WO2024112818A1

WO2024112818A1 - Engineered anti-sars-cov-2 antibodies and uses thereof

Info

Publication number: WO2024112818A1
Application number: PCT/US2023/080792
Authority: WO
Inventors: Davide Corti; Nadine CZUDNOCHOWSKI; Chiara D’AVINO; Susannah Karen DAVIS; Martin Anibal Orecchia; Christopher James Plummer; Christian SALIBA; Martin James SCOTT; Gyorgy Snell
Original assignee: Humabs Biomed Sa; Vir Biotechnology, Inc.; Glaxo Wellcome Uk Limited
Priority date: 2022-11-22
Filing date: 2023-11-21
Publication date: 2024-05-30

Abstract

The instant disclosure provides antibodies and antigen-binding fragments thereof that can bind to a SARS-CoV-2 antigen and, in certain embodiments, are capable of neutralizing a SARS-CoV-2 infection. In certain embodiments, an antibody or antigen-binding fragment of the present disclosure has one or more improved property, such as improved binding and/or neutralization, as compared to an antibody or antigen-binding fragment having the six CDRs of, or the VH and VL amino acid sequences as set forth, in SEQ ID NOs.:30 and 34, respectively, or as set forth in SEQ ID NOs.:22 and 26, respectively. In certain embodiments, the presently disclosed antibodies are capable of binding to S proteins of multiple sarbecoviruses and/or neutralizing infection by multiple sarbecoviruses. In some embodiments, a sarbecovirus is from clade 1a, clade 1b, clade 2, or clade 3. In some embodiments, a sarbecovirus comprises a SARS-CoV-2, a SARS-CoV-2 G504D variant, a SARS-CoV delta variant, a SARS-CoV omicron variant, a SARS-CoV, or any combination thereof. Also provided are polynucleotides that encode an antibody or antigen-binding fragment, vectors and host cells that comprise a polynucleotide, pharmaceutical compositions, and methods of using the presently disclosed antibodies, antigen-binding fragments, polynucleotides, vectors, host cells, and compositions to treat or diagnose a sarbecovirus infection, such as a SARS-CoV-2 infection.

Description

ENGINEERED ANTI-SARS-COV-2 ANTIBODIES AND USES THEREOF

REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

The contents of the electronic sequence listing (444WO_SeqListing.xml; Size: 498,301 bytes; and Date of Creation: November 18, 2023) is herein incorporated by reference in its entirety.

BACKGROUND

A novel betacoronavirus emerged in late 2019. As of March 6, 2023, approximately 676 million cases of infection by this virus (termed, among other names, SARS-CoV-2), were confirmed worldwide, and had resulted in approximately 6.87 million deaths. Modalities for preventing, treating, and diagnosing coronavirus infections, including by emerging SARS-CoV-2 variants, are needed.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows results from an in vitro neutralization of infection assay against WT (Wuhan-1 D614G) and Omicron BQ.1.1 SARS-CoV-2 VSV-pp by S309- N55Q_MLNS antibody (Sotrovimab) and certain engineered variants thereof.

Figures 2A-2B show variable domain sequence alignments of S309- N55Q_MLNS (Sotrovimab) and certain engineered variants thereof (S309-937, S309- 941, S309-1066, S309-2951, S309-2923, S309-3016, and S309-3126).

Figure 2C summarizes neutralization IC50-values of parental S309-N55Q and certain generated variants thereof against BQ.1.1 VSV. S309-846 is specifically indicated.

Figures 3A-3G show variable domain amino acid sequences and example polynucleotide sequences encoding these, of certain engineered variant antibodies of the present disclosure. “VK” indicates the light chain variable domain (kappa).

Figures 4A-4X show variable domain amino acid sequences and (except for Figures 4U-4X) example polynucleotide sequences encoding these, of certain engineered variant antibodies of the present disclosure. “VK” indicates the light chain variable domain (kappa).

Figures 5A-7 show additional characterization of variant antibodies. Figure 5A shows (left) neutralization IC50 values (ng/mL) by twenty-seven engineered variants vs. a panel of SARS or SARS-CoV-2 VSV pseudoviruses and (right) fold-change in neutralization IC50 of the parental antibody (S309-N55Q) versus the engineered variant. Figures 5B-5D show neutralization curves of certain engineered variant antibodies. Figure 5E shows shows (left) neutralization IC50 values (ng/mL) by certain engineered variant antibodies vs. a panel of SARS or SARS-CoV-2 VSV pseudoviruses and (right) fold-change in neutralization IC50 of the parental antibody (S309-N55Q) versus the variant. In the left panel, “BA.5-S8” and “BA.2.275.2-S8” are engineered viruses with mutations in BA.5 or BA.2.75.2 backbones; see e.g. Figure 5A in Cao et al., bioRxiv 2022; doi.org/10.1101/2022.09.15.507787). Figures 6A and 6B show binding affinities (Kd, in nM) of engineered variant antibodies and the parental antibody (as well, in Figure 6B, of purified S309 rlgG) to a panel of Receptor Binding Domains (assessed by SPR). The S8 variant (BA.5 and BA.2.75.2 backbones tested) is described in Cao et al., bioRxiv 2022; doi.org/10.1101/2022.09.15.507787. Figure 7 provides a table summarizing binding affinity and neutralization potency foldimprovement (versus the parental S309-N55Q) of engineered variant antibodies, along with the mutations in the (IMGT + CCG) CDRs of the variants as compared to the parental antibody. The C-terminal amino acid in CDRH2 (“G”) is not shown.

Figure 8 shows binding of certain antibodies to SARS-CoV-2 viruses bearing escape mutations.

Figure 9 shows binding affinity correlations between SARS and SARS-CoV-2 strains based on studies using certain antibodies of the present disclosure.

Figure 10 shows binding (SPR) of certain engineered variant antibodies (selected based on neutralization studies) versus a panel of RBDs.

Figure 11 shows fold-change in binding of the indicated antibodies (S309 N55Q and certain engineered variants) compared to purified S309 IgG. Figure 12 summarizes neutralization IC50-values of parental S309-N55Q and certain engineered variant antibodies against a panel of SARS or SARS-CoV-2 VSV pseudoviruses.

Figures 13A-13B show neutralization IC50 fold change graphs of certain engineered variant antibodies versus the parental antibody S309-N55Q, against BQ.1.1 VSV.

Figures 14A-14G show neutralization activity of certain engineered variant antibodies tested versus Wuhan-Hu-1 (with D614G mutation) and BQ.1.1 VSV viruses. Boxes indicate antibodies that were selected for a second NT test against the following VSV viruses: Wuhan-Hu-1 D614G, BQ.1.1, BA.5, BA.5 S8, BA.5 E340A, BA.5 P337T, BA.2.75.2 S8, BA.2.75-R346T, BA.4.6, BF.7, XBB. l, SARS-CoV.

Figure 15A-15B plot neutralization vs. binding against SARS-CoV-2 and SARS-CoV-1 pseudoviruses for certain engineered S309-N55Q variant antibodies from a first cycle (“la”) and a second cycle (“lb”) of engineering and selection.

Figure 16A-16B plot fold-change improvement in neutralization and binding of certain engineered variant antibodies versus the S309-N55Q parental antibody against SARS-CoV-2 and SARS-CoV-1 pseudoviruses.

Figure 17 shows sensorgram data for antibodies that bound weakly (bottom row) or did not bind (top row) to RBDs with escape mutations.

Figures 18A-18H show graphs of neutralization vs. binding against SARS- CoV-2 and SARS-CoV-1 pseudoviruses for engineered variant antibodies from cycle lb as described for Figures 15A amd 15B. Parental S309 N55Q (supernatant) is indicated.

Figures 19A-19B show neutralizing activity of 56 engineered variant antibodies as fold improvement to the parental S309-N55Q against BA.5.

Figure 20 shows neutralizing activity of certain engineered variant antibodies versus a panel of SARS-CoV-2 and SARS-CoV-1 pseudoviruses (n=32). “VIR-7831” = parental antibody S309-N55Q MLNS aka sotrovimab. Variants 828, 846, and 838 were selected for recombinant expression in mammalian cells. Figure 21A shows neutralization IC50 values (ng/mL) by S309-N55Q and certain engineered variant antibodies vs. a panel of SARS or SARS-CoV-2 VSV pseudoviruses. Figure 21B shows neutralization data for certain engineered variant antibodies, sorted based on BA.5.

Figure 22 shows correlation of neutralizing potency of certain engineered variant antibodies across different SARS-CoV-2 strains. VIR-7831 aka S309-N55Q- MLNS is shown as a comparator. Letter-number combinations (e.g., “C6”, “B5”, “B7”) indicate antibodies according to plate wells as in Figure 21B.

Figure 23 shows (top) neutralization IC50 values and (bottom) corresponding plate wells of certain engineered variant antibodies with high performance from cycle la.

Figure 24 shows SPR experimental setup for assessing binding of certain engineered variant antibodies x 8 RBDs. Well position E12 is the parental antibody. Well position D 12 is a negative control (an anti-RSV antibody).

Figure 25 shows a graph of neutralization vs binding of certain engineered variant antibodies against SARS-CoV-2 and SARS-CoV-1 pseudoviruses. “B07” = variant 846.

Figure 26 shows a graph of correlation between neutralization and affinity for certain engineered variant antibodies plotted as fold-change improvement to parental antibody S309-N55Q.

Figure 27 shows binding affinity correlations between SARS and SARS-CoV-2 strains from studies using certain engineered variant antibodies of the present disclosure.

Figure 28 shows neutralization vs. k-off, neutralization vs. k-on, and neutralization vs. binding (KD) correlation, of certain engineered variant antibodies of the present disclosure. “B07” = variant 846. “E12” = parental antibody S309-N55Q.

Figure 29 shows neutralization data for certain engineered variant antibodies (production cell supernatant) of the present disclosure. Briefly, a VSV pseudovirus neutralization assay was performed using Vero E6 cells. Vero E6 cells were grown in DMEM supplemented with 10% FBS and seeded into clear bottom white 96 well plates (PerkinElmer, 6005688) at a density of 20,000 cells per well. The next day, monoclonal antibodies were serially diluted in pre-warmed complete medium, mixed with pseudoviruses and incubated for 1 h at 37 °C in round bottom polypropylene plates. Medium from cells was aspirated and 50 pl of virus-monoclonal antibody complexes was added to cells, which were then incubated for 1 h at 37 °C. An additional 100 pl of prewarmed complete medium was then added on top of complexes and cells were incubated for an additional 16-24 h. Conditions were tested in duplicate wells on each plate and four wells per plate contained untreated infected cells (defining the 0% of neutralization, ‘MAX RLU’ value) and uninfected cells (defining the 100% of neutralization, ‘MIN RLU’ value). Virus-monoclonal antibody-containing medium was then aspirated from cells and 100 pl of a 1 :2 dilution of SteadyLite Plus (PerkinElmer, 6066759) in PBS with Ca2+ and Mg2+ was added to cells. Plates were incubated for 15 min at room temperature and then were analyzed on the Synergy -Hl (Biotek). The average relative light units (RLUs) of untreated infected wells (MAX RLUave) was subtracted by the average of MIN RLU (MIN RLUave) and used to normalize percentage of neutralization of individual RLU values of experimental data according to the following formula: (1 - (RLUx - MIN RLUave)/ (MAX RLUave - MIN RLUave)) x 100. Data were analyzed and visualized with Prism. IC50 values were calculated from the interpolated value from the log(inhibitor) versus response, using variable slope (four parameters) nonlinear regression with an upper constraint of <100.

Figure 30 shows, in table form, neutralization data for certain engineered variant antibodies of the present disclosure including IC50 values and fold change improvement vs. parental S309-N55Q, as in Figure 29. Antibodies were expressed as recombinant IgGlml7 with M428L and N434S Fc mutations.

Figure 31 summarizes IC50 values for S309-N55Q and certain engineered variant antibodies against the indicated viruses.

Figure 32 summarizes IC50 values for S309-N55Q and certain engineered variant antibodies against the indicated viruses.

Figures 33A and 33B summarize certain engineered variant antibodies of the present disclosure and neutralization by these against SARS-CoV-2 viruses. “CNC” = curve not closed. “NN” = not neutralizing. “ND” = not defined. Shading = selected variants for further assessment based on neutralization potency on BN.1.

Figures 34A-34D show VH and VL amino acid sequences of certain engineered variant antibodies of the present disclosure, and examples of polynucleotide sequences encoding these. Specific amino acid substitution mutations (e.g., N55S) are identified with reference to a corresponding parental antibody sequence (e.g., which has a native N at position 55 in the VH).

Figure 34A shows sequences for S309.887-vl5.1 (comprising S309.887-VH15 (comprising the mutations N55S-N57K-N59Y-A104T-E108Q-L1101) and S309.CLUS- 25-VK1 (comprising the mutations T28M-V29M-S31T-T32N)). In some embodiments, this antibody is provided as IgGlml7,l with M428L and N434S mutations in the Fc. In some embodiments, the light chain further comprises wild-type human IgGKC (Km3).

Figure 34B shows sequences for S309.887-vl9.1 (comprising S309.887-VH19 (comprising the mutations N55S-N57K-N59Y-A104S-E108Q-F114I-N116H) and S309.CLUS-25-VK1 (comprising the mutations T28M-V29M-S31T-T32N)). In some embodiments, this antibody is provided as IgGlml7,l with M428L and N434S mutations in the Fc. In some embodiments, the light chain further comprises wild-type human IgGKC (Km3).

Figure 34C shows sequences for S309.2951-v2.1 (comprising S309.2951-VH2 (comprising the mutations N57K-N59I-A104T-E108Q-L1101) and S309.CLUS-25- VK1 (comprising the mutations T28M-V29M-S31T-T32N)). In some embodiments, this antibody is provided as IgGlml7,l with M428L and N434S mutations in the Fc. In some embodiments, the light chain further comprises wild-type human IgGKC (Km3).

Figure 34D shows sequences for S309.SBD-v8.1.1 (comprising S309.SBD- VH8 (comprising the mutations N55Q-N57K-N59E-A104S-E108Q-F114I-N116H) and S309.CLUS-25-VK1.1 (comprising the mutations M28T-V29M-S31T-T32N)). In some embodiments, this antibody is provided as IgGlml7,l with M428L and N434S mutations in the Fc. In some embodiments, the light chain further comprises wild-type human IgGKC (Km3).

Figure 35 shows amino acid sequences of (top) human IGHG1 (Glml7, 1) with M428L and N434S mutations in the Fc and (bottom) wild-type human IgGKC (Km3), and examples of polynucleotide sequences encoding these. The polynucleotide sequences are codon-optimized for expression in a Chinese Hamster Ovary (CHO) cell line. Any of the presently disclosed antibodies or antigen-binding fragments can comprise these amino acid sequences.

Figure 36 shows sequences for S309. S309.887-v23.1 (comprising S309.887- VH23 (comprising the mutations N55Q-N57K-N59Y-A104S-E108Q-F114I-N116H) and S309.CLUS-25-VK1 (comprising the mutations T28M-V29M-S31T-T32N)). This antibody can be expressed as, for example, IgGlml7,l with M428L and N434S mutations in the Fc. In some embodiments, the light chain further comprises wild-type human IgGKC (Km3).

Figure 37 shows (top) light chain and (bottom) heavy chain amino acid sequences for antibody S309.887-N55Q-rIgGlml7,l-LS (also referred-to herein as S309.887-N55Q-rIgGlml7,l-MLNS).

Figure 38 shows (top) heavy chain (variable and constant region) and (bottom) light chain (variable and constant region) sequences for antibody S309.887-vl9.1- rIgGlml7,l-LS (also referred-to herein as S309.887-vl9.1-rIgGlml7,l-MLNS).

