent with administration of an anti- SARS-COV-2 antibody of the present invention. “Concurrent” administration, for purposes of the present invention, includes, e.g., administration of an anti-SARS-COV-2 antibody and an additional therapeutically active component to a subject in a single dosage form, or in separate dosage forms administered to the subject within about 30 minutes or less of each other. If administered in separate dosage forms, each dosage form may be administered via the same route (e.g., both the anti-SARS-COV-2 antibody and the additional therapeutically active component may be administered intravenously, etc.); alternatively, each dosage form may be administered via a different route e.g., the anti-SARS-COV-2 antibody may be administered intravenously, and the additional therapeutically active component may be administered orally). In any event, administering the components in a single dosage form, in separate dosage forms by the same route, or in separate dosage forms by different routes are all considered “concurrent administration,” for purposes of the present disclosure. For purposes of the present disclosure, administration of an anti-SARS-COV-2 antibody “prior to,” “concurrent with,” or “after” (as those terms are defined hereinabove) administration of an additional therapeutically active component is considered administration of an anti-SARS-COV-2 antibody “in combination with” an additional therapeutically active component.

[0222] The present invention includes pharmaceutical compositions in which an anti-SARS-COV- 2 antibody of the present invention is co-formulated with one or more of the additional therapeutically active component(s) as described elsewhere herein.

[0223] Administration Regimens

[0224] According to certain embodiments, a single dose of an anti-SARS-COV-2 antibody of the invention (or a pharmaceutical composition comprising a combination of an anti-SARS-COV-2 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject in need thereof. According to certain embodiments of the present invention, multiple doses of an anti-SARS-COV-2 antibody (or a pharmaceutical composition comprising a combination of an anti-SARS-COV-2 antibody and any of the additional therapeutically active agents mentioned herein) may be administered to a subject over a defined time course. The methods according to this aspect of the invention comprise sequentially administering to a subject multiple doses of an anti-SARS-COV-2 antibody of the invention. As used herein, “sequentially administering” means that each dose of anti- SARS-COV-2 antibody is administered to the subject at a different point in time, e.g., on different days separated by a predetermined interval (e.g., hours, days, weeks, or months). The present invention includes methods that comprise sequentially administering to the patient a single initial dose of an anti- SARS-COV-2 antibody, followed by one or more secondary doses of the anti-SARS-COV-2 antibody, and optionally followed by one or more tertiary doses of the anti-SARS-COV-2 antibody.

[0225] The terms “initial dose,” “secondary doses,” and “tertiary doses,” refer to the temporal sequence of administration of the anti-SARS-COV-2 antibody of the invention. Thus, the “initial dose” is the dose, which is administered at the beginning of the treatment regimen (also referred to as the “baseline dose”); the “secondary doses” are the doses, which are administered after the initial dose; and the “tertiary doses” are the doses which are administered after the secondary doses. The initial, secondary, and tertiary doses may all contain the same amount of anti-SARS-COV-2 antibody, but generally may differ from one another in terms of frequency of administration. In some embodiments, however, the amount of anti-SARS-COV-2 antibody contained in the initial, secondary and / or tertiary doses varies from one another (e.g., adjusted up or down as appropriate) during the course of treatment. In some embodiments, two or more (e.g., 2, 3, 4, or 5) doses are administered at the beginning of the treatment regimen as “loading doses” followed by subsequent doses that are administered on a less frequent basis (e.g., “maintenance doses”).

[0226] In certain exemplary embodiments of the present invention, each secondary and / or tertiary dose is administered 1 to 48 hours (e.g., 1, 1 %, 2, 2%, 3, 314, 4, 414, 5, 514, 6, 614, 7, 714, 8, 814, 9, 9%, 10, 10’4, 11, 11 %, 12, 12’4, 13, 13'4, 14, 14'4, 15, 15'4, 16, 16’4, 17, 17’4, 18, 18’4, 19, 19'4, 20, 2014, 21, 21 %, 22, 22’4, 23, 23 %, 24, 24%, 25, 25 %, 26, 26%, or more) after the immediately preceding dose. The phrase “the immediately preceding dose,” as used herein, means, in a sequence of multiple administrations, the dose of anti-SARS-COV-2 antibody, which is administered to a patient prior to the administration of the very next dose in the sequence with no intervening doses. The methods, according to this aspect of the invention, may comprise administering to a patient any number of secondary and / or tertiary doses of an anti-SARS-COV-2 antibody. For example, In some embodiments, only a single secondary dose is administered to the patient. In other embodiments, two or more (e.g., 2, 3, 4, 5, 6, 7, 8, or more) secondary doses are administered to the patient. Likewise, In some embodiments, only a single tertiary dose is administered to the patient. In other embodiments, two or more (e.g, 2, 3, 4, 5, 6, 7, 8, or more) tertiary doses are administered to the patient.

[0227] In some embodiments of the invention, the frequency at which the secondary and / or tertiary doses are administered to a patient can vary over the course of the treatment regimen. The frequency of administration may also be adjusted during the course of treatment by a physician depending on the needs of the individual patient following clinical examination.

[0228] Diagnostic Uses of the Antibodies

[0229] The anti-SARS-COV-2 antibodies of the present invention may be used to detect and / or measure SARS-COV-2 in a sample, e.g., for diagnostic purposes. Some embodiments contemplate the use of one or more antibodies of the present invention in assays to detect a SARS-COV-2- associated disease or disorder. Exemplary diagnostic assays for SARS-COV-2 may comprise, e.g., contacting a sample obtained from a patient with an anti-SARS-COV-2 antibody of this disclosure, wherein the anti-SARS-COV-2 antibody is labeled with a detectable label or reporter molecule or used as a capture ligand to selectively isolate SARS-COV-2 from patient samples. Alternatively, an unlabeled anti-SARS-COV-2 antibody can be used in diagnostic applications in combination with a secondary antibody, which is itself detectably labeled. The detectable label or reporter molecule can be a radioisotope, such as H, C, P, S, or I; a fluorescent or chemiluminescent moiety such as fluorescein isothiocyanate, or rhodamine; or an enzyme such as alkaline phosphatase, P- galactosidase, horseradish peroxidase, or luciferase. Specific exemplary assays that can be used to detect or measure SARS-COV-2 in a sample include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescence-activated cell sorting (FACS).

[0230] In another aspect, this disclosure further provides a method for detecting the presence of SARS CoV-2 in a sample comprising the steps of: (i) contacting a sample with the antibody or antigen-binding fragment thereof described herein; and (ii) determining binding of the antibody or antigen-binding fragment to one or more SARS CoV-2 antigens, wherein binding of the antibody to the one or more SARS CoV-2 antigens is indicative of the presence of SARS CoV-2 in the sample.

[0231] In some embodiments, the SARS-CoV-2 antigen comprises the receptor binding domain (RBD) of the S polypeptide.

[0232] In some embodiments, the antibody or antigen-binding fragment thereof is conjugated to a label. In some embodiments, the step of detecting comprises contacting a secondary antibody with the antibody or antigen-binding fragment thereof and wherein the secondary antibody comprises a label. In some embodiments, the label includes a fluorescent label, a chemiluminescent label, a radiolabel, and an enzyme.

[0233] In some embodiments, the step of detecting comprises detecting fluorescence or chemiluminescence. In some embodiments, the step of detecting comprises a competitive binding assay or ELISA.

[0234] In some embodiments, the method further comprises binding the sample to a solid support. In some embodiments, the solid support includes microparticles, microbeads, magnetic beads, and an affinity purification column.

[0235] Samples that can be used in SARS-COV-2 diagnostic assays according to the present invention include any tissue or fluid sample obtainable from a patient, which contains detectable quantities of either SARS-COV-2 protein, or fragments thereof, under normal or pathological conditions. Generally, levels of SARS-COV-2 protein in a particular sample obtained from a healthy patient (e.g., a patient not afflicted with a disease associated with SARS-COV-2) will be measured to initially establish a baseline, or standard, level of SARS-COV-2. This baseline level of SARS-COV-2 can then be compared against the levels of SARS-COV-2 measured in samples obtained from individuals suspected of having a SARS-COV-2-associated condition or symptoms associated with such condition.

[0236] The antibodies specific for SARS-COV-2 protein may contain no additional labels or moieties, or they may contain an N-terminal or C-terminal label or moiety. In one embodiment, the label or moiety is biotin. In a binding assay, the location of a label (if any) may determine the orientation of the peptide relative to the surface upon which the peptide is bound. For example, if a surface is coated with avidin, a peptide containing a N-terminal biotin will be oriented such that the C-terminal portion of the peptide will be distal to the surface.

[0237] Additional Definitions

[0238] To aid in understanding the detailed description of the compositions and methods according to the disclosure, a few express definitions are provided to facilitate an unambiguous disclosure of the various aspects of the disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0239] A “neutralizing antibody” is one that can neutralize the ability of that pathogen to initiate and / or perpetuate an infection in a host and / or in target cells in vitro. “Broadly neutralizing anti- SARS-CoV-2 antibodies” refer to antibodies that neutralize more than one SARS-CoV-2 virus strains / variants in a neutralization assay. A broad neutralizing anti-SARS-CoV-2 antibody may neutralize at least 2, 3, 4, 5, 6, 7, 8, 9 or more different strains / variants of SARS-CoV-2.