Figure 39 shows (top) heavy chain (variable and constant region) and (bottom) light chain (variable and constant region) sequences for antibody S309.887-v23.1- rIgGlml7,l-LS (also referred-to herein as S309.887-v23.1-rIgGlml7,l-MLNS).

DETAILED DESCRIPTION

Provided herein are engineered antibodies and antigen-binding fragments that are capable of binding to a coronavirus antigen (e.g., SARS-CoV-2, such as binding a surface glycoprotein and/or a RBD thereof, as described herein, optionally in a SARS- CoV-2 virion and/or expressed on the surface of a host cell, such as a cell infected by the SARS-CoV-2 coronavirus). In certain embodiments, an antibody or antigen- binding fragment of the present disclosure has one or more improved property, such as binding and/or neutralization, as compared to an antibody or antigen-binding fragment having the six CDR amino acid sequences set forth in SEQ ID NOs.:31-33 and 35-37, or the VH and VL amino acid sequences as set forth in SEQ ID NOs.:30 and 34, respectively (z.e., as compared to an antibody or antigen-binding fragment comprising the six CDRs or the VH and VL amino acid sequences of Sotrovimab, also referred to herein as S309-N55Q or S309-N55Q_MLNS). In certain embodiments, an antibody or antigen-binding fragment of the present disclosure has one or more improved property, such as binding and/or neutralization, as compared to an antibody or antigen-binding fragment having the six CDR amino acid sequences set forth in SEQ ID NOs.:23-25 and 27-29, or the VH and VL amino acid sequences as set forth in SEQ ID NOs.:22 and 26, respectively (z.e., as compared to an antibody or antigen-binding fragment comprising the VH and VL amino acid sequences S309, the parental antibody of Sotrovimab).

Certain embodiments provide engineered variant antibodies and antigen-binding fragments derived from Sotrovimab or S309 (there being only one difference overall (N55Q in VH of Sotrovimab as compared to N55 in S309) between the variable domains of Sotrovimab and those of S309), and in some embodiments possess one or more improved properties compared to Sotrovimab and/or S309. Sotrovimab is an engineered IgGlK variant of a human monoclonal antibody ("S309") identified from a memory B cell obtained from a recovered SARS CoV patient. S309 binds to immobilized SARS CoV-2 RBD and to the ectodomain trimer of the S glycoprotein with sub-picomolar and picomolar avidities, respectively (see Pinto D et al. "Crossneutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody" Nature 583 doi.org/10.1038/s41586-020-2349-y (2020)). S309 is cross-reactive to SARS-CoV and SARS-CoV-2, and potently neutralizes SARS-CoV-2 and SARS-CoV pseudoviruses as well as certain live SARS-CoV-2 viruses. Sotrovimab has been used to treat COVID-19 and received emergency use authorization in the United States for treating COVID-19. In some embodiments, an antibody or antigen-binding fragment of the present disclosure has a similar or improved in vitro neutralization potency (e.g. a lower neutralization IC50 as measured by using a SARS-CoV-2 pseudovirus e.g. VSV) infection assay) against SARS-CoV-2 Wuhan-Hu- 1 (optionally containing the D614G mutation), as compared to an antibody or antigen-binding fragment having HCDRs and the LCDRs of, or the VH and VL amino acid sequences as set forth in SEQ ID NOs.:30 and 34, respectively. In some embodiments, an antibody or antigen-binding fragment of the present disclosure has an improved in vitro neutralization potency e.g. a lower neutralization IC50 as measured by using a SARS-CoV-2 pseudovirus e.g. VSV) infection assay) against SARS-CoV-2 BQ.1.1 (optionally containing the D614G mutation), as compared to an antibody or antigen-binding fragment having HCDRs and the LCDRs of, or the VH and VL amino acid sequences as set forth in SEQ ID NOs.:30 and 34, respectively.

It will be understood that for purposes of comparison, the subject antibody or antigen-binding fragment and the antibody or antigen-binding fragment comprising the CDRs or VH and VL amino acid sequences of Sotrovimab (or S309) will be identical or substantially identical, differing only in their variable domain amino acid sequences. For example, some comparisons may include both the subject antibody or antigenbinding fragment and the antibody or antigen-binding fragment comprising the VH and VL amino acid sequences of Sotrovimab expressed as recombinant human IgGlK with M428L and N434S mutations in the Fc.

In some embodiments, an antibody or antigen-binding fragment of the present disclosure neutralizes infection by Wuhan Hu-1 (optionally containing the D614G mutation) in a pseudovirus (e.g. VSV, using VeroE6 cells as target cells) with an IC50 (ng/mL) value of less than 40, of less than 35, of less than 30, of less than 25, of less than 20, or of about 36, or about 20, or about 17, or about 30, or about 23, or of between 17 and 36, inclusive, or of between 17 and 30, inclusive, or of between 17 and 23, inclusive. By comparison, in this assay, Sotrovimab neutralized Wuhan-Hu- 1 (with D614G) with an IC50 of 25 ng/mL. In some embodiments, an antibody or antigenbinding fragment of the present disclosure neutralizes infection by BQ.1.1 in a pseudovirus (e.g. VSV, using VeroE6 cells as target cells) with an IC50 (ng/mL) value of less than 400, of less than 350, of less than 300, of less than 250, of less than 200, or of about 318, or about 294, or about 289, or about 246, or about 189, or about 180, or of between 180 and 320, inclusive, or of between 180 and 300, inclusive, or of between 180 and 250, inclusive. By comparison, in this assay, Sotrovimab neutralized BQ.1.1 with an IC50 of 2588 ng/mL.

In some embodiments, an antibody, when expressed as recombinant human IgGlml7,l with M428L and N434S mutations in the heavy chain, and a human kappa light chain constant domain, has a neutralization IC50 for Wuhan-Hu-1, for SARS- CoV-2 Delta variant, for SARS-CoV-2 BQ1.1, for SARS-CoV-2 XBB.1.5, for SARS- CoV-2 BN. l, for SARS-CoV-2 CH.1.1, and/or for SARS-CoV-2 BR.2 that is lower than that of S309-N55Q-rIgGlml7-MLNS by the same assay.

In some embodiments, the antibody has a neutralization IC50 that is improved (z.e. is lower than), relative to the neutralization IC50 of S309-N55Q-rIgGlml7-MLNS by the same assay, by, or by about: 1.4-fold, 2.9-fold, 6.3-fold, 3.4-fold, 2.6-fold, 3.9- fold, 5.0-fold, or 3.3-fold, for SARS-CoV-2 Wuhan-Hu-1; 2.6, 3.3, 1.9, 2.1, 3.6, 2.6, or 2.3 for SARS-CoV-2 Delta; 5.1, 16.9, 17.6, 35.9, 4.4, 41.1, 33.1, or 21.7 for SARS- CoV-2 BQ.1.1; 3.8, 5.6, 6.8, 4.6, 6.8, 5.9, or 4.0 for SARS-CoV-2 XBB.1.5; 5.4, 38.5, 48.2, 31.3, 31.7, 35.9, or 42.8 for SARS-CoV-2 BN.l; 3.1, 5.0, 6.4, 4.4, 5.5, 7.4, or 4.3 for SARS-CoV-2 CH.1.1, and/or 3.6, 4.9, 6.8, 4.3, 4.5, 4.4, or 5.0 for SARS-CoV-2 BR2.

For example, as shown in Figure 30, experiments testing neutralization IC50 of the antibody S309.887-vl9.1-rIgGlml7,l-LS showed the following improvements (fold-change) as compared to S309-N55Q-rIgGlml7,l-LS: 3.4 for SARS-CoV-2 Wuhan-Hu-1; 1.9 for SARS-CoV-2 Delta; 35.9 for SARS-CoV-2 BQ.1.1; 6.8 for SARS-CoV-2 XBB.1.5; 48.2 for SARS-CoV-2 BN. l; 6.4 for SARS-CoV-2 CH.1.1, and 6.8 for SARS-CoV-2 BR.2. As shown in Figure 33B, in another set of experiments, S309.887-vl9.1-rIgGlml7,l-LS showed the following improvements (fold-change) as compared to S309-N55Q-rIgGlml7,l-LS: 4.7 for SARS-CoV-2 Wuhan-Hu-1; 63.7 for SARS-CoV-2 BN.l; 8.5 for SARS-CoV-2 XBB.1.5; and 5.4 for SARS-CoV-2 BA.2.75-S8.

S309.887-vl9.1-rIgGlml7,l-LS comprises the following VH sequence: QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWIS TYSGKTYYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYCARDYTRGS WFGQSLIGGIDHWGQGTLVTVSS (SEQ ID NO.:360), and the following VL sequence EIVLTQSPGTLSLSPGERATLSCRASQMMSTNSLAWYQQKPGQAPRLLIYGVSS RASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDTSLTFGGGTKVEIK (SEQ ID NO.:365). The heavy chain amino acid sequence of S309.887-vl9.1- rIgGlml7,l-LS is QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWIS TYSGKTYYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYCARDYTRGS WFGQSLIGGIDHWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKD YFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVI<FNWYVDGVEVHNAI<TI<PREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVLHEALHSHYTQKSLSLSPGK (SEQ ID NO.:404, though it will be understood that the C-terminal lysine or glycine-lysine may be present or absent) and the light chain amino acid sequence of S309.887-vl9.1-rIgGlml7,l-LS is EIVLTQSPGTLSLSPGERATLSCRASQMMSTNSLAWYQQKPGQAPRLLIYGVSS RASGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDTSLTFGGGTKVEIKRT VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQES VTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO.:405).

In certain embodiments, presently disclosed antibodies and antigen-binding fragments can neutralize a coronavirus infection in an in vitro model of infection and/or in a human subject. In certain embedments, presently disclosed antibodies and antigen- binding fragments are capable of binding to and/or neutralizing two, three, or more sarbecoviruses and/or SARS-CoV-2 viruses, such as, for example, a sarbecovirus of clade la, a sarbecovirus of clade lb, a sarbecovirus of clade 2, a sarbecovirus of clade 3, and/or a variant of SARS-CoV-2. In some embodiments, a sarbecovirus is from clade la, clade lb, clade 2, or clade 3. In some embodiments, a sarbecovirus comprises a SARS-CoV-2, a SARS-CoV-2 G504D variant, a SARS-CoV delta variant, a SARS- CoV-2 omicron variant, a SARS-CoV-2 BA.2, a SARS-CoV-2 BA.4, a SARS-CoV-2 BA.5, a SARS-CoV-2 BA.4/5, a SARS-CoV-2 BA.4.6, a ZC45, a BGR08, a SARS- CoV, or any combination thereof.

In certain embodiments, presently disclosed antibodies and antigen-binding fragments can neutralize infection by any one, any two, any three, any four, any five, any six, any seven, or all eight of the following: SARS-CoV-2 Wuhan-Hu- 1; SARS- CoV-2 Delta; SARS-CoV-2 BQ.1.1; SARS-CoV-2 XBB.1.5; SARS-CoV-2 BN.1; SARS-CoV-2 CH.1.1; SARS-CoV-2 BR.2; SARS-CoV-2 BA.2.75-S8.

Amino acid sequences of certain antibodies and antigen-binding fragments are provided herein, as are non-limiting examples of polynucleotide sequences encoding at least a portion of an antibody or antigen-binding fragment. Figures 3A-4X and 34A-39 show variable domain amino acid sequences (and examples of polynucleotide sequences encoding these); in Figures 3A-4X and 34A-39, the CDR amino acid sequences are shown in bold (as determined using a combination of IMGT and CCG definitions, as described further herein). In certain embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3 independently selected from a CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 of any of the antibodies indicated in Figures 3A-4X, 34A-37, 38, and 39. For example, in some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-937, as shown in Figure 3A. In some embodiments, an antibody or antigenbinding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-2951, as shown in Figure 3B. In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309- 1066, as shown in Figure 3C. In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3016, as shown in Figure 3D. In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-941, as shown in Figure 3E. In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3126, as shown in Figure 3F. In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-2923, as shown in Figure 3G.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-877, as shown in Figure 4A.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-887, as shown in Figure 4B.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-2885, as shown in Figure 4C.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-864, as shown in Figure 4D.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-1199, as shown in Figure 4E. In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3072, as shown in Figure 4F.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3048, as shown in Figure 4G.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3047, as shown in Figure 4H.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-1076, as shown in Figure 41.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-880, as shown in Figure 4J.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-1123, as shown in Figure 4K.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3069, as shown in Figure 4L.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-1211, as shown in Figure 4M.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3124, as shown in Figure 4N.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-3089, as shown in Figure 40. In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-928, as shown in Figure 4P.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-866, as shown in Figure 4Q.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-1155, as shown in Figure 4R.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-900, as shown in Figure 4S.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to antibody S309-1187, as shown in Figure 4T.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to the antibody shown in Figure 4U.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to the antibody shown in Figure 4V.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to the antibody shown in Figure 4W.

In some embodiments, an antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally VH and VL amino acid sequences, according to the antibody shown in Figure 4X.

In some embodiments, an antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally the VH and VL amino acid sequences, according to antibody S309.887-vl5.1, as shown in Figure 34A.

In some embodiments, an antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally the VH and VL amino acid sequences, according to antibody S309.887-vl9.1, as shown in Figure 34B.

In some embodiments, an antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally the VH and VL amino acid sequences, according to antibody S309.2951-v2.1, as shown in Figure 34C.

In some embodiments, an antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally the VH and VL amino acid sequences, according to antibody S309.SBD-v8.1, as shown in Figure 34D.

In some embodiments, an antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, and optionally the VH and VL amino acid sequences, according to antibody S309.887-v23.1, as shown in Figure 36.

In some embodiments, an antibody or antigen-binding fragment further comprises the heavy constant domain chain (CH1-CH3) and light chain constant domain (CL) amino acid sequences shown in Figure 35.

In some embodiments, an antibody or antigen-binding fragment comprises the heavy chain and light chain amino acid sequences shown in Figure 37.

In some embodiments, an antibody or antigen-binding fragment comprises the heavy chain (VH + CH1-CH3) and light chain (VL + CL) amino acid sequences shown in Figure 38.

In some embodiments, an antibody or antigen-binding fragment comprises the heavy chain (VH + CH1-CH3) and light chain (VL + CL) amino acid sequences shown in Figure 39. In certain embodiments, an antibody or antigen-binding fragment is provided that comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises the HCDRs, the VH, or the heavy chain amino acid sequence of an antibody as provided herein, and each of the two light chains comprises the LCDRs, the VL, or the light chain amino acid sequence of the antibody.

In certain embodiments, an antibody or antigen-binding fragment is provided that comprises two heavy chains and two light chains, wherein one heavy chain comprises the HCDRs, the VH, or the heavy chain amino acid sequence of an antibody as provided herein, and one light chain comprises the LCDRs, the VL, or the light chain amino acid sequence of the antibody.

Also provided are polynucleotides that encode the antibodies and antigenbinding fragments, vectors, host cells, and related compositions, as well as methods of using the antibodies, nucleic acids, vectors, host cells, and related compositions to treat (e.g., reduce, delay, eliminate, or prevent) a SARS-CoV-2 infection in a subject and/or in the manufacture of a medicament for treating a SARS-CoV-2 infection in a subject.

Provided in certain embodiments are antibodies and antigen-binding fragments that are capable of binding to multiple sarbecoviruses (e.g., a surface glycoprotein, as described herein, of one or more (e.g., one, two, three, four, five, six, or more) different sarbecovirus virions and/or expressed on the surface of a cell infected by two or more sarbecoviruses). In certain embodiments, presently disclosed antibodies and antigenbinding fragments can neutralize infection by two or more sarbecoviruses in an in vitro model of infection and/or in a human subject. Also provided are polynucleotides that encode the antibodies and antigen-binding fragments, vectors, host cells, and related compositions, as well as methods of using the antibodies, nucleic acids, vectors, host cells, and related compositions to treat (e.g., reduce, delay, eliminate, or prevent) infection by two or more sarbecoviruses in a subject and/or in the manufacture of a medicament for treating infection in a subject by two or more sarbecoviruses.

Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure. As used herein, "sarbecovirus" refers to any betacoronavirus within lineage B, and includes lineage B viruses in clade la, clade lb, clade 2, and clade 3. Examples of clade la sarbecoviruses are SARS-CoV and Bat SARS-like coronavirus WIV1 (WIV1). Examples of clade lb sarbecoviruses are SARS-CoV-2 (including Wuhan-Hu- 1 and variants thereof, e.g. a variant comprising a G504D mutation, and/or an Omicron variant, e.g., BA.l, BA.2, BA.5, BA.2.12.1, BA.4. BA.5), RatG13, Pangolin-Guanxi- 2017 (PANG/GX) and Pangolin-Guangdon-2019 (PANG/GD). Examples of clade 2 sarbecoviruses are Bat ZC45 (ZC45), Bat ZXC21 (ZXC21), YN2013, SC2018, SX2011, and RmYN02. Examples of clade 3 sarbecoviruses are BtkY72 and BGR2008.

In some embodiments, an antibody or antigen-binding fragment thereof is capable of binding to: a sarbecovirus of clade la (e.g., SARS-CoV, WIV1, or both); a sarbecovirus of clade lb (e.g., SARS-CoV-2, RatG13, Pangolin-Guanxi-2017 (PANG/GX), Pangolin-Guangdon-209, or any combination thereof); a sarbecovirus of clade 2; and/or a sarbecovirus of clade 3. In some embodiments, an antibody or antigen-binding fragment thereof is capable of binding to a sarbecovirus of clade la, a sarbecovirus of clade lb, a sarbecovirus of clade 2, and a sarbecovirus of clade 3.

In certain embodiments, an antibody or antigen-binding fragment thereof is capable of binding to a SARS-CoV-2 variant; e.g., a G504D variant see e.g. Tortorici et al. Nature 597: 103-108 and supplementary materials (2021), https://doi.org/10.1038/s41586-021-03817-4); a N501Y variant; a Y453F variant; a N439K variant; a K417V variant; a N501 Y-K417N-E484K variant; a E484K variant; a California variant; a Brazilian variant; a Swiss variant; an Omicron variant, or any combination thereof. In certain embodiments, an antibody or antigen-binding fragment thereof is capable of binding to a SARS-CoV-2 Wuhan-Hu- 1 and any one or more of the following: SARS-CoV-2 Delta; SARS-CoV-2 BQ.1.1; SARS-CoV-2 XBB.1.5; SARS-CoV-2 BN.1; SARS-CoV-2 CH.1.1; SARS-CoV-2 BR.2; SARS-CoV-2 BA.2.75-S8. “BA.5-S8” and “BA.2.275.2-S8” are engineered viruses with mutations in BA.5 or BA.2.75.2 backbones; see e.g. Figure 5A in Cao et al., bioRxiv 2022; doi.org/10.1101/2022.09.15.507787. As used herein, "SARS-CoV-2", also referred to herein as "Wuhan seafood market phenomia virus", or "Wuhan coronavirus" or "Wuhan CoV", or "novel CoV", or "nCoV", or "2019 nCoV", or "Wuhan nCoV" is a betacoronavirus believed to be of lineage B (sarbecovirus). SARS-CoV-2 was first identified in Wuhan, Hubei province, China, in late 2019 and spread within China and to other parts of the world by early 2020. Symptoms of SARS-CoV-2 infection include fever, dry cough, and dyspnea. Several SARS-CoV-2 variants have emerged.

The genomic sequence of SARS-CoV-2 isolate Wuhan-Hu- 1 is provided in SEQ ID NO.: 1 (see also GenBank MN908947.3, January 23, 2020), and the amino acid translation of the genome is provided in SEQ ID NO.:2 (see also GenBank QHD43416.1, January 23, 2020). Like other coronaviruses (e.g., SARS-CoV-1), SARS-CoV-2 comprises a "spike" or surface ("S") type I transmembrane glycoprotein containing a receptor binding domain (RBD). RBD is believed to mediate entry of the lineage B SARS coronavirus to respiratory epithelial cells by binding to the cell surface receptor angiotensin-converting enzyme 2 (ACE2). In particular, a receptor binding motif (RBM) in the virus RBD is believed to interact with ACE2.

The amino acid sequence of the SARS-CoV-2 Wuhan-Hu-1 surface glycoprotein is provided in SEQ ID NO.:3. Antibodies and antigen-binding fragments of the present disclosure are capable of binding to a SARS CoV-2 surface glycoprotein (S), such as that of Wuhan-Hu-1. For example, in certain embodiments, an antibody or antigen-binding fragment binds to an epitope in Wuhan-Hu- 1 S protein RBD.

The amino acid sequence of SARS-CoV-2 Wuhan-Hu-1 RBD is provided in SEQ ID NO.:4. SARS-CoV-2 S protein has approximately 73% amino acid sequence identity with SARS-CoV S protein. The amino acid sequence of SARS-CoV-2 RBM is provided in SEQ ID NO.:5. SARS-CoV-2 RBD has approximately 75% to 77% amino acid sequence similarity to SARS-CoV-1 RBD, and SARS-CoV-2 RBM has approximately 50% amino acid sequence similarity to SARS-CoV RBM.

Unless otherwise indicated herein, SARS-CoV-2 Wuhan-Hu-1 refers to a virus comprising the amino acid sequence set forth in any one or more of SEQ ID NOs.:2, 3, and 4, optionally with the genomic sequence set forth in SEQ ID NO.: 1. There have been a number of emerging SARS-CoV-2 variants. Some SARS- CoV-2 variants contain a G504D variant. Some SARS-CoV-2 variants contain an N439K mutation, which has enhanced binding affinity to the human ACE2 receptor (Thomson, E.C., et al., The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity. bioRxiv, 2020). Some SARS-CoV- 2 variants contain an N501 Y mutation, which is associated with increased transmissibility, including the lineages B. l.1.7 (also known as 201/501 Y. VI and VOC 202012/01; (del69-70, dell44, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H mutations)) and B.1.351 (also known as 20H/501Y.V2; L18F, D80A, D215G, R246I, K417N, E484K, N501 Y, D614G, and A701 V mutations), which were discovered in the United Kingdom and South Africa, respectively (Tegally, EL, et al., Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv, 2020: p. 2020.12.21.20248640; Leung, K., et al., Early empirical assessment of the N501 Y mutant strains of SARS-CoV-2 in the United Kingdom, October to November 2020. medRxiv, 2020: p. 2020.12.20.20248581). B.1.351 also include two other mutations in the RBD domain of SARS-CoV2 spike protein, K417N and E484K (Tegally, EL, et al., Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv, 2020: p. 2020.12.21.20248640). Other SARS-CoV-2 variants include the Lineage B.1.1.28, which was first reported in Brazil; the Variant P.l, lineage B.1.1.28 (also known as 20J/501Y.V3), which was first reported in Japan; Variant L452R, which was first reported in California in the United States (Pan American Health Organization, Epidemiological update: Occurrence of variants of SARS-CoV-2 in the Americas, January 20, 2021, available at reliefweb. int/sites/reliefweb. int/files/resources/2021-jan-20-phe-epi-update-SARS- CoV-2.pdf). Other SARS-CoV-2 variants include a SARS CoV-2 of clade 19A; SARS CoV-2 of clade 19B; a SARS CoV-2 of clade 20A; a SARS CoV-2 of clade 20B; a SARS CoV-2 of clade 20C; a SARS CoV-2 of clade 20D; a SARS CoV-2 of clade 20E (EU1); a SARS CoV-2 of clade 20F; a SARS CoV-2 of clade 20G; and SARS CoV-2 Bl.1.207; and other SARS CoV-2 lineages described in Rambaut, A., et al., A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Microbiol 5, 1403-1407 (2020). The Alpha (B.1.1.7), Beta (B.1.351, B.1.351.2, B.1.351.3), Delta (B.1.617.2, AY. l, AY.2, AY.3; see also Mlcochova, P., Kemp, S.A., Dhar, M.S. et al. SARS-CoV-2 B.1.617.2 Delta variant replication and immune evasion. Nature (2021). https://doi.org/10.1038/s41586-021-03944-y), and Gamma (P. l, P.1.1, P.1.2) variants of SARS-CoV-2 circulating in the United States are classified as variants of concern by the U.S. Centers for Disease Control and Prevention (see https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html). Variant BQ.1.1 is described further herein. Other variants include: SARS-CoV-2 XBB.1.5; SARS-CoV-2 BN.1; SARS-CoV-2 CH.1.1; SARS-CoV-2 BR.2; and SARS-CoV-2 BA.2.75-S8. “BA.5-S8” and “BA.2.275.2-S8” are engineered viruses with mutations in BA.5 or BA.2.75.2 backbones; see e.g. Figure 5A in Cao et al., bioRxiv 2022; doi.org/10.1101/2022.09.15.507787. The foregoing SARS-CoV-2 variants, and the amino acid and nucleotide sequences thereof, are incorporated herein by reference.

SARS-CoV is another betacoronavirus of lineage B (sarbecovirus) that causes respiratory symptoms in infected individuals. The genomic sequence of SARS-CoV Urbani strain has GenBank accession number AAP 13441.1.

In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. Also, any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated. As used herein, the term "about" means ± 20% of the indicated range, value, or structure, unless otherwise indicated. It should be understood that the terms "a" and "an" as used herein refer to "one or more" of the enumerated components. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives. As used herein, the terms "include," "have," and "comprise" are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.

"Optional" or "optionally" means that the subsequently described element, component, event, or circumstance may or may not occur, and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which they do not.

In addition, it should be understood that the individual constructs, or groups of constructs, derived from the various combinations of the structures and subunits described herein, are disclosed by the present application to the same extent as if each construct or group of constructs was set forth individually. Thus, selection of particular structures or particular subunits is within the scope of the present disclosure.

The term “consisting essentially of’ is not equivalent to “comprising” and refers to the specified materials or steps of a claim, or to those that do not materially affect the basic characteristics of a claimed subject matter. For example, a protein domain, region, or module (e.g., a binding domain) or a protein “consists essentially of’ a particular amino acid sequence when the amino acid sequence of a domain, region, module, or protein includes extensions, deletions, mutations, or a combination thereof (e.g., amino acids at the amino- or carboxy -terminus or between domains) that, in combination, contribute to at most 20% (e.g., at most 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2% or 1%) of the length of a domain, region, module, or protein and do not substantially affect (z.e., do not reduce the activity by more than 50%, such as no more than 40%, 30%, 25%, 20%, 15%, 10%, 5%, or 1%) the activity of the domain(s), region(s), module(s), or protein (e.g., the target binding affinity of a binding protein).

As used herein, “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, z.e., an a-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.

As used herein, “mutation” refers to a change in the sequence of a nucleic acid molecule or polypeptide molecule as compared to a reference or wild-type nucleic acid molecule or polypeptide molecule, respectively. A mutation can result in several different types of change in sequence, including substitution, insertion or deletion of nucleotide(s) or amino acid(s).

A “conservative substitution” refers to amino acid substitutions that do not significantly affect or alter binding characteristics of a particular protein. Generally, conservative substitutions are ones in which a substituted amino acid residue is replaced with an amino acid residue having a similar side chain. Conservative substitutions include a substitution found in one of the following groups: Group 1 : Alanine (Ala or A), Glycine (Gly or G), Serine (Ser or S), Threonine (Thr or T); Group 2: Aspartic acid (Asp or D), Glutamic acid (Glu or Z); Group 3 : Asparagine (Asn or N), Glutamine (Gin or Q); Group 4: Arginine (Arg or R), Lysine (Lys or K), Histidine (His or H); Group 5: Isoleucine (He or I), Leucine (Leu or L), Methionine (Met or M), Valine (Vai or V); and Group 6: Phenylalanine (Phe or F), Tyrosine (Tyr or Y), Tryptophan (Trp or W). Additionally or alternatively, amino acids can be grouped into conservative substitution groups by similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic grouping may include, for purposes of substitution, Gly, Ala, Vai, Leu, and He. Other conservative substitutions groups include: sulfur-containing: Met and Cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gin; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gin; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, He, Vai, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Additional information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

As used herein, “protein” or “polypeptide” refers to a polymer of amino acid residues. Proteins apply to naturally occurring amino acid polymers, as well as to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, and non-naturally occurring amino acid polymers. Variants of proteins, peptides, and polypeptides of this disclosure are also contemplated. In certain embodiments, variant proteins, peptides, and polypeptides comprise or consist of an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.9% identical to an amino acid sequence of a defined or reference amino acid sequence as described herein.

“Nucleic acid molecule” or “polynucleotide” or “polynucleic acid” refers to a polymeric compound including covalently linked nucleotides, which can be made up of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), which includes mRNA, microRNA, siRNA, viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes cDNA, genomic DNA, and synthetic DNA, either of which may be single or double stranded. If single-stranded, the nucleic acid molecule may be the coding strand or non-coding (anti-sense) strand. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences that encode the same amino acid sequence. Some versions of the nucleotide sequences may also include intron(s) to the extent that the intron(s) would be removed through co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as the result of the redundancy or degeneracy of the genetic code, or by splicing.

In some embodiments, the polynucleotide (e.g. mRNA) comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof. In certain embodiments, the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5-methylcytidine, a 2-thiouridine, or any combination thereof. In some embodiments, the pseudouridine comprises N1 -methylpseudouridine. These features are known in the art and are discussed in, for example, Zhang et al. Front. Immunol., DOI=10.3389/fimmu.2019.00594 (2019); Eyler et al. PNAS 116(46): 23068-23071; DOI: 10.1073/pnas.1821754116 (2019); Nance and Meier, ACS Cent. Set. 2021, 7, 5, 748-756; doi.org/10.1021/acscentsci. lc00197 (2021), and van Hoecke and Roose, J. Translational Med 17:54 (2019); https://doi.org/10.1186/sl2967-019-1804-8, which modified nucleosides and mRNA features are incorporated herein by reference.

Variants of nucleic acid molecules of this disclosure are also contemplated. Variant nucleic acid molecules are at least 70%, 75%, 80%, 85%, 90%, and are preferably 95%, 96%, 97%, 98%, 99%, or 99.9% identical a nucleic acid molecule of a defined or reference polynucleotide as described herein, or that hybridize to a polynucleotide under stringent hybridization conditions of 0.015M sodium chloride, 0.0015M sodium citrate at about 65-68°C or 0.015M sodium chloride, 0.0015M sodium citrate, and 50% formamide at about 42°C. Nucleic acid molecule variants retain the capacity to encode a binding domain thereof having a functionality described herein, such as binding a target molecule.

“Percent sequence identity” refers to a relationship between two or more sequences, as determined by comparing the sequences. Preferred methods to determine sequence identity are designed to give the best match between the sequences being compared. For example, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment). Further, non-homologous sequences may be disregarded for comparison purposes. The percent sequence identity referenced herein is calculated over the length of the reference sequence, unless indicated otherwise. Methods to determine sequence identity and similarity can be found in publicly available computer programs. Sequence alignments and percent identity calculations may be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. Within the context of this disclosure, it will be understood that where sequence analysis software is used for analysis, the results of the analysis are based on the “default values” of the program referenced. “Default values” mean any set of values or parameters which originally load with the software when first initialized.

The term “isolated” means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring nucleic acid or polypeptide present in a living animal is not isolated, but the same nucleic acid or polypeptide, separated from some or all of the co-existing materials in the natural system, is isolated. Such nucleic acid could be part of a vector and/or such nucleic acid or polypeptide could be part of a composition (e.g., a cell lysate), and still be isolated in that such vector or composition is not part of the natural environment for the nucleic acid or polypeptide. "Isolated" can, in some embodiments, also describe an antibody, antigen binding fragment, polynucleotide, vector, host cell, or composition that is outside of a human body.

The term “gene” means the segment of DNA or RNA involved in producing a polypeptide chain; in certain contexts, it includes regions preceding and following the coding region (e.g., 5' untranslated region (UTR) and 3' UTR) as well as intervening sequences (introns) between individual coding segments (exons).

A “functional variant” refers to a polypeptide or polynucleotide that is structurally similar or substantially structurally similar to a parent or reference compound of this disclosure, but differs slightly in composition (e.g., one base, atom or functional group is different, added, or removed), such that the polypeptide or encoded polypeptide is capable of performing at least one function of the parent polypeptide with at least 50% efficiency, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide. In other words, a functional variant of a polypeptide or encoded polypeptide of this disclosure has “similar binding,” “similar affinity” or “similar activity” when the functional variant displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide, such as an assay for measuring binding affinity (e.g., Biacore® or tetramer staining measuring an association (Ka) or a dissociation (KD) constant).

As used herein, a “functional portion” or “functional fragment” refers to a polypeptide or polynucleotide that comprises only a domain, portion or fragment of a parent or reference compound, and the polypeptide or encoded polypeptide retains at least 50% activity associated with the domain, portion or fragment of the parent or reference compound, preferably at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% level of activity of the parent polypeptide, or provides a biological benefit (e.g., effector function). A “functional portion” or “functional fragment” of a polypeptide or encoded polypeptide of this disclosure has “similar binding” or “similar activity” when the functional portion or fragment displays no more than a 50% reduction in performance in a selected assay as compared to the parent or reference polypeptide (preferably no more than 20% or 10%, or no more than a log difference as compared to the parent or reference with regard to affinity).

As used herein, the term “engineered,” “recombinant,” or “non-natural” refers to an organism, microorganism, cell, nucleic acid molecule, or vector that includes at least one genetic alteration or has been modified by introduction of an exogenous or heterologous nucleic acid molecule, wherein such alterations or modifications are introduced by genetic engineering (i.e., human intervention). Genetic alterations include, for example, modifications introducing expressible nucleic acid molecules encoding functional RNA, proteins, fusion proteins or enzymes, or other nucleic acid molecule additions, deletions, substitutions, or other functional disruption of a cell’s genetic material. Additional modifications include, for example, non-coding regulatory regions in which the modifications alter expression of a polynucleotide, gene, or operon.

As used herein, “heterologous” or “non-endogenous” or “exogenous” refers to any gene, protein, compound, nucleic acid molecule, or activity that is not native to a host cell or a subject, or any gene, protein, compound, nucleic acid molecule, or activity native to a host cell or a subject that has been altered. Heterologous, non-endogenous, or exogenous includes genes, proteins, compounds, or nucleic acid molecules that have been mutated or otherwise altered such that the structure, activity, or both is different as between the native and altered genes, proteins, compounds, or nucleic acid molecules. In certain embodiments, heterologous, non-endogenous, or exogenous genes, proteins, or nucleic acid molecules (e.g., receptors, ligands, etc.) may not be endogenous to a host cell or a subject, but instead nucleic acids encoding such genes, proteins, or nucleic acid molecules may have been added to a host cell by conjugation, transformation, transfection, electroporation, or the like, wherein the added nucleic acid molecule may integrate into a host cell genome or can exist as extra-chromosomal genetic material (e.g., as a plasmid or other self-replicating vector). The term “homologous” or “homolog” refers to a gene, protein, compound, nucleic acid molecule, or activity found in or derived from a host cell, species, or strain. For example, a heterologous or exogenous polynucleotide or gene encoding a polypeptide may be homologous to a native polynucleotide or gene and encode a homologous polypeptide or activity, but the polynucleotide or polypeptide may have an altered structure, sequence, expression level, or any combination thereof. A non-endogenous polynucleotide or gene, as well as the encoded polypeptide or activity, may be from the same species, a different species, or a combination thereof.

In certain embodiments, a nucleic acid molecule or portion thereof native to a host cell will be considered heterologous to the host cell if it has been altered or mutated, or a nucleic acid molecule native to a host cell may be considered heterologous if it has been altered with a heterologous expression control sequence or has been altered with an endogenous expression control sequence not normally associated with the nucleic acid molecule native to a host cell. In addition, the term “heterologous” can refer to a biological activity that is different, altered, or not endogenous to a host cell. As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a fusion protein, or any combination thereof. As used herein, the term “endogenous” or “native” refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or a subject.

The term “expression”, as used herein, refers to the process by which a polypeptide is produced based on the encoding sequence of a nucleic acid molecule, such as a gene. The process may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post- translational modification, or any combination thereof. An expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

The term “operably linked” refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (z.e., the coding sequence is under the transcriptional control of the promoter). “Unlinked” means that the associated genetic elements are not closely associated with one another and the function of one does not affect the other.

As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as separate nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a protein (e.g., a heavy chain of an antibody), or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of referenced heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, not the number of separate nucleic acid molecules introduced into a host cell. The term “construct” refers to any polynucleotide that contains a recombinant nucleic acid molecule (or, when the context clearly indicates, a fusion protein of the present disclosure). A (polynucleotide) construct may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A “vector” is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, a RNA vector or a linear or circular DNA or RNA molecule that may include chromosomal, non-chromosomal, semi-synthetic or synthetic nucleic acid molecules. Vectors of the present disclosure also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther. 5:108, 2003: Mates et al., Nat. Genet. 41'.753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide to a cell genome (e.g, viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

As used herein, “expression vector” or “vector” refers to a DNA construct containing a nucleic acid molecule that is operably linked to a suitable control sequence capable of effecting the expression of the nucleic acid molecule in a suitable host. Such control sequences include a promoter to effect transcription, an optional operator sequence to control such transcription, a sequence encoding suitable mRNA ribosome binding sites, and sequences which control termination of transcription and translation. The vector may be a plasmid, a phage particle, a virus, or simply a potential genomic insert. Once transformed into a suitable host, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself or deliver the polynucleotide contained in the vector into the genome without the vector sequence. In the present specification, “plasmid,” “expression plasmid,” “virus,” and “vector” are often used interchangeably.

The term “introduced” in the context of inserting a nucleic acid molecule into a cell, means “transfection”, “transformation,” or “transduction” and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell wherein the nucleic acid molecule may be incorporated into the genome of a cell (e.g, chromosome, plasmid, plastid, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

In certain embodiments, polynucleotides of the present disclosure may be operatively linked to certain elements of a vector. For example, polynucleotide sequences that are needed to effect the expression and processing of coding sequences to which they are ligated may be operatively linked. Expression control sequences may include appropriate transcription initiation, termination, promoter, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (z.e., Kozak consensus sequences); sequences that enhance protein stability; and possibly sequences that enhance protein secretion. Expression control sequences may be operatively linked if they are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.

In certain embodiments, the vector comprises a plasmid vector or a viral vector (e.g., a lentiviral vector or a y-retroviral vector). Viral vectors include retrovirus, adenovirus, parvovirus (e.g., adeno-associated viruses), coronavirus, negative strand RNA viruses such as ortho-myxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g., measles and Sendai), positive strand RNA viruses such as picornavirus and alphavirus, and double-stranded DNA viruses including adenovirus, herpesvirus (e.g., Herpes Simplex virus types 1 and 2, Epstein-Barr virus, cytomegalovirus), and poxvirus (e.g., vaccinia, fowlpox, and canarypox). Other viruses include, for example, Norwalk virus, togavirus, flavivirus, reoviruses, papovavirus, hepadnavirus, and hepatitis virus. Examples of retroviruses include avian leukosis-sarcoma, mammalian C-type, B-type viruses, D type viruses, HTLV-BLV group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et al., Eds., Lippincott-Raven Publishers, Philadelphia, 1996).

“Retroviruses” are viruses having an RNA genome, which is reverse-transcribed into DNA using a reverse transcriptase enzyme, the reverse-transcribed DNA is then incorporated into the host cell genome. “Gammaretrovirus” refers to a genus of the retroviridae family. Examples of gammaretroviruses include mouse stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

“Lentiviral vectors” include HIV-based lentiviral vectors for gene delivery, which can be integrative or non-integrative, have relatively large packaging capacity, and can transduce a range of different cell types. Lentiviral vectors are usually generated following transient transfection of three (packaging, envelope, and transfer) or more plasmids into producer cells. Like HIV, lentiviral vectors enter the target cell through the interaction of viral surface glycoproteins with receptors on the cell surface. On entry, the viral RNA undergoes reverse transcription, which is mediated by the viral reverse transcriptase complex. The product of reverse transcription is a double-stranded linear viral DNA, which is the substrate for viral integration into the DNA of infected cells.

In certain embodiments, the viral vector can be a gammaretrovirus, e.g., Moloney murine leukemia virus (MLV)-derived vectors. In other embodiments, the viral vector can be a more complex retrovirus-derived vector, e.g., a lentivirus-derived vector. HIV-l-derived vectors belong to this category. Other examples include lentivirus vectors derived from HIV-2, FIV, equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells for transducing mammalian host cells with viral particles containing transgenes are known in the art and have been previous described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6.321939, 2011; Zhao et al., J. Immunol. 174 AM5, 2005; Engels et al., Hum. Gene Ther. 77: 1155, 2003; Frecha et al., Mol. Ther. 18.Y1 , 2010; and Verhoeyen et al ., Methods Mol. Biol. 506.91 , 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors also can be used for polynucleotide delivery including DNA viral vectors, including, for example adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSVs), including amplicon vectors, replication-defective HSV and attenuated HSV (Krisky et al., Gene Ther. 5: 1517, 1998).

Other vectors that can be used with the compositions and methods of this disclosure include those derived from baculoviruses and a-viruses. (Jolly, D J. 1999. Emerging Viral Vectors, pp 209-40 in Friedmann T. ed. The Development of Human Gene Therapy. New York: Cold Spring Harbor Lab), or plasmid vectors (such as sleeping beauty or other transposon vectors).

When a viral vector genome comprises a plurality of polynucleotides to be expressed in a host cell as separate transcripts, the viral vector may also comprise additional sequences between the two (or more) transcripts allowing for bicistronic or multi ci str onic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral 2A peptide, or any combination thereof.

Plasmid vectors, including DNA-based antibody or antigen-binding fragmentencoding plasmid vectors for direct administration to a subject, are described further herein.

As used herein, the term “host” refers to a cell or microorganism targeted for genetic modification with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present disclosure).

A host cell may include any individual cell or cell culture which may receive a vector or the incorporation of nucleic acids or express proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, simian cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other material by use of a viral vector, transformation via calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other methods. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989). In the context of a SARS-CoV-2 infection (or infection by another sarbecovirus), a “host” refers to a cell or a subject infected with the SARS-CoV-2 coronavirus (or other sarbecovirus).

“Antigen” or “Ag”, as used herein, refers to an immunogenic molecule that provokes an immune response. This immune response may involve antibody production, activation of specific immunologically-competent cells, activation of complement, antibody dependent cytotoxicicity, or any combination thereof. An antigen (immunogenic molecule) may be, for example, a peptide, glycopeptide, polypeptide, glycopolypeptide, polynucleotide, polysaccharide, lipid, or the like. It is readily apparent that an antigen can be synthesized, produced recombinantly, or derived from a biological sample. Exemplary biological samples that can contain one or more antigens include tissue samples, stool samples, cells, biological fluids, or combinations thereof. Antigens can be produced by cells that have been modified or genetically engineered to express an antigen. Antigens can also be present in a sarbecovirus, e.g. SARS-CoV-2 coronavirus (e.g., a surface glycoprotein or portion thereof), such as present in a virion, or expressed or presented on the surface of a cell infected by SARS- CoV-2.

The term “epitope” or “antigenic epitope” includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule, such as an immunoglobulin, or other binding molecule, domain, or protein. Epitopic determinants generally contain chemically active surface groupings of molecules, such as amino acids or sugar side chains, and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. Where an antigen is or comprises a peptide or protein, the epitope can be comprised of consecutive amino acids (e.g., a linear epitope), or can be comprised of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope), or non-contiguous amino acids that are in close proximity irrespective of protein folding. Antibodies, Antigen-Binding Fragments, and Compositions

In one aspect, the present disclosure provides an (e.g. isolated) anti-SARS-CoV- 2 antibody, or an antigen-binding fragment thereof. It will be understood that “anti- SARS-CoV-2 antibody” refers to an antibody that is capable of binding to a SARS- CoV-2 antigen (e.g., a surface glycoprotein, a RBD), though, as provided herein, certain embodiments provide antibodies and antigen-binding fragments that are capable of binding to SARS-CoV-2 (e.g., Wuhan-Hu-1 and/or one or more other SARS-CoV-2 strains or variants, such as BQ.1.1) and/or to one or more other sarbecoviruses.

In certain embodiments, the antibody or antigen-binding fragment is capable of binding to a sarbecovirus of clade la, clade lb, clade 2, and/or clade 3. In certain embodiments, the antibody or antigen-binding fragment is capable of binding to a surface glycoprotein of any one or more of SARS-CoV-2, SARS-CoV-2 G504D (optionally Wuhan-Hu-1 comprising G504D, BA.5 comprising D504G, or both), SARS-CoV-2 delta variant, SARS-CoV-2 omicron variant, SARS-CoV-2 BA.2, SARS- CoV-2 BA.5, SARS-CoV-2 BA.4-5 (also expressed as SARS-CoV-2 BA.4/5), SARS- CoV-2 BA.4, SARS-CoV-2 BA.4.6, ZC45, BGR08, and SARS-CoV (also referred-to as a SARS-CoV-1). In certain embodiments, an antibody or antigen-binding fragment thereof is capable of binding to a SARS-CoV-2 Wuhan-Hu- 1 and any one or more of the following: SARS-CoV-2 Delta; SARS-CoV-2 BQ.1.1; SARS-CoV-2 XBB.1.5; SARS-CoV-2 BN.1; SARS-CoV-2 CH.1.1; SARS-CoV-2 BR.2; SARS-CoV-2 BA.2.75-S8. “BA.5-S8” and “BA.2.275.2-S8” are engineered viruses with mutations in BA.5 or BA.2.75.2 backbones; see e.g. Figure 5A in Cao et al., bioRxiv 2022; doi.org/10.1101/2022.09.15.507787.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure associates with or unites with a coronavirus (e.g. SARS-CoV-2) surface glycoprotein epitope or antigen comprising the epitope, while not significantly associating or uniting with any other molecules or components in a sample.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure associates with or unites (e.g., binds) to a SARS-CoV-2 surface glycoprotein epitope, and can also associate with or unite with an epitope from another coronavirus e.g., SARS-CoV) present in the sample, but not significantly associating or uniting with any other molecules or components in the sample. In other words, in certain embodiments, an antibody or antigen binding fragment of the present disclosure is cross-reactive for SARS-CoV-2 and one or more additional coronavirus.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of binding to a surface glycoprotein of two or more sarbecoviruses. In some embodiments, the two or more sarbecoviruses are selected from: clade la sarbecoviruses and/or clade lb sarbecoviruses; clade 2 sarbecoviruses; clade 3 sarbecoviruses; or naturally occuring variants thereof, and any combination thereof. In certain embodiments, the antibody or antigen-binding fragment is capable of binding to a surface glycoprotein of two or more sarbecoviruses; e.g., capable of binding when a sarbecovirus S protein is expressed on a cell surface of a host cell and/or on a sarbecovirus virion. In certain embodiments, the two or more sarbecoviruses are selected from SARS-CoV, WIV1, SARS-CoV2, SARS-CoV-2 G504D, Anlongl l2, YN2013, SX2011, SC2018, PANG/GD, PANG/GX, RatG13, ZXC21, ZC45, RmYN02, BGR2008 (aka BGR08), BtkY72, and naturally occurring variants thereof. In some embodiments, the two or more sarbecoviruses include one or more of SARS-CoV-2 variants P.l, B. l.1.7, B.1.429, B.1.351, B.1.617.2, BA.l, BA.2, BA.2.12.1, BAA, BA.5, BA.4/5, and BAA.6. In some embodiments, the two or more sarbecoviruses include one or more SARS-CoV-2 variants having S protein mutations N501 Y, Y453F, N439K, K417V, E484K, or any combination thereof. In some embodiments, an antibody or antigen-binding fragment is capable of binding to a SARS-CoV-2 G504D variant. In some embodiments, an antibody or antigen-binding fragment is capable of binding to a SARS-CoV-2 B.1.617.2. In some embodiments, an antibody or antigen-binding fragment is capable of binding to a SARS-CoV (also referred-to as a SARS-CoV-1).