[0240] The term “antibody” as referred to herein includes whole antibodies and any antigenbinding fragment or single chains thereof. Whole antibodies are glycoproteins comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CHI, CH2, and CH3. Each light chain is comprised of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The heavy chain variable region CDRs and FRs are HFR1, HCDR1, HFR2, HCDR2, HFR3, HCDR3, HFR4. The light chain variable region CDRs and FRs are LFR1, LCDR1, LFR2, LCDR2, LFR3, LCDR3, LFR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.

[0241] The term “antigen-binding fragment or portion” of an antibody (or simply “antibody fragment or portion”), as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., a spike or S protein of SARS-CoV-2 virus). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed within the term “antigenbinding fragment or portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL, and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fab' fragment, which is essentially a Fab with part of the hinge region (see, FUNDAMENTAL IMMUNOLOGY (Paul ed., 3rd ed. 1993)); (iv) a Fd fragment consisting of the VH and CHI domains; (v) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (vi) a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consists of a VH domain; (vii) an isolated CDR; and (viii) a nanobody, a heavy chain variable region containing a single variable domain and two constant domains. Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv or scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain antibodies are also intended to be encompassed within the term “antigen-binding fragment or portion” of an antibody. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0242] An “isolated antibody,” as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to a spike or S protein of SARS-CoV-2 virus is substantially free of antibodies that specifically bind antigens other than the neuraminidase). An isolated antibody can be substantially free of other cellular material and / or chemicals. The terms “monoclonal antibody” or “monoclonal antibody composition” as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0243] The term “human antibody” is intended to include antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region also is derived from human germline immunoglobulin sequences. The human antibodies of the invention can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term “human antibody,” as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.

[0244] The term “human monoclonal antibody” refers to antibodies displaying a single binding specificity, which have variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibodies can be produced by a hybridoma that includes a B cell obtained from a transgenic nonhuman animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0245] The term “recombinant human antibody,” as used herein, includes all human antibodies that are prepared, expressed, created, or isolated by recombinant means, such as (a) antibodies isolated from an animal (e.g., a mouse) that is transgenic or transchromosomal for human immunoglobulin genes or a hybridoma prepared therefrom (described further below), (b) antibodies isolated from a host cell transformed to express the human antibody, e.g., from a transfectoma, (c) antibodies isolated from a recombinant, combinatorial human antibody library, and (d) antibodies prepared, expressed, created or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. In some embodiments, however, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0246] The term “isotype” refers to the antibody class (e. , IgM or IgGl) that is encoded by the heavy chain constant region genes. The phrases “an antibody recognizing an antigen” and “an antibody specific for an antigen” are used interchangeably herein with the term “an antibody which binds specifically to an antigen.”

[0247] The term “human antibody derivatives” refers to any modified form of the human antibody, e.g., a conjugate of the antibody and another agent or antibody. The term “humanized antibody” is intended to refer to antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made within the human framework sequences.

[0248] The term “chimeric antibody” is intended to refer to antibodies in which the variable region sequences are derived from one species, and the constant region sequences are derived from another species, such as an antibody in which the variable region sequences are derived from a mouse antibody, and the constant region sequences are derived from a human antibody. The term can also refer to an antibody in which its variable region sequence or CDR(s) is derived from one source (e.g., an IgAl antibody), and the constant region sequence or Fc is derived from a different source (e.g., a different antibody, such as an IgG, IgA2, IgD, IgE or IgM antibody).

[0249] The invention encompasses isolated or substantially purified nucleic acids, peptides, polypeptides, or proteins. In the context of the present invention, an “isolated” nucleic acid, DNA or RNA molecule or an “isolated” polypeptide is a nucleic acid, DNA molecule, RNA molecule, or polypeptide that exists apart from its native environment and is therefore not a product of nature. An isolated nucleic acid, DNA molecule, RNA molecule, or polypeptide may exist in a purified form or may exist in a non-native environment such as, for example, a transgenic host cell. A “purified” nucleic acid molecule, peptide, polypeptide, or protein, or a fragment thereof, is substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized. In one embodiment, an “isolated” nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived. A protein, peptide, or polypeptide that is substantially free of cellular material includes preparations of protein, peptide, or polypeptide having less than about 30%, 20%, 10%, or 5% (by dry weight) of contaminating protein. When the protein of the invention, or biologically active portion thereof, is recombinantly produced, preferably culture medium represents less than about 30%, 20%, 10%, or 5% (by dry weight) of chemical precursors or non-protein-of-interest chemicals.

[0250] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, pegylation, or any other manipulation, such as conjugation with a labeling component. As used herein, the term “amino acid” includes natural and / or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.

[0251] A peptide or polypeptide “fragment” as used herein refers to a less than full-length peptide, polypeptide, or protein. For example, a peptide or polypeptide fragment can have is at least about 3, at least about 4, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40 amino acids in length, or single unit lengths thereof. For example, fragment may be 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, or more amino acids in length. There is no upper limit to the size of a peptide fragment. However, in some embodiments, peptide fragments can be less than about 500 amino acids, less than about 400 amino acids, less than about 300 amino acids, or less than about 250 amino acids in length. Preferably the peptide fragment can elicit an immune response when used to inoculate an animal. A peptide fragment may be used to elicit an immune response by inoculating an animal with a peptide fragment in combination with an adjuvant, a peptide fragment that is coupled to an adjuvant, or a peptide fragment that is coupled to arsanilic acid, sulfanilic acid, an acetyl group, or a picryl group. A peptide fragment can include a nonamide bond and can be a peptidomimetic. As used herein, the term “conjugate” or “conjugation” or “linked” as used herein refers to the attachment of two or more entities to form one entity. A conjugate encompasses both peptide- small molecule conjugates as well as peptide-protein / peptide conjugates.

[0252] The term “recombinant,” as used herein, refers to antibodies or antigen-binding fragments thereof of the invention created, expressed, isolated, or obtained by technologies or methods known in the art as recombinant DNA technology, which include, e.g., DNA splicing and transgenic expression. The term refers to antibodies expressed in a non-human mammal (including transgenic non-human mammals, e.g., transgenic mice), or a cell (e.g., CHO cells) expression system or isolated from a recombinant combinatorial human antibody library.

[0253] A “nucleic acid” or “polynucleotide” refers to a DNA molecule (for example, but not limited to, a cDNA or genomic DNA) or an RNA molecule (for example, but not limited to, an mRNA), and includes DNA or RNA analogs. A DNA or RNA analog can be synthesized from nucleotide analogs. The DNA or RNA molecules may include portions that are not naturally occurring, such as modified bases, modified backbone, deoxyribonucleotides in an RNA, etc. The nucleic acid molecule can be single-stranded or double-stranded.

[0254] The term “substantial identity” or “substantially identical,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of the nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or GAP, as discussed below. A nucleic acid molecule having substantial identity to a reference nucleic acid molecule may, in certain instances, encode a polypeptide having the same or substantially similar amino acid sequence as the polypeptide encoded by the reference nucleic acid molecule.

[0255] As applied to polypeptides, the term “substantial similarity” or “substantially similar” means that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap weights, share at least 90% sequence identity, even more preferably at least 95%, 98% or 99% sequence identity. Preferably, residue positions, which are not identical, differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g, charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, which is herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include 1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic- hydroxyl side chains: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; 5) basic side chains: lysine, arginine, and histidine; 6) acidic side chains: aspartate and glutamate, and 7) sulfur-containing side chains: cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysinearginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443 45, herein incorporated by reference. A “moderately conservative” replacement is any change having a nonnegative value in the PAM250 log-likelihood matrix.

[0256] Sequence similarity for polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as GAP and BESTFIT, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA with default or recommended parameters; a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra). Another preferred algorithm when comparing a sequence of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, especially BLASTP or TBLASTN, using default parameters. See, e.g, Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and (1997) Nucleic Acids Res. 25:3389- 3402, each of which is herein incorporated by reference.

[0257] As used herein, the term “affinity” refers to the strength of the sum total of noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity, which reflects a 1 : 1 interaction between members of a binding pair (e.g., antibody and antigen). The affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (KD). Affinity can be measured by common methods known in the art, including those described herein.

[0258] The term “specifically binds,” or “binds specifically to,” or the like, refers to an antibody that binds to a single epitope, e.g., under physiologic conditions., but which does not bind to more than one epitope. Accordingly, an antibody that specifically binds to a polypeptide will bind to an epitope that is present on the polypeptide, but which is not present on other polypeptides. Specific binding can be characterized by an equilibrium dissociation constant of at least about 1x10-8 M or less (e.g., a smaller KD denotes a tighter binding). Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. As described herein, antibodies have been identified by surface plasmon resonance, e.g., BIACORE™, which bind specifically to a spike or S protein of a SARS-CoV-2 virus.

[0259] Preferably, the antibody binds to a spike or S protein with “high affinity,” namely with a KD of 1 X 10-7 M or less, more preferably 5 x 10-8 M or less, more preferably 3 x 10-8 M or less, more preferably 1 x 10-8 M or less, more preferably 5 x 10-9 M or less or even more preferably 1 x 10-9 M or less, as determined by surface plasmon resonance, e.g., BIACORE. The term “does not substantially bind” to a protein or cells, as used herein, means does not bind or does not bind with a high affinity to the protein or cells, i.e., binds to the protein or cells with a KD of 1 x 10-6 M or more, more preferably 1 x 10-5 M or more, more preferably 1 x 10-4 M or more, more preferably 1 x 10-3 M or more, even more preferably 1 x 10-2 M or more.

[0260] The term “Kassoc” or “Ka,” as used herein, is intended to refer to the association rate of a particular antibody-antigen interaction, whereas the term “Kdis” or “Kd,” as used herein, is intended to refer to the dissociation rate of a particular antibody-antigen interaction. The term “KD,” as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values for antibodies can be determined using methods well established in the art. A preferred method for determining the KD of an antibody is by using surface plasmon resonance, preferably using a biosensor system such as a BIACORE system.

[0261] Antibodies that “compete with another antibody for binding to a target” refer to antibodies that inhibit (partially or completely) the binding of the other antibody to the target. Whether two antibodies compete with each other for binding to a target, i.e., whether and to what extent one antibody inhibits the binding of the other antibody to a target, may be determined using known competition experiments. In some embodiments, an antibody competes with, and inhibits binding of another antibody to a target by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100%. The level of inhibition or competition may be different depending on which antibody is the “blocking antibody” (i.e. , the cold antibody that is incubated first with the target). Competition assays can be conducted as described, for example, in Ed Harlow and David Lane, Cold Spring Harb Protoc; 2006 or in Chapter 11 of “Using Antibodies” by Ed Harlow and David Lane, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA 1999. Competing antibodies bind to the same epitope, an overlapping epitope or to adjacent epitopes (e.g., as evidenced by steric hindrance). Other competitive binding assays include: solid phase direct or indirect radioimmunoassay (RIA), solid phase direct or indirect enzyme immunoassay (EIA), sandwich competition assay (see Stahli el al., Methods in Enzymology 9:242 (1983)); solid phase direct biotin-avidin EIA (see Kirkland el al., J. Immunol. 137:3614 (1986)); solid phase direct labeled assay, solid phase direct labeled sandwich assay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Press (1988)); solid phase direct label RIA using 1-125 label (see Morel el al., Mol. Immunol. 25(1):7 (1988)); solid phase direct biotin-avidin EIA (Cheung el al., Virology 176:546 (1990)); and direct labeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).

[0262] The term “epitope” as used herein refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different areas on an antigen and may have different biological effects. The term “epitope” also refers to a site on an antigen to which B and / or T cells respond. It also refers to a region of an antigen that is bound by an antibody. Epitopes may be defined as structural or functional. Functional epitopes are generally a subset of the structural epitopes and have those residues that directly contribute to the affinity of the interaction. Epitopes may also be conformational, that is, composed of nonlinear amino acids. In some embodiments, epitopes may include determinants that are chemically active surface groupings of molecules such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and, In some embodiments, may have specific three-dimensional structural characteristics, and / or specific charge characteristics. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immune-precipitation assays, wherein overlapping or contiguous peptides from a spike or S protein are tested for reactivity with a given antibody. Methods of determining spatial conformation of epitopes include techniques in the art and those described herein, for example, x-ray crystallography and 2-dimensional nuclear magnetic resonance (see, e.g., Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996)).

[0263] The term “epitope mapping” refers to the process of identification of the molecular determinants for antibody-antigen recognition.

[0264] The term “binds to an epitope” or “recognizes an epitope” with reference to an antibody or antibody fragment refers to continuous or discontinuous segments of amino acids within an antigen. Those of skill in the art understand that the terms do not necessarily mean that the antibody or antibody fragment is in direct contact with every amino acid within an epitope sequence.

[0265] The term “binds to the same epitope” with reference to two or more antibodies means that the antibodies bind to the same, overlapping, or encompassing continuous or discontinuous segments of amino acids. Those of skill in the art understand that the phrase “binds to the same epitope” does not necessarily mean that the antibodies bind to or contact exactly the same amino acids. The precise amino acids that the antibodies contact can differ. For example, a first antibody can bind to a segment of amino acids that is completely encompassed by the segment of amino acids bound by a second antibody. In another example, a first antibody binds one or more segments of amino acids that significantly overlap the one or more segments bound by the second antibody. For the purposes herein, such antibodies are considered to “bind to the same epitope.”

[0266] As used herein, the term “immune response” refers to a biological response within a vertebrate against foreign agents, which response protects the organism against these agents and diseases caused by them. An immune response is mediated by the action of a cell of the immune system (for example, a T lymphocyte, B lymphocyte, natural killer (NK) cell, macrophage, eosinophil, mast cell, dendritic cell, or neutrophil) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines, and complement) that results in selective targeting, binding to, damage to, destruction of, and / or elimination from the vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. An immune reaction includes, e.g., activation or inhibition of a T cell, e.g., an effector T cell or a Th cell, such as a CD4+ or CD8+ T cell, or the inhibition of a Treg cell.

[0267] The term “detectable label” as used herein refers to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, chromophores, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin, avidin, streptavidin or haptens), intercalating dyes and the like. The term “fluorescer” refers to a substance or a portion thereof that is capable of exhibiting fluorescence in the detectable range.

[0268] In many embodiments, the terms “subject” and “patient” are used interchangeably irrespective of whether the subject has or is currently undergoing any form of treatment. As used herein, the terms “subject” and “subjects” may refer to any vertebrate, including, but not limited to, a mammal (e.g., cow, pig, camel, llama, horse, goat, rabbit, sheep, hamsters, guinea pig, cat, dog, rat, and mouse, a non-human primate (for example, a monkey, such as a cynomolgus monkey, chimpanzee, etc.) and a human). The subject may be a human or a non-human. In more exemplary aspects, the mammal is a human. As used herein, the expression “a subject in need thereof’ or “a patient in need thereof’ means a human or non-human mammal that exhibits one or more symptoms or indications of disorders (e.g., neuronal disorders, autoimmune diseases, and cardiovascular diseases), and / or who has been diagnosed with inflammatory disorders. In some embodiments, the subject is a mammal. In some embodiments, the subject is human. As used herein, the term “disease” is intended to be generally synonymous and is used interchangeably with the terms “disorder” and “condition” (as in medical condition), in that all reflect an abnormal condition (e.g., inflammatory disorder) of the human or animal body or of one of its parts that impairs normal functioning, is typically manifested by distinguishing signs and symptoms, and causes the human or animal to have a reduced duration or quality of life.

[0269] As used herein, the term “treating” or “treatment” of any disease or disorder refers in one embodiment, to ameliorating the disease or disorder (z.c., arresting or reducing the development of the disease or at least one of the clinical symptoms thereof). In another embodiment, “treating” or “treatment” refers to ameliorating at least one physical parameter, which may not be discernible by the patient. In yet another embodiment, “treating” or “treatment” refers to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. In yet another embodiment, “treating” or “treatment” refers to preventing or delaying the onset or development or progression of the disease or disorder.

[0270] The terms “prevent,” “preventing,” “prevention,” “prophylactic treatment” and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

[0271] The terms “decrease,” “reduced,” “reduction,” “decrease,” or “inhibit” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “reduced,” “reduction,” “decrease,” or “inhibit” means a decrease by at least 10% as compared to a reference level, for example, a decrease by at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% decrease (e.g., absent level as compared to a reference sample), or any decrease between 10-100% as compared to a reference level.

[0272] As used herein, the term “agent” denotes a chemical compound, a mixture of chemical compounds, a biological macromolecule (such as a nucleic acid, an antibody, a protein or portion thereof, e.g. , a peptide), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues. The activity of such agents may render it suitable as a “therapeutic agent,” which is a biologically, physiologically, or pharmacologically active substance (or substances) that acts locally or systemically in a subject.

[0273] As used herein, the terms “therapeutic agent,” “therapeutic capable agent,” or “treatment agent” are used interchangeably and refer to a molecule or compound that confers some beneficial effect upon administration to a subject. The beneficial effect includes enablement of diagnostic determinations; amelioration of a disease, symptom, disorder, or pathological condition; reducing or preventing the onset of a disease, symptom, disorder, or condition; and generally counteracting a disease, symptom, disorder or pathological condition.

[0274] The term “therapeutic effect” is art-recognized and refers to a local or systemic effect in animals, particularly mammals, and more particularly humans caused by a pharmacologically active substance.