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure associates with or unites with a sarbecovirus surface glycoprotein epitope or antigen comprising the epitope, while not significantly associating or uniting with any other molecules or components in a sample. In some embodiments, the epitope is comprised in a SI subunit of a spike (S) protein. In further embodiments, the epitope is comprised in a receptor binding domain (RBD) of a S protein.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure associates with or unites (e.g., binds) to a first sarbecovirus surface glycoprotein epitope, and can also associate with or unite with an epitope from another sarbecovirus present in the sample, but not significantly associating or uniting with any other molecules or components in the sample. In other words, in certain embodiments, an antibody or antigen binding fragment of the present disclosure is cross-reactive against and specifically binds to two or more sarbecoviruses.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure specifically binds to a sarbecovirus surface glycoprotein, such as a SARS- CoV-2 surface glycoprotein. As used herein, “specifically binds” refers to an association or union of an antibody or antigen-binding fragment to an antigen with an affinity or K_a (z.e., an equilibrium association constant of a particular binding interaction with units of 1/M) equal to or greater than 10⁵ M'¹ (which equals the ratio of the on-rate [K_on] to the off rate [K_Off] for this association reaction), while not significantly associating or uniting with any other molecules or components in a sample. Alternatively, affinity may be defined as an equilibrium dissociation constant (Ka) of a particular binding interaction with units of M (e.g., 10'⁵ M to 10'¹³ M). Antibodies may be classified as “high-affinity” antibodies or as “low-affinity” antibodies. “High-affinity” antibodies refer to those antibodies having a K_a of at least 10⁷M^-1, at least 10⁸ M'¹, at least 10⁹ M'¹, at least IO¹⁰ M'¹, at least 10¹¹ M'¹, at least 10¹² M'¹, or at least 10¹³ M'¹. “Low-affinity” antibodies refer to those antibodies having a K_a of up to 10⁷M^-1, up to 10⁶ M'¹, up to 10⁵ M'¹. Alternatively, affinity may be defined as an equilibrium dissociation constant (Kd) of a particular binding interaction with units of M (e.g., 10'⁵ M to 10'¹³ M).

In some contexts, antibody and antigen-binding fragments may be described with reference to affinity and/or to avidity for antigen. Unless otherwise indicated, avidity refers to the total binding strength of an antibody or antigen-binding fragment thereof to antigen, and reflects binding affinity, valency of the antibody or antigen- binding fragment (e.g., whether the antibody or antigen-binding fragment comprises one, two, three, four, five, six, seven, eight, nine, ten, or more binding sites), and, for example, whether another agent is present that can affect the binding (e.g., a noncompetitive inhibitor of the antibody or antigen-binding fragment).

A variety of assays are known for identifying antibodies of the present disclosure that bind a particular target, as well as determining binding domain or binding protein affinities, such as Western blot, ELISA (e.g., direct, indirect, or sandwich), analytical ultracentrifugation, spectroscopy, and surface plasmon resonance (Biacore®) analysis (see, e.g., Scatchard et al., Ann. N.Y. Acad. Sci. 51:660, 1949; Wilson, Science 295:2103, 2002; Wolff et al., Cancer Res. 53:2560, 1993; and U.S. Patent Nos. 5,283,173, 5,468,614, or the equivalent). Assays for assessing affinity or apparent affinity or relative affinity are also known.

In certain examples, binding can be determined by recombinantly expressing a sarbecovirus antigen, such as a SARS-CoV-2 antigen in a host cell (e.g., by transfection) and immunostaining the (e.g., fixed, or fixed and permeabilized) host cell with antibody and analyzing binding by flow cytometery (e.g., using a ZE5 Cell Analyzer (BioRad®) and FlowJo software (TreeStar). In some embodiments, positive binding can be defined by differential staining by antibody of SARS-CoV-2 -expressing cells versus control (e.g., mock) cells.

In some embodiments, an antibody or antigen-binding fragment of the present disclosure binds to a sarbecovirus spike protein (i.e., from two or more sarbecoviruses) expressed on the surface of a host cell (e.g., an Expi-CHO cell), as determined by flow cytometry.

In some embodiments an antibody or antigen-binding fragment of the present disclosure binds to a sarbecovirus S protein, such as a SARS-CoV-2 S protein, as measured using biolayer interferometry.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing infection by a sarbecovirus, such as e.g. SARS- CoV-2, SARS-CoV, SARS-CoV-2 delta variant, SARS-CoV-2 omicron variant e.g. a SARS CoV-2 omicron subtype such as BQ.1.1, BA.2, BA.12.1, BA.4, or BA.5) and/or SARS-CoV-2 G504D variant. In certain embodiments, an antibody or antigen-binding fragment thereof is capable of neutralizing infection by a SARS-CoV-2 Wuhan-Hu- 1 and by any one or more of the following: SARS-CoV-2 Delta; SARS-CoV-2 BQ.1.1; SARS-CoV-2 XBB.1.5; SARS-CoV-2 BN.l; SARS-CoV-2 CH.1.1; SARS-CoV-2 BR.2; SARS-CoV-2 BA.2.75-S8.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing infection by two or more sarbecoviruses. As used herein, a “neutralizing antibody” (or “neutralizing antigen-binding fragment”) is one that can neutralize, z.e., prevent, inhibit, reduce, impede, or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms “neutralizing antibody” and “an antibody that neutralizes” or “antibodies that neutralize” are used interchangeably herein. In any of the presently disclosed embodiments, the antibody or antigen-binding fragment is capable of preventing and/or neutralizing a SARS-CoV-2 infection (and/or infection by another sarbecovirus) in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.

In some embodiments, an antibody or antigen-binding fragment is capable of neutralizing an infection by a clade 1 (e.g. clade la, clade lb, or both) sarbecovirus, a clade 2 sarbecovirus, a clade 3 sarbecovirus, or any combination thereof. In some embodiments, an antibody or antigen-binding fragment is capable of neutralizing an infection by any one or more, any two or more, any three or more, any four or more, any five or more, any six or more, any seven or more, any eight or more, any nine or more, any ten or more, any eleven or more, or all twelve of: a SARS-CoV-2 Wuhan- Hu-1; a SARS-CoV-2 G504D; a SARS-CoV; a SARS-CoV-2 delta variant, a SARS- CoV-2 omicron variant; a SARS-CoV-2 BA.2; a SARS-CoV-2 BA.2.12.1; a SARS- CoV-2 BA.4; a SARS-CoV-2 BA.4/5; a SARS-CoV-2 BA.5; a SARS-CoV-2 BA.4.6; a BGR08; and a ZC45. In any of the presently disclosed embodiments, the antibody or antigen-binding fragment is capable of preventing and/or neutralizing a sarbecovirus (e.g. SARS-CoV-2, SARS-CoV-2 variant, SARS-CoV, ZC45, BGR08) infection in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human. Terms understood by those in the art of antibody technology are each given the meaning acquired in the art, unless expressly defined differently herein. For example, the term “antibody” refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as any antigen-binding portion or fragment of an intact antibody that has or retains the ability to bind to the antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab'2 fragment. Thus, the term “antibody” herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including fragment antigen binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rlgG) fragments, single chain antibody fragments, including single chain variable fragments (scFv), and single domain antibodies (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multi specific, e.g., bi specific antibodies, diabodies, triabodies, tetrabodies, tandem di-scFv, and tandem tri-scFv. Unless otherwise stated, the term “antibody” should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or sub-class, including IgG and sub-classes thereof (IgGl (e.g., IgGlm, 17), IgG2, IgG3, IgG4), IgM, IgE, IgA, and IgD.

The terms “VL” or “VL” and “VH” or “VH” refer to the variable binding region from an antibody light chain and an antibody heavy chain, respectively. In certain embodiments, a VL is a kappa (K) class (also “VK” herein). In certain embodiments, a VL is a lambda (X) class. The variable binding regions comprise discrete, well-defined sub-regions known as “complementarity determining regions” (CDRs) and “framework regions” (FRs). The terms “complementarity determining region,” and “CDR,” are synonymous with “hypervariable region” or “HVR,” and refer to sequences of amino acids within antibody variable regions, which, in general, together confer the antigen specificity (and at least in part and in some cases substantially, the binding affinity) of the antibody, wherein consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from one another in primary structure by a framework region. There are three CDRs in each variable region (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRHs and CDRLs, respectively). In certain embodiments, an antibody VH comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2- HCDR2-FR3-HCDR3-FR4; and an antibody VL comprises four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. In general, the VH and the VL together form the antigen-binding site through their respective CDRs.

As used herein, a “variant” of a CDR refers to a functional variant of a CDR sequence having up to 1-3 amino acid substitutions (e.g., conservative or nonconservative substitutions), deletions, or combinations thereof.

Numbering of CDR and framework regions may be according to any known method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Kabat et al., “Sequences of Proteins of Immunological Interest,” US Dept. Health and Human Services, Public Health Service National Institutes of Health, 1991, 5^th ed.; Chothia and Lesk, J. Mol. Biol. 796:901-917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and Pliickthun, J. Mol. Bio. 309:657-670 (2001)). Other CDR numbering schemes include North (described in “A New Clustering of Antibody CDR Loop Conformations”, Journal of Molecular Biology, 406, 228-256 (2011)), AbM, and Martin (also referred to as Enhanced Chothia).

Equivalent residue positions can be annotated and for different molecules to be compared using Antigen receptor Numbering And Receptor Classification (ANARCI) software tool (2016, Bioinformatics 15:298-300). Also for IMGT numbering, a variable domain (or variable domain-containing) amino acid sequence of interest can be annotated using IMGT V-QUEST (www.imgt.org/IMGT_vquest/analysis; see also Brochet e/ a/. NucL Acids Res. 36 W503-508, doi: 10.1093/nar/gkn316 (2008)). In certain embodiments, antibody CDRs and amino acid numbering of variable regions are according to the system developed by the Chemical Computing Group ("CCG"); e.g., using Molecular Operating Environment (MOE) software (www.chemcomp.com). Accordingly, identification of CDRs of a variable domain (VH or VL) sequence as provided herein according to one numbering scheme is not exclusive of an antibody comprising CDRs of the same variable domain as determined using a different numbering scheme.

In some embodiments, CDRs of a variable domain or region (VH or VL) sequence as provided herein are a combination of any two or more of the following numbering schemes: Kabat, Chothia, IMGT, CCG, AHo, AbM, Martin, North, and EU.

In some embodiments, CDRs of a variable domain or region (VH or VL) sequence as provided herein are a combination of IMGT CDR definitions and one or more V-region amino acid flanking the IMGT CDR. In Example 1, antibody V-region amino acids flanking an IMGT CDR were identified, using the CCG system described above, as potential sites for engineering to improve binding affinity and/or neutralization of the antibody or antigen-binding fragment against SARS-CoV-2, relative to the parental antibody (comprising VH SEQ ID NO.:30, VL SEQ ID NO.:34).

Table 1 shows the VH and VL amino acid sequences of SEQ ID NOs.:30 and 34 (i.e. the VH and VL amino acid sequences of Sotrovimab); the IMGT CDRs are shown in bold and extended CDR sequences (IMGT + CCG) are underlined. For CDRH3 and CDRL3, the IMGT and IMGT+CCG defintions are the same. In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises one or more substitution mutation in CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and/or CDRL3 (IMGT + CCG), relative to SEQ ID NOs.:30 and 34, respectively. S309 differs from Sotrovimab in the variable domains only by a native N at position 55 in the VH, whereas Sotrovimab comprises a Q at that position.

Table 1. SEQ ID NOs.:30 and 34 (VH and VL of Sotrovimab), with IMGT CDRs shown in bold and IMGT + CCG CDRs underlined

In certain embodiments, an antibody or an antigen-binding fragment of the present disclosure comprises a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and/or a CDRL3 of an antibody as set forth in Table 2. It will be appreciated that CDRs of the different antibodies set forth in Table 2 can be combined, and/or can be combined with a corresponding CDR (CCG + IMGT) of S309 or Sotrovimab, to provide an antibody or antigen-binding fragment comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3. Table 2. CDR (IMGT+CCG) Amino Acid SEQ ID NOs. of Certain Variant

Antibodies of the Present Disclosure.

In some embodiments, an antibody or antigen-binding fragment comprises: (i) the CDRH1 amino acid sequence of any one of SEQ ID NOs.:39, 49, 59, 69, 79, 89, 99, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, and 391; (ii) the CDRH2 amino acid sequence of any one of SEQ ID NOs.:40, 50, 60, 70, 80, 90, 100, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, and 392; (iii) the CDRH3 amino acid sequence of any one of SEQ ID NOs.:41, 51, 61, 71, 81, 91, 101, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, and 393; (iv) the CDRL1 amino acid sequence of any one of SEQ ID NOs.: 44, 54, 64, 74, 84, 94, 104, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, and 396; (v) the CDRL2 amino acid sequence of any one of SEQ ID NOs.:45, 55, 65, 75, 85, 95, 105, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, and 397; and (vi) the CDRL3 amino acid sequence of any one of SEQ ID NOs.:46, 56, 66, 76, 86, 96, 106, 118, 128, 138, 148, 158, 168, 178, 198, 208, 218, 228, 238, 248, 258, 268, 278, 288, 298, 308, 318, 328, 338, 348, 358, 368, 378, 388, and 398. Alternatively, it will be appreciated that an antibody or antigen-binding fragment can comprise one or more of these CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences, in combination of one or more corresponding CDR sequence from SEQ ID NO.:30, SEQ ID NO.:22, SEQ ID NO.:38, and SEQ ID NO.:34. In other words, also contemplated are antibodies and antigen-binding fragments that comprise a CDRH1, a CDRH2, a CDRH3, a CDRL1, a CDRL2, and a CDRL3, wherein at least one of the CDRs is according to a variant antibody of the present disclosure and at least one of the CDRs (IMGT + CCG) is according to antibody S309 or Sotrovimab. In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises CDRH1, CDRH3, CDRL1, CDRL2, and CDRL3 of an exemplary antibody as shown in Table 2, and comprises a variant CDRH2 wherein the sequence ISTYNG is replaced by the amino acid sequence ISTYSG or the amino acid sequence ISTYQG. In some embodiments, an antibody or antigen-binding fragment comprises a variant of a disclosed CDRH2, wherein a NG amino acid motif is replaced by a different amino acid motif; e.g., XG wherein X is not N, or NX, wherein X is not G. Without wishing to be bound by theory, a NG motif can be labile for deamination at the N, which may be undesirable in some contexts.