[0275] The term “effective amount,” “effective dose,” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve a desired effect. A “therapeutically effective amount” or “therapeutically effective dosage” of a drug or therapeutic agent is any amount of the drug that, when used alone or in combination with another therapeutic agent, promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom -free periods, or a prevention of impairment or disability due to the disease affliction. A “prophylactically effective amount” or a “prophylactically effective dosage” of a drug is an amount of the drug that, when administered alone or in combination with another therapeutic agent to a subject at risk of developing a disease or of suffering a recurrence of disease, inhibits the development or recurrence of the disease. The ability of a therapeutic or prophylactic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.

[0276] Doses are often expressed in relation to bodyweight. Thus, a dose which is expressed as [g, mg, or other unit] / kg (or g, mg etc.) usually refers to [g, mg, or other unit] “per kg (or g, mg etc.) body weight,” even if the term “bodyweight” is not explicitly mentioned.

[0277] As used herein, the term “composition” or “pharmaceutical composition” refers to a mixture of at least one component useful within the invention with other components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of one or more components of the invention to an organism.

[0278] As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the composition, and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0279] As used herein, the term “pharmaceutically acceptable carrier” includes a pharmaceutically acceptable salt, pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting a compound(s) of the present invention within or to the subject such that it may perform its intended function. Typically, such compounds are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each salt or carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose, and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen- free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; diluent; granulating agent; lubricant; binder; disintegrating agent; wetting agent; emulsifier; coloring agent; release agent; coating agent; sweetening agent; flavoring agent; perfuming agent; preservative; antioxidant; plasticizer; gelling agent; thickener; hardener; setting agent; suspending agent; surfactant; humectant; carrier; stabilizer; and other non-toxic compatible substances employed in pharmaceutical formulations, or any combination thereof. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of one or more components of this disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions.

[0280] “Combination” therapy, as used herein, unless otherwise clear from the context, is meant to encompass administration of two or more therapeutic agents in a coordinated fashion and includes, but is not limited to, concurrent dosing. Specifically, combination therapy encompasses both co-administration (e.g., administration of a co-formulation or simultaneous administration of separate therapeutic compositions) and serial or sequential administration, provided that administration of one therapeutic agent is conditioned in some way on the administration of another therapeutic agent. For example, one therapeutic agent may be administered only after a different therapeutic agent has been administered and allowed to act for a prescribed period of time. See, e.g. , Kohrt et al. (2011) Blood 117:2423.

[0281] As used herein, the term “co-administration” or “co-administered” refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co- administration of two or more agents / therapies is concurrent. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents / therapies used may vary.

[0282] As used herein, the term “contacting,” when used in reference to any set of components, includes any process whereby the components to be contacted are mixed into the same mixture (for example, are added into the same compartment or solution), and does not necessarily require actual physical contact between the recited components. The recited components can be contacted in any order or any combination (or sub-combination) and can include situations where one or some of the recited components are subsequently removed from the mixture, optionally prior to addition of other recited components. For example, “contacting A with B and C” includes any and all of the following situations: (i) A is mixed with C, then B is added to the mixture; (ii) A and B are mixed into a mixture; B is removed from the mixture, and then C is added to the mixture; and (iii) A is added to a mixture of B and C.

[0283] “Sample,” “test sample,” and “patient sample” may be used interchangeably herein. The sample can be a sample of serum, urine plasma, amniotic fluid, cerebrospinal fluid, cells, or tissue. Such a sample can be used directly as obtained from a patient or can be pre-treated, such as by fdtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art. The terms “sample” and “biological sample” as used herein generally refer to a biological material being tested for and / or suspected of containing an analyte of interest, such as antibodies. The sample may be any tissue sample from the subject. The sample may comprise protein from the subject.

[0284] As used herein, the term “zzz vitro” refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0285] As used herein, the term “zzz vivo” refers to events that occur within a multi-cellular organism, such as a non-human animal.

[0286] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise.

[0287] As used herein, the terms “including,” “comprising,” “containing,” or “having” and variations thereof are meant to encompass the items listed thereafter and equivalents thereof as well as additional subject matter unless otherwise noted.

[0288] As used herein, the phrases “in one embodiment,” “in various embodiments,” “in some embodiments,” and the like are used repeatedly. Such phrases do not necessarily refer to the same embodiment, but they may unless the context dictates otherwise.

[0289] As used herein, the terms “and / or” or “ / ” means any one of the items, any combination of the items, or all of the items with which this term is associated.

[0290] As used herein, the word “substantially” does not exclude “completely,” e.g., a composition that is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition of the invention.

[0291] As used herein, the term “each,” when used in reference to a collection of items, is intended to identify an individual item in the collection but does not necessarily refer to every item in the collection. Exceptions can occur if explicit disclosure or context clearly dictates otherwise.

[0292] As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value). Unless indicated otherwise herein, the term “about” is intended to include values, e.g., weight percents, proximate to the recited range that are equivalent in terms of the functionality of the individual ingredient, the composition, or the embodiment.

[0293] As disclosed herein, a number of ranges of values are provided. It is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither, or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0294] The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0295] All methods described herein are performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. In regard to any of the methods provided, the steps of the method may occur simultaneously or sequentially. When the steps of the method occur sequentially, the steps may occur in any order, unless noted otherwise. In cases in which a method comprises a combination of steps, each and every combination or sub-combination of the steps is encompassed within the scope of the disclosure, unless otherwise noted herein.

[0296] Each publication, patent application, patent, and other reference cited herein is incorporated by reference in its entirety to the extent that it is not inconsistent with the present disclosure. Publications disclosed herein are provided solely for their disclosure prior to the filing date of the present invention. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed.

[0297] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

[0298] EXAMPLES

[0299] EXAMPLE 1

[0300] This example describes the materials, methods, and instrumentation used in EXAMPLE 2.

[0301] Study participants.

[0302] Participants were healthy adults that had been vaccinated with 3 or 4 doses of an mRNA vaccine (mRNA- 1273 (Moderna) or BNT162b2 (Pfizer)). The group of participants were categorized into two study groups as age was of interest: Elderly (75-91 years) and younger (23- 66years). The elderly participants were followed-up for a blood sample 8.5- or 3- months after receiving their third or fourth dose of the mRNA vaccine, separately. The younger participants were followed-up at 6.5 months for a blood sample after the third dose. All participants provided written informed consent before participation in the study and the study was conducted in accordance with Good Clinical Practice. The study was performed in compliance with all relevant ethical regulations and the protocol (DRO-1006) for studies with human participants was approved by the Institutional Review Board of the Rockefeller University. For detailed participant characteristics see Table 1.

[0303] Blood samples processing and storage

[0304] Venous blood samples were collected into Heparin and Serum-gel monovette tubes by standard phlebotomy at The Rockefeller University. Peripheral Blood Mononuclear Cells (PBMCs) obtained from samples collected were further purified as previously reported by gradient centrifugation and stored in liquid nitrogen in the presence of Fetal Calf Serum (FCS) and Dimethyl sulfoxide (DMSO) (Gaebler et al., 2021; Robbiani et al., 2020). Heparinized serum and plasma samples were aliquoted and stored at -20°C or less. Prior to experiments, aliquots of plasma samples were heat-inactivated (56°C for 1 hour) and then stored at 4°C.

[0305] Table 1. Individual participant characteristics

[0306] Docket No. 070413.20802

[0307] 77

[0308] 159904310.2

[0309] Docket No. 070413.20802

[0310] 78

[0311] 159904310.2

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[0313] Table 1. (continued)

[0314] 79

[0315] 159904310.2

[0316] Docket No. 070413.20802

[0317] 80

[0318] 159904310.2

[0319] Docket No. 070413.20802

[0320] Table 1. (continued)

[0321] 81

[0322] 159904310.2

[0323] Docket No. 070413.20802

[0324] 82

[0325] 159904310.2

[0326] Docket No. 070413.20802

[0327] Table 1. (continued)

[0328] 83

[0329] 159904310.2

[0330] Docket No. 070413.20802

[0331] 84

[0332] 159904310.2

[0333] Docket No. 070413.20802

[0334] 85

[0335] 159904310.2

[0336] ELISAs

[0337] Enzyme-Linked Immunosorbent Assays (ELISAs)(Amanat et al., 2020) were performed to evaluate antibodies binding to SARS-CoV-2 wild-type (Wuhan-Hu-1) RBD, Omicron (BA.4 / 5) RBD, Omicron (XBB) RBD and Omicron (XBB.1.5) RBD protein by coating of high- binding 96-half-well plates (Coming 3690) with 50 pl per well of a Ipg / ml indicated protein solution in Phosphate-buffered Saline (PBS) 1 hour at room temperature. Plates were washed 6 times with washing buffer (1 x PBS with 0.05% Tween-20 (Sigma-Aldrich) and incubated with 170 pl per well blocking buffer (1 x PBS with 1% BSA and 0.05% Tween-20 (Sigma) and 0. ImM EDTA) for 1 hour at room temperature. Immediately after blocking, plasma samples or monoclonal antibodies were added in PBS and incubated for 1 hour at room temperature. Plasma samples were assayed at a 1 :66 starting dilution and 10 additional 3-fold serial dilutions. 10 pg / ml starting concentration was used to test monoclonal antibodies followed by 10 additional 4- fold serial dilutions. Plates were washed 6 times with washing buffer and then incubated with anti-human IgG secondary antibody conjugated to horseradish peroxidase (HRP) (Jackson Immuno Research 109-036-088 109-035-129 and Sigma A0295) in blocking buffer at a 1:5,000 dilution. Plates were developed by addition of the HRP substrate, 3,3’,5,5’-Tetramethylbenzidine (TMB) (ThermoFisher) for 10 minutes (plasma samples and monoclonal antibodies). 50 pl of 1 M H2SO4was used to stop the reaction and absorbance was measured at 450 nm with an ELISA microplate reader (FLUOStar® Omega, BMG Labtech) with Omega and Omega MARS software for analysis. A positive control (For anti-RBD ELISA, plasma from participant COV72(Robbiani et al., 2020), diluted 66.6-fold and ten additional threefold serial dilutions in PBS; for anti -Omicron ELISA, plasma from B040(Wang et al., 2022b) was used as a control) was added to every assay plate for normalization for plasma samples. The average of its signal was used for normalization of all the other values on the same plate with Excel software before calculating the half-maximal binding titer using four-parameter nonlinear regression (GraphPad Prism V9.1). Negative controls of pre-pandemic plasma samples from healthy donors were used for validation (for more details please see(Robbiani et al., 2020)). For monoclonal antibodies, the ELISA half-maximal concentration (ECso) was determined using four-parameter nonlinear regression (GraphPad Prism V9.1). EC50S above 1000 ng / mL were considered non-binders.

[0338] SARS-CoV-2 pseudotyped reporter virus The plasmid PSARS-COV-2-S I9 expressing a C-terminally truncated SARS-CoV-2 spike protein based on Wuhan-Hu-1 spike has been described before (Robbiani et al., 2020; Schmidt et al., 2020). Variant pseudoviruses resembling SARS-CoV-2 variants Omicron BA. l, Omicron BA.2, Omicron BA.4 / 5, Omicron BA.2.75.2 and Omicron XBB.1.5 have been described before (Schmidt et al., 2022; Wang et al., 2022a; Wang et al., 2022b) and were generated by introduction of substitutions using synthetic gene fragments (IDT) or overlap extension PCR mediated mutagenesis and Gibson assembly. Specifically, the variant-specific deletions and substitutions introduced were:

[0339] Omicron BA. l : A67V, A69-70, T95I, G142D, 143 -145, A211, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493K, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, H679K, P681H, N764K, D796Y, N856K, Q954H, N969H, N969K, L981F

[0340] Omicron BA.2: T19I, L24S, del25-27, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, S477N, T478K, E484A, Q493R, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K

[0341] Omicron BA.4 / 5: T19I, L24S, del25-27, del69-70, G142D, V213G, G339D, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, L452R, S477N, T478K, E484A, F486V, Q498R, N501 Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K

[0342] Omicron BA.2.75.2: T19I, L24S, del25-27, G142D, K147E, W152R, F157L, I210V, V213G, G257S, G339H, R346T, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, G446S, N460K, S477N, T478K, E484A, F486S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K, DI 199N

[0343] Omicron XBB.1.5: T19I, L24S, del25-27, V83A, G142D, del 144, H146Q, Q183E, V213E, G252V, G339H, R346T, L368I, S371F, S373P, S375F, T376A, D405N, R408S, K417N, N440K, V445P, G446S, N460K, S477N, T478K, E484A, F486P, F490S, Q498R, N501Y, Y505H, D614G, H655Y, N679K, P681H, N764K, D796Y, Q954H, N969K

[0344] Deletions / substitutions corresponding to variants of concern listed above were incorporated into a spike protein that also includes the R683G substitution, which disrupts the furin cleavage site and increases particle infectivity. Neutralizing activity against mutant pseudoviruses were compared to a wildtype (WT) SARS-CoV-2 spike sequence (NC_045512), carrying R683G.

[0345] SARS-CoV-2 pseudotyped particles were generated as previously described (Robbiani et al., 2020; Schmidt et al., 2020). Briefly, 293T (CRL-11268) cells were obtained from ATCC, and the cells were transfected with pNL4-3AEnv-nanoluc and PSARS-COV-2-SAI9, particles were harvested 48 hours post-transfection, filtered and stored at -80°C.

[0346] Pseudotyped virus neutralization assay

[0347] Pre-pandemic negative control plasma from healthy donors, plasma from individuals who received three or fourth dose of an mRNA vaccine, or monoclonal antibodies were five-fold serially diluted and incubated with SARS-CoV-2 pseudotyped virus for 1 hour at 37 °C. The mixture was subsequently incubated with HT1080 / Ace2 cl 14 cells for 48 hours after which cells were washed with PBS and lysed with Luciferase Cell Culture Lysis 5* reagent (Promega). Nanoluc Luciferase activity in lysates was measured using the Nano-Gio® Luciferase Assay System (Promega) with the CLARIOstar® Microplate Multimode Reader (BMG). The relative luminescence units were normalized to those derived from cells infected with SARS-CoV-2 pseudotyped virus(Wang et al., 2021c) in the absence of plasma or monoclonal antibodies. The half-maximal neutralization titers for plasma (NT50) or half-maximal inhibitory concentrations for monoclonal antibodies (IC50) were determined using four-parameter nonlinear regression (least squares regression method without weighting; constraints: top=l, bottom=0) (GraphPad Prism).

[0348] Biotinylation of viral protein for use in flow cytometry

[0349] Purified and Avi-tagged SARS-CoV-2 WT RBD was biotinylated using the Biotin- Protein Ligase-BIRA kit according to manufacturer’s instructions (Avidity) as described before (Robbiani et al., 2020). Ovalbumin (Sigma, A5503-1G) was biotinylated using the EZ-Link™ Sulfo-NHS-LC-Biotinylation kit according to the manufacturer’s instructions (Thermo Scientific). Biotinylated ovalbumin was conjugated to streptavidin-BV711 for single-cell sorts (BD biosciences, 563262). WT RBD was conjugated to streptavidin-PE (BD Biosciences, 554061) and streptavidin-AF647 (Biolegend, 405237) for single-cell sorting. Flow cytometry and single cell sorting Single-cell sorting by flow cytometry was described previously(Robbiani et al., 2020). Simply, peripheral blood mononuclear cells (PBMC) were enriched for B cells by negative selection using a pan-B-cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec, 130-101-638). The enriched B cells were incubated in Flourescence-Activated Cellsorting (FACS) buffer (1 x PBS, 2% FCS, 1 mM ethylenediaminetetraacetic acid (EDTA)) with the following anti -human antibodies (all at 1 :200 dilution): anti-CD20-PECy7 (BD Biosciences, 335793), anti-CD3-APC-eFluro 780 (Invitrogen, 47-0037-41), anti-CD8-APC-eFluor 780 (Invitrogen, 47-0086-42), anti-CD16-APC-eFluor 780 (Invitrogen, 47-0168-41), anti -CD 14- APC-eFluor 780 (Invitrogen, 47-0149-42), as well as Zombie NIR (BioLegend, 423105) and fluorophore-labeled RBD and ovalbumin (Ova) for 30 min on ice. Single CD3 CD8 CD14 CD16" CD20 Ova WT RBD-PE+- WT RBD-AF647+B cells were sorted into individual wells of 96- well plates containing 4 pl of lysis buffer (0.5 x PBS, 10 mM Dithiothreitol (DTT), 3,000 units / ml RNasin Ribonuclease Inhibitors (Promega, N2615) per well using a FACS Aria III and FACSDiva software (Becton Dickinson) for acquisition and FlowJo for analysis. The sorted cells were frozen on dry ice, and then stored at -80 °C or immediately used for subsequent RNA reverse transcription. For B cell phenotype analysis, in addition to above antibodies, B cells were also stained with following anti-human antibodies (all at 1 :200 dilution): anti-IgD-BV650 (BD, 740594), anti-CD27-BV786 (BD biosciences, 563327), anti-CD19-BV605 (Biolegend, 302244), anti-CD71-PerCP-Cy5.5 (Biolegend, 334114), anti-IgG-PECF594 (BD, 562538), anti-IgM- AF700 (Biolegend, 314538), anti-IgA-Viogreen (Miltenyi Biotec, 130-113-481), anti-CDl lc- BV711(BD biosciences, 563130), anti-CD21- PerCPCy5.5 (Biolegend, 354908), anti-CD38- BV421 (BD biosciences, 562445).