In certain embodiments, an antibody or antigen-binding fragment comprises the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of SEQ ID NOs.: (i) 39, 40, 41, 44, 45, and 46, respectively; (ii) 49, 50, 51, 54, 55, and 56, respectively; (iii) 59, 60, 61, 64, 65, and 66, respectively; (iv) 69, 70, 71, 74, 75, and 76, respectively; (v) 79, 80, 81, 84, 85, and 86, respectively; (vi) 89, 90, 91, 94, 95, and 96, respectively; (vii) 99, 100, 101, 104, 105, and 106, respectively; (viii) 111, 112, 113, 116, 117, and 118, respectively; (ix) 121, 122, 123, 126, 127, and 128, respectively; (x) 131, 132, 133, 136, 137, and 138, respectively; (xi) 141, 142, 143, 146, 147, and 148, respectively; (xi) 151, 152, 153, 156, 157, and 158, respectively; (xii) 161, 162, 163, 166, 167, and 168, respectively; (xiii) 171, 172, 173, 176, 177, and 178, respectively; (xiv) 181, 182, 183, 186, 187, and 188, respectively; (xv) 191, 192, 193, 196, 197, and 198, respectively; (xvi) 201, 202, 203, 206, 207, and 208, respectively; (xvii) 211, 212, 213, 216, 217, and 218, respectively; (xviii) 221, 222, 223, 226, 227, and 228, respectively; (xix) 231, 232, 233, 236, 237, and 238, respectively; (xx) 241, 242, 243, 246, 247, and 248, respectively; (xxi) 251, 252, 253, 256, 257, and 258, respectively; (xxii) 261, 262, 263, 266, 267, and 268, respectively; (xxiii) 271, 272, 273, 276, 277, and 278, respectively; (xxiv) 281, 282, 283, 286, 287, and 288, respectively; (xxiv) 291, 292, 293, 296, 297, and 298, respectively; (xxv) 301, 302, 303, 306, 307, and 308, respectively; (xxvi) 311, 312, 313, 316, 317, and 318, respectively; (xxvii) 321, 322, 323, 326, 327, and 328, respectively; (xxviii) 331, 332, 333, 336, 337, and 338, respectively, (xxix) 341, 342, 343, 346, 347, and 348, respectively, (xxx) 351, 352, 353, 356, 357, and 358, respectively, (xxxi) 361, 362, 363, 366, 367, and 368, respectively, (xxxii) 371, 372, 373, 376, 377, and 378, respectively, (xxxiii) 381, 382, 383, 386, 387, and 388, respectively, or (xxxiv) 391, 392, 393, 396, 397, and 398, respectively.

In some embodiments, an isolated antibody or antigen-binding fragment is provided that comprises: (i) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 39, 40, and 41, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 44, 45, and 46; (ii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 49, 50, and 51, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 54, 55, and 56; (iii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 59, 60, and 61, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 64, 65, and 66; (iv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 69, 70, and 71, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 74, 75, and 76; (v) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 79, 80, and 81, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 84, 85, and 86; (vi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 89, 90, and 91, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 94, 95, and 96; (vii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 99, 100, and 101, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 104, 105, and 106; (viii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 111, 112, and 113, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:l 16, 117, and 118; (ix) in a VH, the amino acid sequences set forth in SEQ ID N0s.:121, 122, and 123, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 126, 127, and 128; (x) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 131, 132, and 133, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:136, 137, and 138; (xi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 141, 142, and 143, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:146, 147, and 148; (xi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:151, 152, and 153, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:156, 157, and 158; (xii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 161, 162, and 163, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:166, 167, and 168; (xiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:171, 172, and 173, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:176, 177, and 178; (xiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 181, 182, and 183, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:186, 187, and 188; (xv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 191, 192, and 193, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:196, 197, and 198; (xvi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:201, 202, and 203, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:206, 207, and 208; (xvii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:211, 212, and 213, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:216, 217, and 218; (xviii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:221, 222, and 223, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:226, 227, and 228; (xix) in a VH, the amino acid sequences set forth in SEQ ID NOs.:231, 232, and 233, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:236, 237, and 238; (xx) in a VH, the amino acid sequences set forth in SEQ ID NOs.:241, 242, and 243, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:246, 247, and 248; (xxi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:251, 252, and 253, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:256, 257, and 258; (xxii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:261, 262, and 263, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:266, 267, and 268; (xxiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:271, 272, and 273, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:276, 277, and 278; (xxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:281, 282, and 283, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:286, 287, and 288; (xxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:291, 292, and 293, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:296, 297, and 298; (xxv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:301, 302, and 303, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:306, 307, and 308; (xxvi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:311, 312, and 313, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:316, 317, and 318; (xxvii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:321, 322, and 323, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:326, 327, and 328; (xxviii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:331, 332, and 333, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:336, 337, and 338, (xxix) in a VH, the amino acid sequences set forth in SEQ ID NOs.:341, 342, and 343, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:346, 347, and 348, (xxx) in a VH, the amino acid sequences set forth in SEQ ID NOs.:351, 352, and 353, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:356, 357, and 358, (xxxi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:361, 362, and 363, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:366, 367, and 368, (xxxii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:371, 372, and 373, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:376, 377, and 378, (xxxiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:381, 382, and 383, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:386, 387, and 388, or (xxxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:391, 392, and 393, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:396, 397, and 398.

In further embedments, the antibody or antigen-binding fragment comprises a VH having at least 85% identity (z.e., at least 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to a VH amino acid sequence provided in Table 3, and/or a VL having at least 85% identity (i.e., at least 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to a VL amino acid sequence provided in Table 3. In still further embedments, the antibody or antigen-binding fragment comprises a VH having at least 90% identity identity to a VH amino acid sequence provided in Table 3, and/or a VL having at least 90% identity to a VL amino acid sequence provided in Table 3. In still further embedments, the antibody or antigenbinding fragment comprises a VH having at least 92% identity identity to a VH amino acid sequence provided in Table 3, and/or a VL having at least 92% identity to a VL amino acid sequence provided inTable 3. In still further embedments, the antibody or antigen-binding fragment comprises a VH having at least 95% identity identity to a VH amino acid sequence provided inTable 3, and/or a VL having at least 95% identity to a VL amino acid sequence provided in Table 3. In still further embedments, the antibody or antigen-binding fragment comprises a VH having at least 97% identity identity to a VH amino acid sequence provided in Table 3, and/or a VL having at least 97% identity to a VL amino acid sequence provided in Table 3. In still further embedments, the antibody or antigen-binding fragment comprises a VH having at least 99% identity identity to a VH amino acid sequence provided in Table 3, and/or a VL having at least 99% identity to a VL amino acid sequence provided in Table 3.

In some embodiments, the antibody or antigen-binding fragment comprises a VH amino acid sequence selected from the VH amino acid sequences provided in Table 3, and a VL amino acid sequence selected from the VL amino acid sequences provided in Table 3. In some embodiments, the antibody or antigen-binding fragment comprises a VH amino acid sequence selected from the VH amino acid sequences provided in Table 3, and the VL amino acid sequence of SEQ ID NO.:34. In some embodiments, the antibody or antigen-binding fragment comprises a VL amino acid sequence selected from the VL amino acid sequences provided in Table 3, and the VH amino acid sequence of SEQ ID NO.:30. In some embodiments, the antibody or antigen-binding fragment comprises a VL amino acid sequence selected from the VL amino acid sequences provided in Table 3, and the VH amino acid sequence of SEQ ID NO.:22. In some embodiments, the antibody or antigen-binding fragment comprises a VH amino acid sequence selected from the VH amino acid sequences provided in Table 3, and the VL amino acid sequence of SEQ ID NO.:26.

Table 3. Variable Domain Amino Acid Sequences of Certain Antibodies of the Present Disclosure.

In some embodiments, the antibody or antigen-binding fragment comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs.:22, 30, 38, 48, 58, 68, 78, 98, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310,

320, 330, 340, 350, 360, 370, 380, and 390, and the VL comprises an amino acid sequence having at least 85% identity to any one of SEQ ID NOs.:34, 43, 53, 63, 78, 83, 93, 103, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, and 395. In some embodiments, the antibody or antigen-binding fragment comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs.:22, 30, 38, 48, 58, 68, 78, 98, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, and 390, and the VL comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs.:34, 43, 53, 63, 78, 83, 93, 103, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, and 395. In some embodiments, the antibody or antigen-binding fragment comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs.:22, 30, 38, 48, 58, 68, 78, 98, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, and 390, and the VL comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs.:34, 43, 53, 63, 78, 83, 93, 103, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, and 395. In some embodiments, the antibody or antigen-binding fragment comprises a VH and a VL, wherein the VH comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs.:22, 30, 38, 48, 58, 68, 78, 98, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, and 390, and the VL comprises an amino acid sequence having at least 99% identity to any one of SEQ ID NOs.:34, 43, 53, 63, 78, 83, 93, 103, 115, 125, 135, 145, 155, 165, 175, 185, 195, 205, 215, 225, 235, 245, 255, 265, 275, 285, 295, 305, 315, 325, 335, 345, 355, 365, 375, 385, and 395.

In some embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:38 and the VL amino acid sequence of SEQ ID NO.:43. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:48 and the VL amino acid sequence of SEQ ID NO.: 53. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:58 and the VL amino acid sequence of SEQ ID NO.:63. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:68 and the VL amino acid sequence of SEQ ID NO.:73. In other embodiments, the antibody or antigenbinding fragment comprises the VH amino acid sequence of SEQ ID NO.: 78 and the VL amino acid sequence of SEQ ID NO.: 83. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:88 and the VL amino acid sequence of SEQ ID NO.:93. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:98 and the VL amino acid sequence of SEQ ID NO.: 103. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.: 110 and the VL amino acid sequence of SEQ ID NO.: 115. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO. : 120 and the VL amino acid sequence of SEQ ID NO. : 125. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.: 130 and the VL amino acid sequence of SEQ ID NO. : 135. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO. : 140 and the VL amino acid sequence of SEQ ID NO. : 145. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.: 150 and the VL amino acid sequence of SEQ ID NO.: 155. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.: 160 and the VL amino acid sequence of SEQ ID NO.: 165. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.: 170 and the VL amino acid sequence of SEQ ID NO.: 175. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.: 180 and the VL amino acid sequence of SEQ ID NO.: 185. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO. : 190 and the VL amino acid sequence of SEQ ID NO. : 195. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:200 and the VL amino acid sequence of SEQ ID NO.:205. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:210 and the VL amino acid sequence of SEQ ID NO.:215. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:220 and the VL amino acid sequence of SEQ ID NO.:225. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:230 and the VL amino acid sequence of SEQ ID NO.:235. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO. :240 and the VL amino acid sequence of SEQ ID NO. :245. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:250 and the VL amino acid sequence of SEQ ID NO.:255. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:260 and the VL amino acid sequence of SEQ ID NO.:265. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:270 and the VL amino acid sequence of SEQ ID NO.:275. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:280 and the VL amino acid sequence of SEQ ID NO.:285. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:290 and the VL amino acid sequence of SEQ ID NO.:295. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:300 and the VL amino acid sequence of SEQ ID NO.:305. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:310 and the VL amino acid sequence of SEQ ID NO.:315. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:320 and the VL amino acid sequence of SEQ ID NO.:325. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:330 and the VL amino acid sequence of SEQ ID NO.:335. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:340 and the VL amino acid sequence of SEQ ID NO.:345. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:350 and the VL amino acid sequence of SEQ ID NO.:355. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:360 and the VL amino acid sequence of SEQ ID NO.:365. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:370 and the VL amino acid sequence of SEQ ID NO.:375. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:380 and the VL amino acid sequence of SEQ ID NO.:385. In other embodiments, the antibody or antigen-binding fragment comprises the VH amino acid sequence of SEQ ID NO.:390 and the VL amino acid sequence of SEQ ID NO.:395.

In certain embodiments, an antibody or antigen-binding fragment further comprises a CH1-CH3 amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO.:6, SEQ ID NO.:7, SEQ ID NO.:108, or SEQ ID NO.: 109, and/or a CL amino acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% to SEQ ID NO.:9 or 8, and preferably according to SEQ ID NO.:9.

In certain embodiments, an antibody or antigen-binding fragment further comprises a CH1-CH3 amino acid sequence according to SEQ ID NO.:6, SEQ ID NO.:7, SEQ ID NO.: 108, or SEQ ID NO.: 109, and/or a CL amino acid sequence according to SEQ ID NO.:9 or 8, and preferably according to SEQ ID NO.:9.

In some embodiments, an antibody comprises two heavy chains and two light chains, wherein each of the two heavy chains comprises or consists of the same VH+CH1-CH3 amino acid sequence and each of the two light chains comprises or consists of the same VL+CL amino acid sequence.

It will be understood that, for example, production in a mammalian cell line can remove one or more C-terminal lysine (or glycine-lysine motif) of an antibody heavy chain (see, e.g., Liu et al. mAbs 6(5): 1145-1154 (2014)). Accordingly, an antibody or antigen-binding fragment of the present disclosure can comprise a heavy chain, a CH1- CH3, a CH3, or an Fc polypeptide wherein a C-terminal lysine residue (or glycinelysine motif) is present or is absent; in other words, encompassed are embodiments where the C-terminal residue of a heavy chain, a CH1-CH3, or an Fc polypeptide is not a lysine or wherein a C-terminal glycine-lysine motif is not present, and embodiments where a lysine is the C-terminal residue or whereni a C-terminal glycine-lysine motif is present. In certain embodiments, a composition comprises a plurality of an antibody and/or an antigen-binding fragment of the present disclosure, wherein one or more antibody or antigen-binding fragment does not comprise a lysine residue or glycine- lysine motif at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein one or more antibody or antigen-binding fragment comprises a lysine residue or glycine-lysine motif at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide.

A "Fab" (fragment antigen binding) is the part of an antibody that binds to antigens and includes the variable region and CHI of the heavy chain linked to the light chain via an inter-chain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, z.e., it has a single antigen-binding site. Pepsin treatment of an antibody yields a single large F(ab')2 fragment that roughly corresponds to two disulfide linked Fab fragments having divalent antigen-binding activity and is still capable of cross-linking antigen. Both the Fab and F(ab')2 are examples of "antigenbinding fragments." Fab' fragments differ from Fab fragments by having additional few residues at the carboxy terminus of the CHI domain including one or more cysteines from the antibody hinge region. Fab'-SH is the designation herein for Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments originally were produced as pairs of Fab' fragments that have hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

Fab fragments may be joined, e.g., by a peptide linker, to form a single chain Fab, also referred to herein as "scFab". In these embodiments, an inter-chain disulfide bond that is present in a native Fab may not be present, and the linker serves in full or in part to link or connect the Fab fragments in a single polypeptide chain. A heavy chain- derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VH + CHI, or "Fd") and a light chain-derived Fab fragment (e.g., comprising, consisting of, or consisting essentially of VL + CL) may be linked in any arrangement to form a scFab. For example, a scFab may be arranged, in N-terminal to C-terminal direction, according to (heavy chain Fab fragment - linker - light chain Fab fragment) or (light chain Fab fragment - linker - heavy chain Fab fragment). Peptide linkers and exemplary linker sequences for use in scFabs are discussed in further detail herein.

"Fv" is a small antibody fragment that contains a complete antigen-recognition and antigen-binding site. This fragment generally consists of a dimer of one heavy- and one light-chain variable region domain in tight, non-covalent association. However, even a single variable domain (or half of an Fv comprising only three CDRs specific for an antigen) has the ability to recognize and bind antigen, although typically at a lower affinity than the entire binding site.

"Single-chain Fv" also abbreviated as "sFv" or "scFv", are antibody fragments that comprise the VH and VL antibody domains connected into a single polypeptide chain. In some embodiments, the scFv polypeptide comprises a polypeptide linker disposed between and linking the VH and VL domains that enables the scFv to retain or form the desired structure for antigen binding. Such a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In certain embodiments, the antibody or antigen-binding fragment comprises a scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to the VL domain. In particular embodiments, a scFv comprises a VH domain linked to a VL domain by a peptide linker, which can be in a VH-linker- VL orientation or in a VL-linker-VH orientation. Any scFv of the present disclosure may be engineered so that the C-terminal end of the VL domain is linked by a short peptide sequence to the N-terminal end of the VH domain, or vice versa (z.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some embodiments, a linker may be linked to an N-terminal portion or end of the VH domain, the VL domain, or both. Peptide linker sequences may be chosen, for example, based on: (1) their ability to adopt a flexible extended conformation; (2) their inability or lack of ability to adopt a secondary structure that could interact with functional epitopes on the first and second polypeptides and/or on a target molecule; and/or (3) the lack or relative lack of hydrophobic or charged residues that might react with the polypeptides and/or target molecule. Other considerations regarding linker design (e.g., length) can include the conformation or range of conformations in which the VH and VL can form a functional antigen-binding site. In certain embodiments, peptide linker sequences contain, for example, Gly, Asn and Ser residues. Other near neutral amino acids, such as Thr and Ala, may also be included in a linker sequence. Other amino acid sequences which may be usefully employed as linker include those disclosed in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Pat. No. 4,935,233, and U.S. Pat. No. 4,751,180. Other illustrative and non-limiting examples of linkers may include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys- Val-Asp (SEQ ID NO: 19) (Chaudhary et al., Proc. Natl. Acad. Sci. USA 87: 1066- 1070 (1990)) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg- Ser-Leu-Asp (SEQ ID NO: 20) (Bird et al., Science 242:423-426 (1988)) and the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 21) when present in a single iteration or repeated 1 to 5 or more times, or more; see, e.g., SEQ ID NO: 17. Any suitable linker may be used, and in general can be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 15 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments, or modules connected by the linker), and will preferably be biologically inert and/or have a low risk of immunogenicity in a human. Exemplary linkers include those comprising or consisting of the amino acid sequence set forth in any one or more of SEQ ID NOs: 10-21. In certain embodiments, the linker comprises or consists of an amino acid sequence having at least 75% (i.e., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 10-21. scFv can be constructed using any combination of the VH and VL sequences or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein.

In some embodiments, linker sequences are not required; for example, when the first and second polypeptides have non-essential N-terminal amino acid regions that can be used to separate the functional domains and prevent steric interference.

During antibody development, DNA in the germline variable (V), joining (J), and diversity (D) gene loci may be rearranged and insertions and/or deletions of nucleotides in the coding sequence may occur. Somatic mutations may be encoded by the resultant sequence, and can be identified by reference to a corresponding known germline sequence. In some contexts, somatic mutations that are not critical to a desired property of the antibody (e.g., binding to a SARS-CoV-2 antigen), or that confer an undesirable property upon the antibody (e.g., an increased risk of immunogenicity in a subject administered the antibody), or both, may be replaced by the corresponding germline-encoded amino acid, or by a different amino acid, so that a desirable property of the antibody is improved or maintained and the undesirable property of the antibody is reduced or abrogated. Thus, in some embodiments, the antibody or antigen-binding fragment of the present disclosure comprises at least one more germline-encoded amino acid in a variable region as compared to a parent antibody or antigen-binding fragment, provided that the parent antibody or antigen binding fragment comprises one or more somatic mutations.

In certain embodiments, an antibody or antigen-binding fragment comprises an amino acid modification (e.g., a substitution mutation) to remove an undesired risk of oxidation, deamidation, and/or isomerization.

Also provided herein are variant antibodies that comprise one or more amino acid alterations in a variable region (e.g., VH, VL, framework or CDR) as compared to a presently disclosed antibody, wherein the variant antibody is capable of binding to a SARS-CoV-2 antigen. In certain embodiments, an antibody or antigen-binding fragment of the present disclosure is monospecific (e.g., binds to a single epitope) or is multispecific (e.g., binds to multiple epitopes and/or target molecules). Antibodies and antigen binding fragments may be constructed in various formats. Exemplary antibody formats disclosed in Spiess et al., Mol. Immunol. 67(2):95 (2015), and in Brinkmann and Kontermann, mAbs 9(2): 182-212 (2017), which formats and methods of making the same are incorporated herein by reference and include, for example, Bispecific T cell Engagers (BiTEs), DARTs, Knobs-Into-Holes (KIH) assemblies, scFv-CH3-KIH assemblies, KIH Common Light-Chain antibodies, TandAbs, Triple Bodies, TriBi Minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, Intrabodies, CrossMabs, Dual Action Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IgG, Charge Pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LUZ-Y assemblies, Fcabs, Kk-bodies, orthogonal Fabs, DVD-Igs (e.g., US Patent No. 8,258,268, which formats are incorporated herein by reference in their entirety), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)- IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVLIgG (four-in-one), as well as so-called FIT-Ig (e.g., PCT Publication No. WO 2015/103072, which formats are incorporated herein by reference in their entirety), so- called WuxiBody formats (e.g., PCT Publication No. WO 2019/057122, which formats are incorporated herein by reference in their entirety), and so-called In-Elbow-Insert Ig formats (lELIg; e.g., PCT Publication Nos. WO 2019/024979 and WO 2019/025391, which formats are incorporated herein by reference in their entirety).

In certain embodiments, the antibody or antigen-binding fragment comprises two or more of VH domains, two or more VL domains, or both (z.e., two or more VH domains and two or more VL domains). In particular embodiments, an antigen-binding fragment comprises the format (N-terminal to C-terminal direction) VH-linker-VL- linker-VH-linker-VL, wherein the two VH sequences can be the same or different and the two VL sequences can be the same or different. Such linked scFvs can include any combination of VH and VL domains arranged to bind to a given target, and in formats comprising two or more VH and/or two or more VL, one, two, or more different eptiopes or antigens may be bound. It will be appreciated that formats incorporating multiple antigen-binding domains may include VH and/or VL sequences in any combination or orientation. For example, the antigen-binding fragment can comprise the format VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH, or VL-linker- VH-linker- VH-linker- VL .

Monospecific or multispecific antibodies or antigen-binding fragments of the present disclosure constructed comprise any combination of the VH and VL sequences and/or any combination of the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 sequences disclosed herein. A bispecific or multispecific antibody or antigenbinding fragment may, in some embodiments, comprise one, two, or more antigenbinding domains (e.g., a VH and a VL) of the instant disclosure. Two or more binding domains may be present that bind to the same or a different SARS-CoV-2 epitope, and a bispecific or multispecific antibody or antigen-binding fragment as provided herein can, in some embodiments, comprise a further SARS-CoV-2 binding domain, and/or can comprise a binding domain that binds to a different antigen or pathogen altogether.

In any of the presently disclosed embodiments, the antibody or antigen-binding fragment can be multispecific; e.g., bispecific, trispecific, or the like.

In certain embodiments, the antibody or antigen-binding fragment comprises a Fc polypeptide, or a fragment thereof. The "Fc" fragment or Fc polypeptide comprises the carboxy -terminal portions (i.e., the CH2 and CH3 domains of IgG) of both antibody H chains held together by disulfides. Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g., B cell receptor); and B cell activation. As discussed herein, modifications (e.g., amino acid substitutions) may be made to an Fc domain in order to modify (e.g., improve, reduce, or ablate) one or more functionality of an Fc-containing polypeptide (e.g., an antibody of the present disclosure). Such functions include, for example, Fc receptor (FcR) binding, antibody half-life modulation (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Amino acid modifications that modify (e.g., improve, reduce, or ablate) Fc functionalities include, for example, the T250Q/M428L, M252Y/S254T/T256E, H433K/N434F, M428L/N434S, E233P/L234V/L235A/G236 + A327G/A330S/P331S, E333A, S239D/A330L/I332E, P257I/Q311, K326W/E333S, S239D/I332E/G236A, N297Q, K322A, S228P, L235E + E318A/K320A/K322A, L234A/L235A (also referred to herein as “LALA”), and L234A/L235A/P329G mutations, which mutations are summarized and annotated in "Engineered Fc Regions", published by InvivoGen (2011) and available online at invivogen.com/PDF/review/review-Engineered-Fc-Regions- invivogen.pdf?utm_source=review&utm_medium=pdf&utm_ campaign=review&utm_content=Engineered-Fc-Regions, and are incorporated herein by reference.

For example, to activate the complement cascade, the Clq protein complex can bind to at least two molecules of IgGl or one molecule of IgM when the immunoglobulin molecule(s) is attached to the antigenic target (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R., described (MoL Immunol. 22 (1985) 161-206) that the heavy chain region comprising amino acid residues 318 to 337 is involved in complement fixation. Duncan, A. R., and Winter, G. (Nature 332 (1988) 738-740), using site directed mutagenesis, reported that Glu318, Lys320 and Lys322 form the binding site to Clq. The role of Glu318, Lys320 and Lys 322 residues in the binding of Clq was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement mediated lysis.

For example, FcR binding can be mediated by the interaction of the Fc moiety (of an antibody) with Fc receptors (FcRs), which are specialized cell surface receptors on cells including hematopoietic cells. Fc receptors belong to the immunoglobulin superfamily, and shown to mediate both the removal of antibody-coated pathogens by phagocytosis of immune complexes, and the lysis of erythrocytes and various other cellular targets (e.g., tumor cells) coated with the corresponding antibody, via antibody dependent cell mediated cytotoxicity (ADCC; Van de Winkel, J. G., and Anderson, C. L., J. Leukoc. Biol. 49 (1991) 511-524). FcRs are defined by their specificity for immunoglobulin classes; Fc receptors for IgG antibodies are referred to as FcyR, for IgE as FcsR, for IgA as FcaR and so on and neonatal Fc receptors are referred to as FcRn. Fc receptor binding is described for example in Ravetch, J. V., and Kinet, J. P., Annu. Rev. Immunol. 9 (1991) 457-492; Capel, P. J., et al., Immunomethods 4 (1994) 25-34; de Haas, M., et al., J Lab. Clin. Med. 126 (1995) 330-341; and Gessner, J. E., et al., Ann. Hematol. 76 (1998) 231-248.

Cross-linking of receptors by the Fc domain of native IgG antibodies (FcyR) triggers a wide variety of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity, and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. Fc moi eties providing crosslinking of receptors (e.g., FcyR) are contemplated herein. In humans, three classes of FcyR have been characterized to-date, which are: (i) FcyRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcyRII (CD32), which binds complexed IgG with medium to low affinity, is widely expressed, in particular on leukocytes, is believed to be a central player in antibody-mediated immunity, and which can be divided into FcyRIIA, FcyRIIB and FcyRIIC, which perform different functions in the immune system, but bind with similar low affinity to the IgG-Fc, and the ectodomains of these receptors are highly homologuous; and (iii) FcyRIII (CD 16), which binds IgG with medium to low affinity and has been found in two forms: FcyRIIIA, which has been found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is believed to mediate ADCC; and FcyRIIIB, which is highly expressed on neutrophils.

FcyRIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and seems able to activate the killing process. FcyRIIB seems to play a role in inhibitory processes and is found on B-cells, macrophages and on mast cells and eosinophils. Importantly, it has been shown that 75% of all FcyRIIB is found in the liver (Ganesan, L. P. et al., 2012: “FcyRIIb on liver sinusoidal endothelium clears small immune complexes,” Journal of Immunology 189: 4981-4988). FcyRIIB is abundantly expressed on Liver Sinusoidal Endothelium, called LSEC, and in Kupffer cells in the liver and LSEC are the major site of small immune complexes clearance (Ganesan, L. P. et al., 2012: FcyRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

In some embodiments, the antibodies disclosed herein and the antigen-binding fragments thereof comprise an Fc polypeptide or fragment thereof for binding to FcyRIIb, in particular an Fc region, such as, for example IgG-type antibodies. Moreover, it is possible to engineer the Fc moiety to enhance FcyRIIB binding by introducing the mutations S267E and L328F as described by Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933. Thereby, the clearance of immune complexes can be enhanced (Chu, S., et al., 2014: Accelerated Clearance of IgE In Chimpanzees Is Mediated By Xmab7195, An Fc-Engineered Antibody With Enhanced Affinity For Inhibitory Receptor FcyRIIb. Am J Respir Crit, American Thoracic Society International Conference Abstracts). In some embodiments, the antibodies of the present disclosure, or the antigen binding fragments thereof, comprise an engineered Fc moiety with the mutations S267E and L328F, in particular as described by Chu, S. Y. et al., 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933.

On B cells, FcyRIIB may function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcyRIIB is thought to inhibit phagocytosis as mediated through FcyRIIA. On eosinophils and mast cells, the B form may help to suppress activation of these cells through IgE binding to its separate receptor.

Regarding FcyRI binding, modification in native IgG of at least one of E233- G236, P238, D265, N297, A327 and P329 reduces binding to FcyRI. IgG2 residues at positions 233-236, substituted into corresponding positions IgGl and IgG4, reduces binding of IgGl and IgG4 to FcyRI by 10³-fold and eliminated the human monocyte response to antibody-sensitized red blood cells (Armour, K. L., et al. Eur. J. Immunol. 29 (1999) 2613-2624).

Regarding FcyRII binding, reduced binding for FcyRIIA is found, e.g., for IgG mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292 and K414.

Two allelic forms of human FcyRIIA are the "H131" variant, which binds to IgGl Fc with high affinity, and the "R131" variant, which binds to IgGl Fc with low affinity. See, e.g., Bruhns et al., Blood 773:3716-3725 (2009).

Regarding FcyRIII binding, reduced binding to FcyRIIIA is found, e.g., for mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338 and D376. Mapping of the binding sites on human IgGl for Fc receptors, the above-mentioned mutation sites, and methods for measuring binding to FcyRI and FcyRIIA, are described in Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604.

Two allelic forms of human FcyRIIIA are the "Fl 58" variant, which binds to IgGl Fc with low affinity, and the “VI 58” variant, which binds to IgGl Fc with high affinity. See, e.g., Bruhns et al., Blood 773:3716-3725 (2009).

Regarding binding to FcyRII, two regions of native IgG Fc appear to be involved in interactions between FcyRIIs and IgGs, namely (i) the lower hinge site of IgG Fc, in particular amino acid residues L, L, G, G (234 - 237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, in particular a loop and strands in the upper CH2 domain adjacent to the lower hinge region, e.g., in a region of P331 (Wines, B.D., et al., I. Immunol. 2000; 164: 5313 - 5318). Moreover, FcyRI appears to bind to the same site on IgG Fc, whereas FcRn and Protein A bind to a different site on IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B.D., et al., I. Immunol. 2000; 164: 5313 - 5318).

Also contemplated are mutations that increase binding affinity of an Fc polypeptide or fragment thereof of the present disclosure to a (i.e., one or more) Fey receptor (e.g., as compared to a reference Fc polypeptide or fragment thereof or containing the same that does not comprise the mutation(s)). See, e.g., Delillo and Ravetch, Cell 161(5): 1035-1045 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016), the Fc mutations and techniques of which are incorporated herein by reference.

In any of the herein disclosed embodiments, an antibody or antigen-binding fragment can comprise a Fc polypeptide or fragment thereof comprising a mutation selected from G236A; S239D; A330L; and I332E; or a combination comprising any two or more of the same; e.g., S239D/I332E; S239D/A330L/I332E; G236A/S239D/I332E; G236A/A330L/I332E (also referred to herein as "GAALIE"); or G236A/S239D/A330L/I332E. In some embodiments, the Fc polypeptide or fragment thereof does not comprise S239D. In some embodiments, the Fc polypeptide or fragment thereof comprises S at position 239.