[0350] Antibody sequencing, cloning and expression

[0351] Antibodies were identified and sequenced as described previously (Robbiani et al., 2020; Wang et al., 2021a). In brief, RNA from single cells was reverse transcribed (SuperScript III Reverse Transcriptase, Invitrogen, 18080-044), and the cDNA was stored at -20 °C or used for subsequent amplification of the variable IGH, IGL and IGK genes by nested PCR and Sanger sequencing. Sequence analysis was performed using MacVector. Amplicons from the first PCR reaction were used as templates for sequence- and ligation-independent cloning into antibody expression vectors. Recombinant monoclonal antibodies were produced and purified as previously described (Robbiani et al., 2020). During this process, several non-native mutations were introduced into the heavy and light chain fragments. Principally, the kappa (light) chain fragments were amplified using primers that reached into the framework region of the antibodies. The kappa primer introduces non-native amino acids into the kappa chain. In addition, the heavy chain was truncated at the C-terminus for the purpose of antibody production, being one amino acid shorter than what usually is present in IgG heavy chain constant regions 10* genomics

[0352] All procedures were performed while maintaining cells at 4°C. B cells were negatively selected from PBMCs with a pan-B-cell isolation kit. lOx Genomics V(D)J libraries were generated with the Chromium Single Cell 5' Library & Gel Bead Kit (lOx Genomics, cat. PN- 1000014) and Chromium Single Cell V(D)J Enrichment Kit, Human B Cell (lOx Genomics, cat. PN-1000016) as described in the lOx Genomics protocol. The 5’ expression library was sequenced with NovaSeq 6000 SI (100 cycles) (Illumina, cat. 20012865) and the V(D)J library was sequenced with NextSeq 500 / 550 Mid Output Kit v2.5 (300 Cycles) (Illumina, cat. 20024905).

[0353] Single-cell RNA-Seq processing

[0354] The UMI quantification and BCR clonotype assembly were performed using CellRanger (v7.1.0) and analyzed in R with Seurat (v4.3.0). Cells with a mitochondrial proportion greater than 10% and / or a feature count <200 or >2,500 were discarded. Sample batches were combined, normalized and scaled with SCTransform. Based on their gene expression profile, single cells were visualized in a lower dimensional space using Uniform Manifold Approximation and Projection (UMAP) clustering.

[0355] Biolayer interferometry

[0356] Biolayer interferometry assays were performed as previously described(Robbiani et al., 2020). In brief, the Octet Red instrument (ForteBio) was used at 30 °C with shaking at 1,000 r.p.m. Epitope binding assays were performed with protein A biosensor (ForteBio 18-5010), following the manufacturer’s protocol “classical sandwich assay” as follows: (1) Sensor check: sensors immersed 30 sec in buffer alone (buffer ForteBio 18-1105), (2) Capture 1stAb: sensors immersed 10 min with Abl at 10 pg / mL, (3) Baseline: sensors immersed 30 sec in buffer alone, (4) Blocking: sensors immersed 5 min with IgG isotype control at 10 pg / mL. (5) Baseline: sensors immersed 30 sec in buffer alone, (6) Antigen association: sensors immersed 5 min with RBD at 10 pg / mL. (7) Baseline: sensors immersed 30 sec in buffer alone. (8) Association Ab2: sensors immersed 5 min with Ab2 at 10 pg / mL. Curve fitting was performed using the Fortebio Octet Data analysis software (ForteBio). Affinity measurement of anti-SARS-CoV-2 IgGs binding were corrected by subtracting the signal obtained from traces performed with IgGs in the absence of WT RBD. The kinetic analysis using protein A biosensor (as above) was performed as follows: (1) baseline: 60sec immersion in buffer. (2) loading: 200sec immersion in a solution with IgGs 10 pg / ml. (3) baseline: 200sec immersion in buffer. (4) Association: 300sec immersion in solution with WT RBD at 20, 10 or 5 pg / ml (5) dissociation: 600sec immersion in buffer. Curve fitting was performed using a fast 1 : 1 binding model and the Data analysis software (ForteBio). Mean KD values were determined by averaging all binding curves that matched the theoretical fit with an R2value > 0.8.

[0357] Computational analyses of antibody sequences

[0358] Antibody sequences were trimmed based on quality and annotated using Igblastn v.1.14. with IMGT domain delineation system. Annotation was performed systematically using Change- O toolkit v.0.4.540 (Gupta et al., 2015). Clonality of heavy and light chain was determined using DefineClones.py implemented by Change-0 vO.4.5 (Gupta et al., 2015). The script calculates the Hamming distance between each sequence in the data set and its nearest neighbor. Distances are subsequently normalized and to account for differences injunction sequence length, and clonality is determined based on a cut-off threshold of 0.15. Heavy and light chains derived from the same cell were subsequently paired, and clonotypes were assigned based on their V and J genes using in-house R and Perl scripts. All scripts and the data used to process antibody sequences are publicly available on GitHub (github.com / stratust / igpipeline / tree / igpipeline2_timepoint_v2).

[0359] The frequency distributions of human V genes in anti-SARS-CoV-2 antibodies from this study was compared to 131,284,220 IgH and IgL sequences generated by(Soto et al., 2019) and downloaded from cAb-Rep(Guo et al., 2019), a database of human shared BCR clonotypes available at cab-rep.c2b2.columbia.edu / . We selected the IgH and IgL sequences from the database that are partially coded by the same V genes and counted them according to the constant region. The frequencies shown in FIGs. 8A-8I are relative to the source and isotype analyzed. We used the two-sided binomial test to check whether the number of sequences belonging to a specific IGHV or IGLV gene in the repertoire is different according to the frequency of the same IgV gene in the database. Adjusted p-values were calculated using the false discovery rate (FDR) correction. Significant differences are denoted with stars. Nucleotide somatic hypermutation and Complementarity-Determining Region (CDR3) length were determined using in-house R and Perl scripts. For somatic hypermutations, IGHV and IGLV nucleotide sequences were aligned against their closest germlines using Igblastn and the number of differences was considered to correspond to nucleotide mutations. The average number of mutations for V genes was calculated by dividing the sum of all nucleotide mutations across all participants by the number of sequences used for the analysis.

[0360] EXAMPLE 2

[0361] The memory B cell responses to three or four doses of an mRNA vaccine (mRNA-1273 (Modema) or BNT162b2 (Pfizer)) was studied in a cohort of 45 individuals, recruited between March 24, 2022, and September 7, 2022. The cohort was divided into two groups: (1) elderly individuals (n=31; median age = 77 years) who were vaccinated with 3 or 4 doses of a wild-type (WT) mRNA vaccine, 50% of whom were female; (2) younger individuals (n=14) who received 3 mRNA vaccine doses (WT), 68% of whom were female (for details see Table 1). None of the participants had a history of SARS-CoV-2 infection (FIG. 5A) and none experienced serious adverse events after vaccination. The vaccination and blood collection schedule for the two groups is depicted in FIG. 1A.

[0362] Plasma antibody binding and neutralization

[0363] Plasma IgG responses to the SARS-CoV-2 Wuhan-Hu- 1 (WT)- and Omicron BA.4 / 5- Receptor binding domain (RBD) were measured by enzyme-linked immunosorbent assay (ELISA)(Muecksch et al., 2022) 6.5-9.5 months after a third dose irrespective of whether they received a fourth vaccine dose. There was no significant difference in IgG binding titers to WT or Omicron BA.4 / 5 between the elderly and younger individuals whether they received 3 or 4 vaccine doses (FIGs. IB and 1C, and FIGs. 5B-5D and Table 1).

[0364] Plasma neutralizing activity was measured using HIV-1 pseudotyped with the WT SARS-CoV-2 spike protein (Cho et al., 2021; Wang et al., 2021c). The geometric mean half- maximal neutralizing titer (NT50) for elderly individuals after the third dose was equivalent to that of the younger individuals (FIG. ID and Table 1). Although it did not reach statistical significance, there was a small increase in the geometric mean NT50 against WT in the elderly after the fourth vaccine dose, and a significant 2.8-fold increase compared to younger individuals who received a third dose (Bar-On et al., 2022; Regev-Yochay et al., 2022) (p=0.007, FIG. ID). Finally, there was no correlation between age and plasma neutralizing titers (FIG. 5D).

[0365] Plasma neutralizing activity was also assessed against Omicron BA. l, BA.2, BA.4 / 5 and BA.2.75.2 and XBB.1.5 variants. The geometric mean plasma NT50S against the variants were not different between the elderly and younger individuals who received a third mRNA vaccine dose (FIGs. IE- II). However, the elderly who received a fourth dose showed a 3.2-fold increase in Omicron BA.4 / 5 neutralizing titers, when compared to younger vaccinees after 3 vaccine doses (FIG. 1G). Notably, Omicron XBB.1.5 showed the highest neutralization resistance of all variants tested. Despite advanced age, the plasma from elderly individuals who had at least three mRNA vaccine doses showed comparable neutralizing activity to plasma from younger individuals against all variants tested.

[0366] Memory B cells

[0367] The memory B cell compartment in younger mRNA vaccinated individuals contains a diverse collection of B cells that when challenged can produce antibodies which neutralize a variety of different viral variants (Goel et al., 2021; Goel et al., 2022; Kim et al., 2022; Muecksch et al., 2022; Sette and Crotty, 2022; Turner et al., 2021; Wang et al., 2022a; Wang et al., 2021b). To examine the memory B cell compartment in the elderly we initially performed flow cytometry experiments using phycoerythrin (PE) and Alexa-Fluor-647 (AF647) labeled WT RBDs (FIG. 6A). Elderly and younger individuals showed similar relative percentages RBD- specific Memory B cells (MBCs) (FIG. 2A). However, the elderly had a smaller absolute number of B cells (p=0.029, fig. S2B) and a higher relative proportion of atypical or age-associated B cells (ABCs) (Cancro, 2020)(p=0.0007, FIGs. 6C and 6D). There was no correlation between the sampling interval and the frequency of ABCs (FIGs. 6E and 6FF). Thus, the absolute number of circulating RBD-specific memory B cells found in the elderly was significantly lower than in the younger cohort (p=0.008, FIG. 2B).

[0368] To compare the antibodies produced by memory B cells in the two cohorts, we obtained 567 and 519 paired heavy and light chain antibody sequences from 7 elderly and 5 younger individuals, respectively (FIG. 2C and FIGs. 6G-6I). Individuals in both groups showed expanded clones of memory B cells that expressed closely related IGHV and IGLV genes (FIGs. 2C and 2D). However, the anti-RBD memory repertoire was less diverse in the elderly in part due to a relative increase in the number of clonally related sequences (p=0.030, FIG. 2E; p=0.045, FIG. 2F). VH1-69, VH3-30, VH4-39 and VH4-30 were over-represented, and VH4-31, VH3-13 and VH3-9 under-represented among the elderly vaccinees (FIGs. 7A-7C). The biased B cell receptor (BCR) repertoire in the elderly was associated with more restricted overall V gene family member usage and increased clonality among randomly collected circulating B cells in elderly individuals (FIGs. 7D-7H). Thus, the RBD-specific memory B cell antibody repertoire in elderly vaccinees is smaller, and less diverse than that found in younger individuals.

[0369] To examine the specificity of the MBC antibodies we cloned and expressed 255 monoclonal antibodies (mAbs). We selected one representative mAb from each clone of expanded memory B cells, and at least 15 mAbs from individual memory B cells detected only once in each participant (Table 2). 91 and 164 monoclonal antibodies were obtained from younger and elderly vaccinees, respectively (Table 2). Each of the antibodies was tested for binding to WT-, Omicron BA. 4 / 5-, XBB- or XBB.1 ,5-RBDs by ELISA. All antibodies bound to WT RBD, and there was no significant difference in the ECsos among the groups (Fig. 3A and fig. S4A). The fraction of antibodies that bound to XBB.1.5 RBD was significantly smaller in elderly than younger vaccinees (FIG. 3B, p=0.0003), while no difference in the fraction of Omicron BA.4 / 5 or Omicron XBB-RBD binders between the two groups (FIG. 3B and FIGs. 8B-8D). Given the similarities in binding activity between the antibodies from third and fourth dose elderly vaccine recipients the two groups were pooled for subsequent analyses.

[0370] Memory B cell antibodies elicited after the 1st or 2nd vaccine dose typically target the Angiotensin-converting enzyme 2 (ACE2) binding surface of the RBD (Class 1 and 2) (Barnes et al., 2020; Brouwer et al., 2020; Cho et al., 2021; Dugan et al., 2021; Lan et al., 2020; Robbiani et al., 2020; Wang et al., 2021c). By contrast, antibodies recovered from memory B cells after the third vaccine dose are more likely to target less accessible and more conserved regions of the RBD (non-Class 1 and 2) (Muecksch et al., 2022). To analyze which epitopes are targeted by the memory antibodies isolated from elderly vaccinees we performed Biolayer Interferometry (BLI) competition experiments with 5 antibodies that bind to different epitopes on the RBD (Cl 05, C144, C135, C2172, C5078 for Class 1, 2, 3, 4, 5 respectively) (Barnes et al., 2020; He et al., 2022; Muecksch et al., 2022; Rogers et al., 2020). There was a modest but significant difference in the epitopes targeted by the memory antibodies obtained from the two groups which was accounted for by an increased representation of Class 1 / 2 and decreased representation of class 2 / 3 / 5 antibodies in the elderly (p = 0.048, FIG. 3C, and FIG. 8F). The difference is consistent with a relative increase representation of VH1 -69 and VH3-3O among RBD-binding antibodies in the elderly (Cl / 2: p=0.045, C2 / 3 / 5: p=0.033; FIG. 8G).

[0371] The antibodies obtained from the elderly individuals showed only a slight reduction in affinity for WT RBD than the antibodies obtained from the younger individuals (KD: 1.7 vs 3.3nM; p=0.001, FIG. 3D). The decrease in affinity was mainly associated with antibodies obtained from clonally expanded B cells (p=0.002, FIG. 3E) which also accumulated fewer somatic hypermutations in the elderly (p=0.01, FIG. 8H). While the somatic mutations in Classl / 2 antibodies were similar between the two cohorts, non-Class 1 / 2 antibodies from the elderly carried fewer somatic mutations than the young group. Thus, the results suggest that the elderly have less adaptability in their memory antibody responses (p=0.01, FIG. 81).

[0372] Neutralization potency and Breadth

[0373] 253 RBD binding antibodies were tested for neutralizing activity in a SARS-CoV-2 pseudotype-neutralization assay using WT, Omicron BA.l and BA.4 / 5 SARS-CoV-2 spikes(Wang et al., 2022b). Although there was no significant difference in antibody potency against WT or variant pseudoviruses between elderly and younger individuals (FIGs. 4A and 4B, and FIGs. 9A and 9B, and Table 2), the epitopes targeted by the neutralizing antibodies were different in the 2 groups. Neutralizing antibodies obtained from the elderly were slightly biased to recognize Class 1 and 2 epitopes irrespective of their breadth of activity against different variants (FIGs. 4C and 4D). In contrast, neutralizing antibodies that recognize Class 4- and 5- epitopes were enriched in younger individuals (FIG. 4C and FIGs. 9C and 9D).

[0374] Discussion

[0375] Memory B cells are essential contributors to rapid antibody production upon pathogen challenge (Kurosaki et al., 2015; Victora and Nussenzweig, 2022; Weisel and Shlomchik, 2017). Several clinical trials of passive antibody therapy have demonstrated that early administration of significant quantities of neutralizing antibodies is essential for averting the serious consequences of SARS-CoV-2 infection in susceptible individuals (Cohen, 2022; Focosi et al., 2022; Gupta et al., 2021; Hammond et al., 2022; Montgomery et al., 2022; Weinreich et al., 2021). The data provided herein indicate that elderly individuals that received a third or fourth dose of mRNA vaccine developed smaller absolute numbers of SARS-CoV-2 RBD-specific memory B cells that express a more limited antibody repertoire than younger vaccinees. This limitation along with a more limited T cell repertoire in elderly individuals (Britanova et al., 2014; Goronzy and Weyand, 2019; Sun et al., 2022) could contribute to a blunted neutralizing antibody response to infection and increased risk of serious outcomes in the elderly (Cerqueira-Silva et al., 2022).

[0376] Aging is associated with several different defects that impact humoral immunity. These include decreased B cell production in the bone marrow (Labrie et al., 2004; Miller and Allman, 2003; Stephan et al., 1998; Zharhary, 1988), smaller numbers of circulating memory B cells (Frasca et al., 2011; Paganelli et al., 1992), more limited germinal center responses (Kosco et al., 1989; Luscieti et al., 1980; Sage et al., 2015; Shankwitz et al., 2020; Szakal et al., 1990), and alterations in signaling in both B and T cells (Frasca et al., 2020; Mogilenko et al., 2022) as well as reduced number of innate immune cells (Sohrabi et al., 2021). Each of these could contribute to our observation that the elderly develop fewer anti-SARS-CoV-2 RBD-specific memory B cells.

[0377] Despite the overall decrease in the number of memory B cells, the neutralizing activity of individual memory antibodies in the elderly was not significantly different from that of the younger cohort. However, the memory repertoire in the elderly continued to be dominated by Class 1 / 2 antibodies after three vaccine doses while younger individuals evolved to produce memory that was focused on other epitope classes that include more conserved regions of the RBD that remain less mutated in current circulating variants (Cao et al., 2022; Muecksch et al., 2022). This observation is consistent with the finding that the immune response to influenza is also less adaptable in elderly individuals (Henry et al ., 2019). Why the focus of the anti-RBD response fails to diversify in the older individuals has not been determined, but could be a combination of the more limited B cell numbers, altered signaling and germinal center responses. In addition, diversification by epitope masking may be impaired due to lower levels of serum antibodies produced after the 1stand 2ndvaccine doses (Collier et al., 2021; Schaefer-Babajew et al., 2023; Tas et al., 2022; Walsh et al., 2020)

[0378] Vaccination for influenza is specifically tailored to the elderly by increasing the dose (Grohskopf et al., 2022). Although increased dose or a shorter interval between vaccinations for the elderly is not currently recommended practice, the data herein suggests that modifying SARS-CoV-2 vaccine regimens specifically for this at-risk population should be considered especially if they enhance memory B and T cell responses.

[0379] The foregoing examples and description of the preferred embodiments should be taken as illustrating, rather than as limiting the present invention as defined by the claims. As will be readily appreciated, numerous variations and combinations of the features set forth above can be utilized without departing from the present invention as set forth in the claims. Such variations are not regarded as a departure from the scope of the invention, and all such variations are intended to be included within the scope of the following claims. All references cited herein are incorporated by reference in their entireties.

[0380] Table 2 Sequences, RBD binding and neutralization of cloned recombinant antibodies

[0381] Table 2 (Continued). RBD binding and neutralization of cloned recombinant antibodies.

[0382]

[0383]

[0384]

[0385]

[0386] Table 3. Sequences of antibody C5078

[0387] CDR sequences can be determined using any of suitable numbering systems (e.g., Kabat,

[0388] Chothia, Enhanced Chothia, IMGT, and AHoAbM).

[0389] References

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[0411] Ill

Claims

CLAIMSWhat is claimed is:

1. An isolated anti-SARS-CoV-2 antibody or antigen-binding fragment thereof that binds specifically to a SARS-CoV-2 antigen.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the SARS-CoV-2 antigen comprises a spike polypeptide, and preferably wherein the SARS-CoV-2 antigen comprises a spike polypeptide of a human or an animal SARS-CoV-2.

3. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the SARS-CoV-2 antigen comprises the receptor binding domain (RBD).

4. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising HCDR1, HCDR2, and HCDR3 of a heavy chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 2 and LCDR1, LCDR2, and LCDR3 of a light chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 4.

5. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising a heavy chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising an amino acid sequence with at least 75% identity to the amino acid sequence of SEQ ID NO: 4.

6. The antibody or antigen-binding fragment thereof of any one of the preceding claims, comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 4.

7. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is capable of neutralizing a plurality of SARS-CoV-2 strains.

8. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is a multivalent antibody comprising(a) a first target binding site that specifically binds to an epitope within a spike polypeptide, and(b) a second target binding site that binds to an epitope on a different epitope on the spike polypeptide or a different molecule.

9. The antibody or antigen-binding fragment thereof of claim 8, wherein the multivalent antibody is a bivalent or bispecific antibody.

10. The antibody or the antigen-binding fragment thereof of any one of the preceding claims, further comprising a variant Fc constant region.

11. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a monoclonal antibody.

12. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a chimeric antibody, a humanized antibody, or humanized monoclonal antibody.

13. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody is a single-chain antibody, Fab or Fab2 fragment.

14. The antibody or antigen-binding fragment thereof of any one of the preceding claims, wherein the antibody or antigen-binding fragment thereof is detectably labeled or conjugated to a toxin, a therapeutic agent, a polymer, a receptor, an enzyme, or a receptor ligand.

15. The antibody or the antigen-binding fragment thereof of claim 14, wherein the polymer is polyethylene glycol (PEG).

16. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of any one of the preceding claims and optionally a pharmaceutically acceptable carrier or excipient.

17. The pharmaceutical composition of claim 16, wherein the pharmaceutical comprises two or more of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15.

18. The pharmaceutical composition of any one of claims 16 to 17, further comprising a second therapeutic agent.

19. The pharmaceutical composition of claim 18, wherein the second therapeutic agent comprises an anti-inflammatory drug or an antiviral compound.

20. The pharmaceutical composition of claim 19, wherein: (a) the antiviral compound comprises: a nucleoside analog, a peptide, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA dependent RNA polymerase; or (b) the antiviral compound is selected from: acyclovir, gancyclovir, vidarabine, foscamet, cidofovir, amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, and an interferon, and preferably wherein the interferon is an interferon- a or an interferon- .

21. Use of the pharmaceutical composition of any one of claims 16 to 20 in the preparation of a medicament for the diagnosis, prophylaxis, treatment, or combination thereof of a condition resulting from a SARS-CoV-2.

22. A nucleic acid molecule encoding a polypeptide chain of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15.

23. A vector comprising the nucleic acid molecule of claim 22.

24. A cultured host cell comprising the vector of claim 23.

25. A method of preparing an antibody, or antigen-binding portion thereof, comprising: obtaining the cultured host cell of claim 24; culturing the cultured host cell in a medium under conditions permitting expression of a polypeptide encoded by the vector and assembling of an antibody or fragment thereof; and purifying the antibody or fragment from the cultured cell or the medium of the cell.

26. A kit comprising a pharmaceutically acceptable dose unit of the antibody or antigenbinding fragment thereof of any one of claims 1 to 15 or the pharmaceutical composition of any one of claims 16 to 20.

27. A method of neutralizing SARS-CoV-2 in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or a therapeutically effective amount of the pharmaceutical composition of any one of claims 16 to 20.

28. A method of preventing or treating a SARS-CoV-2 infection, comprising administering to a subject in need thereof a therapeutically effective amount of the antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or a therapeutically effective amount of the pharmaceutical composition of any one of claims 16 to 20.

29. A method of neutralizing SARS-CoV-2 in a subject, comprising administering to a subject in need thereof a therapeutically effective amount of a first antibody or antigen-binding fragment thereof and a second antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or a therapeutically effective amount of the pharmaceutical composition of any one of claims 16 to 20, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen binding fragment thereof exhibit synergistic activity.

30. A method of preventing or treating a SARS-CoV-2 infection, comprising administering to a subject in need thereof a therapeutically effective amount of a first antibody or antigenbinding fragment thereof and a second antibody or antigen-binding fragment thereof of any one of claims 1 to 15 or a therapeutically effective amount of the pharmaceutical composition of any one of claims 16 to 20, wherein the first antibody or antigen-binding fragment thereof and the second antibody or antigen binding fragment thereof exhibit synergistic activity.

31. The method of any one of claims 27 to 28, further comprising administering to the subject a therapeutically effective amount of a second therapeutic agent or therapy.

32. The method of claim 31 , wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising a HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 and 4 and the second therapeutic agent or therapy comprises an antibody or antigen-binding fragment thereof comprising a HCVR / LCVR amino acid sequence pair, wherein the antibody or antigen-binding fragment thereof is different from the second therapeutic agent or therapy.

33. The method of any one of claims 31 to 32, wherein the antibody or antigen-binding fragment thereof is administered before, after, or concurrently with the second therapeutic agent or therapy.

34. The method of any one of claims 31 to 33, wherein the second therapeutic agent comprises an anti-inflammatory drug or an antiviral compound.

35. The method of claim 34, wherein: (a) the antiviral compound comprises: a nucleoside analog, a peptide, an oligopeptide, a polypeptide, a protease inhibitor, a 3C-like protease inhibitor, a papain-like protease inhibitor, or an inhibitor of an RNA dependent RNA polymerase; or (b) the antiviral compound is selected from: acyclovir, gancyclovir, vidarabine, foscamet, cidofovir, amantadine, ribavirin, trifluorothymidine, zidovudine, didanosine, zalcitabine, and an interferon, and preferably wherein the interferon is an interferon-a or an interferon-p.

36. The method of any one of claims 29 to 30, wherein the first antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region comprising a HCVR / LCVR amino acid sequence pair of SEQ ID NOs: 2 and 4 and the second therapeutic agent or therapy comprises an antibody or antigen-binding fragment thereof comprising a HCVR / LCVR amino acid sequence pair, wherein the antibody or antigen-binding fragment thereof is different from the second antibody or antigen-binding fragment thereof.

37. The method of any one of claims 29 to 30 and 36, wherein the first antibody or antigenbinding fragment thereof is administered before, after, or concurrently with the second antibody or antigen-binding fragment thereof.

38. The method of any one of claims 27 to 37, wherein the antibody or antigen-binding fragment thereof is administered to the subject intravenously, subcutaneously, or intraperitoneally.

39. The method of claim 28 or 30, wherein the antibody or antigen-binding fragment thereof is administered prophylactically or therapeutically.

40. A method for detecting the presence of SARS CoV-2 in a sample comprising the steps of: contacting a sample with the antibody or antigen-binding fragment thereof of any one of claims 1 to 15; and determining binding of the antibody or antigen-binding fragment to one or more SARS CoV-2 antigens, wherein binding of the antibody to the one or more SARS CoV-2 antigens is indicative of the presence of SARS CoV-2 in the sample.

41. The method of claim 40, wherein the SARS-CoV-2 antigen comprises a spike polypeptide of a human or an animal SARS-CoV-2, and preferably wherein the SARS-CoV-2 antigen comprises the receptor binding domain (RBD).

42. The method of any one of claims 40 to 41, wherein the antibody or antigen-binding fragment thereof is conjugated to a label, and preferably wherein the label is selected from a fluorescent label, a chemiluminescent label, a radiolabel, and an enzyme.

43. The method of any one of claims 40 to 42, wherein the step of detecting comprises contacting a secondary antibody with the antibody or antigen-binding fragment thereof and wherein the secondary antibody comprises a label, preferably wherein the step of detecting comprises detecting fluorescence or chemiluminescence, and further preferably wherein the step of detecting comprises a competitive binding assay or ELISA.

44. The method of any one of claims 40 to 43, wherein the sample is a blood sample, and preferably wherein the method comprises binding the sample to a solid support, preferablywherein the solid support is selected from microparticles, microbeads, magnetic beads, and an affinity purification column.