In certain embodiments, the Fc polypeptide or fragment thereof may comprise or consist of at least a portion of an Fc polypeptide or fragment thereof that is involved in binding to FcRn binding. In certain embodiments, the Fc polypeptide or fragment thereof comprises one or more amino acid modifications that improve binding affinity for (e.g., enhance binding to) FcRn (e.g., at a pH of about 6.0) and, in some embodiments, thereby extend in vivo half-life of a molecule comprising the Fc polypeptide or fragment thereof (e.g., as compared to a reference Fc polypeptide or fragment thereof or antibody that is otherwise the same but does not comprise the modification(s)). In certain embodiments, the Fc polypeptide or fragment thereof comprises or is derived from a IgG Fc and a half-life-extending mutation comprises any one or more of: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I Q31 II; D376V; T307A; E380A (EU numbering). In certain embodiments, a half-life-extending mutation comprises M428L/N434S (also referred to herein as "MLNS" and "LS"). In certain embodiments, a half-life-extending mutation comprises M428L/N434A (also referred to herein as "MLNA" and "LA"). In certain embodiments, a half-life-extending mutation comprises M252Y/S254T/T256E. In certain embodiments, a half-life-extending mutation comprises T250Q/M428L. In certain embodiments, a half-life-extending mutation comprises P257I/Q311I. In certain embodiments, a half-life-extending mutation comprises P257I/N434H. In certain embodiments, a half-life-extending mutation comprises D376V/N434H. In certain embodiments, a half-life-extending mutation comprises T307A/E380A/N434A.

In some embodiments, an antibody or antigen-binding fragment includes a Fc moiety that comprises the substitution mutations M428L/N434S or M428L/N434A. In some embodiments, an antibody or antigen-binding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations G236A/A330L/I332E. In certain embodiments, an antibody or antigen-binding fragment includes a (e.g., IgG) Fc moiety that comprises a G236A mutation, an A330L mutation, and a I332E mutation (GAALIE), and does not comprise a S239D mutation (e.g., comprises a native S at position 239). In particular embodiments, an antibody or antigen-binding fragment includes an Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S (or M428L/N434A) and G236A/A330L/I332E, and optionally does not comprise S239D. In certain embodiments, an antibody or antigenbinding fragment includes a Fc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S (or M428L/N434A) and G236 A/S239D/A330L/I332E.

An antibody or antigen-binding fragment of the present disclosure may be of any allotype or combination of allotypes. “Allotype” refers to the allelic variation found among the IgG subclasses. For example, an allotype may comprise Glml (or Glm(a)), Glm2 (or Glm(x)), Glm3 (or Glm(f)), Glml7 (or Gm(z))m, Glm27, and/or Glm28 (Glm27 and Glm28 have been described as “alloallotypes”). The Glm3 and Glml7 allotypes are located at the same position in the CHI domain (position 214 according to EU numbering). Glm3 comprises R214 (EU), while Glml7 comprises K214 (EU). The Glml allotype is located in the CH3 domain (at positions 356 and 358 (EU)) and refers to the replacements E356D and M358L. The Glm2 allotype refers to a replacement of the alanine in position 431 (EU) by a glycine. Glm allotypes, alloallotypes, and features thereof are known in the art and described at, for example, www.imgt.org/IMGTrepertoire/Proteins/allotypes/human/IGH/IGHC/Glm_allotypes.ht ml and Lefranc, M.-P. and Lefranc, G. Human Gm, Km and Am allotypes and their molecular characterization: a remarkable demonstration of polymorphism In: B. Tait, F. Christiansen (Eds.), Immunogenetics, chap. 34, Humana Press, Springer, New York, USA. Methods Mol. Biol. 2012; 882, 635-680. PMID: 22665258, LIGM: 406, the contents and allotypes and allotype information of which are incorporated herein by reference.

The Glml allotype may be combined, for example, with the Glm3, Glml7, Glm27, Glm2, and/or Glm28 allotype. In some embodiments, an allotype is Glm3 with no Glml (Glm3,-1). In some embodiments, an allotype is Glml7,l allotype. In some embodiments, an allotype is Glm3,l. In some embodiments, an allotype is Glml7 with no Glml (Glml7,-1). Optionally, these allotypes may be combined (or not combined) with the Glm2, Glm27 or Glm28 allotype. For example, an allotype may be Glml7,l,2.

In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a Glm3 allotype. In some embodiments, an antibody or antigenbinding fragment of the present disclosure comprises a Glm3, allotype and comprises M428L and N434S mutations in CH3, as described further herein. In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a Glml7, 1 allotype. In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a Glml7, 1 allotype and comprises M428L and N434S or M428L and N434A mutations in CH3, as described further herein.

In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a CH1-CH3 amino acid sequence as set forth in any one of SEQ ID Nos.:6, 7, 108, and 109.

In certain embodiments, the antibody or antigen-binding fragment comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or the antibody or antigen-binding fragment is partially or fully aglycosylated and/or is partially or fully afucosylated. Host cell lines and methods of making partially or fully aglycosylated or partially or fully afucosylated antibodies and antigen-binding fragments are known (see, e.g., PCT Publication No. WO 2016/181357; Suzuki et al. Clin. Cancer Res. 73(6):1875-82 (2007); Huang et al. MAbs 6: 1-12 (2018)). Fucosylation of an Fc polypeptide or fragment thereof, or of an antibody, can be effected by introducing amino acid mutations to introduce or disrupt a fucosylation site; by expressing the antibody or antigen-binding fragment thereof in a host cell which has been genetically engineered to lack the ability (or have an inhibited or compromised ability) to fucosylate the antibody or antigen-binding fragment thereof; by expressing the antibody or antigen-binding fragment thereof under conditions in which a host cell is impaired in its ability to fucosylate the antibody or antigen-binding fragment thereof (e.g., in the presence of 2- fluoro-L-fucose (2FF)), or the like.

In certain embodiments, an antibody or antigen-binding fragment: is afucosylated; has been produced in a host cell that is incapable of fucosylation or that is inhibited in its ability to fucosylate the antibody or antigen-binding fragment thereof; has been produced under conditions that inhibit fucosylation thereof by a host cell; or any combination thereof.

In certain embodiments, an antibody or antigen-binding fragment comprises an amino acid mutation that (1) inhibits fucosylation as compared to a reference antibody or antigen-binding fragment (i.e. that is otherwise the same as the antibody or antigenbinding fragment), respectively, and/or (2) that abrogates a fucosylation site that is present in the reference antibody or antigen-binding fragment, respectively. In certain embodiments, the antibody or antigen-binding fragment is capable of eliciting continued protection in vivo in a subject even once no detectable levels of the antibody or antigen-binding fragment can be found in the subject (i.e., when the antibody or antigen-binding fragment has been cleared from the subject following administration). Such protection is referred to herein as a vaccinal effect. Without wishing to be bound by theory, it is believed that dendritic cells can internalize complexes of antibody and antigen and thereafter induce or contribute to an endogenous immune response against antigen. In certain embodiments, an antibody or antigen-binding fragment comprises one or more modifications, such as, for example, mutations in the Fc comprising G236A, A330L, and I332E, that are capable of activating dendritic cells that may induce, e.g., T cell immunity to the antigen. In certain embodiments, an antibody or antigen-binding fragment comprises a Fc variant (shown in the below table as fucosylated, unless otherwise indicated) as shown in the following table; see also International Application PCT/US2022/030556. In certain embodiments, an antibody or antigen-bindign fragment of the present disclosure comprises a Fc polypeptide comprising a mutation or combination of mutations disclosed in International Application PCT/US2022/030556, which mutations and combinations of mutations (and fucosylated and afucosylated antibodies and antigen-binding fragments comprising the same) are incorporated herein by reference.

Table of Certain Fc Variants and Properties Thereof

Additional features of disclosed Fc variant-containing antibodies and antigenbinding fragments are described herein.

In some embodiments, an antibody or antigen-binding fragment comprises, in an (e.g., human) IgGl heavy chain, the amino acid mutation(s) set forth in any one of (i)- (xviii): (i) G236A, L328V, and Q295E; (ii) G236A, P230A, and Q295E; (iii) G236A, R292P, and I377N; (iv) G236A, K334A, and Q295E; (v) G236S, R292P, and Y300L; (vi) G236A and Y300L; (vii) G236A, R292P, and Y300L; (viii) G236S, G420V, G446E, and L309T; (ix) G236A and R292P; (x) R292P and Y300L; (xi) G236A and R292P; (xii) Y300L; (xiii) E345K, G236S, L235Y, and S267E; (xiv) E272R, L309T, S219Y, and S267E; (xv) G236Y; (xvi) G236W; (xvii) F243L, G446E, P396L, and S267E; (xviii) G236A, S239D, and H268E, wherein the numbering of amino acid residues is according to the EU index as set forth in Kabat. In certain embodiments, the antibody or antigen-binding fragment is afucosylated. In some embodiments, the antibody or antigen-binding fragment further comprises one or more mutation that enhances binding to a human FcRn, such as M428L and N434S mutations or M428L and N434A mutations (EU numbering) or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody or antigen-binding fragment is afucosylated. In some embodiments, the IgGl heavy chain comprises a CH1-CH3 or a CH2-CH3 or a hinge-CH2-CH3, wherein the CH1-CH3 or CH2-CH3 or hinge-CH2-CH3 has at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity (or similarity) to a wild-type human IgGl CH1-CH3 or CH2-CH3 or hinge-CH2-CH3, respectively.

It will be understood that two or more amino acid substitutions present in a variant can be expressed in a variety of ways, for example, as G236A_Y300L, or as G236A/Y300L. Moreover, a mutation or combination mutation may be referenced using a short form including the original amino acid(s) and the amino acid(s) resulting from the substitution(s). For example, G236A may be described as “GA” or “236A”; G236A_Y300L may be described as “GAYL”; G236A_L328V_Q295E may be described as “GALVQE”; G236A R292P Y300L may be described as “GARPYL”, G236A R292P I377N may be described as “GARPIN”, or the like.

In certain embodiments, a variant of an Fc polypeptide comprises only the specified or recited amino acid mutations (e.g., substitutions), and does not comprise any further amino acid substitutions or mutations; e.g., relative to the reference polypeptide (e.g., a wild-type Fc polypeptide or fragment thereof). For example, in some embodiments, a variant Fc polypeptide comprising the amino acid substitutions G236A Y300L does not comprise any other amino acid substitutions; i.e., comprises an amino acid sequence that is wild-type except for G236A and Y300L. In some embodiments, a variant of an Fc polypeptide does not comprise R292P, does not comprise Y300L, or both.

In any of the presently disclosed embodiments, a variant of an Fc polypeptide or fragment thereof can be derived from or comprise a human Fc polypeptide or fragment thereof, and/or can be derived from or comprise a human IgGl, a human IgG2, a human IgG3, or a human IgG4 isotype. In this context, the expression “derived from” means that the variant is the same as the referenced polypeptide or isotype, except for the specified modification(s) (e.g., amino acid substitution(s)). By way of example, a variant Fc polypeptide which comprises a wild-type human IgGl Fc amino acid sequence except for the amino acid substitution mutations G236A L328V Q295E (and, optionally, other amino acid substitutions) can be said to be “derived from” wild-type human IgGl Fc. In any of the presently disclosed embodiments, a polypeptide, CH2, Fc, Fc fragment, antibody, or antigen-binding fragment may comprise human Ig sequence, such as human IgGl sequence. In some embodiments, the polypeptide, CH2, Fc, Fc fragment, antibody, or antigen-binding fragment can comprise a native or wildtype human Ig sequence with the exception of the described mutation(s), or can comprise a human Ig (e.g. IgG) sequence that contains one or more additional mutations.

In certain embodiments, an antibody of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a valine (V) at EU position 328, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GALVQE”). In some embodiments, the antibody further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the is afucosylated.

In certain other embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG hinge-CH2 polypeptide; or (ii) an IgG hinge-Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 230, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAPAQE”). In some embodiments, the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody or antigen-binding fragment is afucosylated.

In certain other embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and an asparagine (N) at EU position 377. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPIN”). In some embodiments, the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody is afucosylated.

In certain other embodiments, an antibody or antigen-binding fragment comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an alanine (A) at EU position 334, and a glutamic acid (E) at EU position 295. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAKAQE”). In some embodiments, the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody or antigen-binding fragment is afucosylated.

In certain other embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a serine (S) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSRPYL”). In some embodiments, the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody or antigen-binding fragment is afucosylated.

In certain other embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPYL”). In some embodiments, the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody or antigen-binding fragment is afucosylated.

In certain other embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAYL”). In some embodiments, the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody or antigen-binding fragment is afucosylated.

In certain other embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of: (i) an IgG CH2 polypeptide; or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, and a glutamic acid (E) at EU position 268. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GASDHE”). In some embodiments, the antibody or antigen-binding fragment further comprises the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein. In certain embodiments, the antibody or antigen-binding fragment is afucosylated.

Also provided is an antibody or antigen-binding fragment of the present disclosure comprising a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236 and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GAYL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, a proline (P) at EU position 292, and a leucine (L) at EU position 300. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARPYL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of an IgG Fc polypeptide, wherein the variant comprises a serine (S) at EU position 236, a valine (V) at EU position 420, a glutamic acid (E) at EU position 446, and a threonine (T) at EU position 309. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSGVGELT”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide, wherein the variant comprises an alanine (A) at EU position 236 and a proline (P) at EU position 292. In some embodiments, the antibody or antigen-binding fragment comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GARP”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide, wherein the variant comprises a proline (P) at EU position 292 and a leucine (L) at EU position 300, and wherein, optionally, variant and, further optionally, the antibody has increased binding to a human FcyRIIIa with as compared to the binding of a reference antibody to the human FcyRIIIa, wherein, optionally, the binding is as determined using an electrochemiluminescence assay, further optionally Meso Scale Discovery. In some embodiments, the antibody or antigen-binding fragment comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or IgG Fc polypeptide (“RPYL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a leucine (L) at EU position 300. In some embodiments, the IgG CH2 polypeptide or IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“YL”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a lysine (K) at EU position 345, a serine (S) at EU position 236, tyrosine (Y) at EU position 235, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GSEKLYSE”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG hinge-CH2 polypeptide or (ii) an IgG hinge-Fc polypeptide or a fragment thereof, wherein the variant comprises an arginine (R) at EU position 272, a threonine (T) at EU position 309, a tyrosine (Y) at EU position 219, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG hinge-CH2 polypeptide or an IgG hinge-Fc polypeptide or a fragment thereof comprises an (e.g. otherwise wild-type) IgGl hinge-CH2 polypeptide or IgG hinge-Fc polypeptide or a fragment thereof (“SYSEERLT”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a tyrosine (Y) at EU position 236. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g, otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GY”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a tryptophan (W) at EU position 236. In some embodiments, the IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof comprises an (e.g, otherwise wild-type) IgGl CH2 polypeptide or Fc polypeptide or fragment thereof (“GW”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, wherein the IgG Fc polypeptide or fragment thereof, and optionally the polypeptide, is afucosylated, and wherein, further optionally, the variant comprises a leucine (L) at EU position 330 and a glutamic acid (E) at EU postion 332, wherein, still further optionally, the variant does not comprise an aspartic acid (D) at EU position 239, and, even further optionally, comprises a serine (S) at EU position 239. In some embodiments, the IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof comprises an (e.g., otherwise wild-type) IgGl CH2 polypeptide or Fc polypeptide or fragment thereof (“GA-afuc” or “GAALIE-afuc”, respectively). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present.

Also provided is an antibody or antigen-binding fragment of the present disclosure that comprises a variant of an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises a leucine (L) at EU position 243, a glutamic acid (E) at EU position 446, a leucine (L) at EU position 396, and a glutamic acid (E) at EU position 267. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“FLSEPLGE”). In certain further embodiments, the mutations M428L and N434S, or the mutations M428L and N434A, or any other mutation(s) that enhance binding to a human FcRn, such as those described herein, are present. In certain embodiments, the antibody or antigen-binding fragment is afucoyslated.

Also provided is an antibody or antigen-binding fragment that comprises a variant of: (i) an IgG CH2 polypeptide or (ii) an IgG Fc polypeptide or a fragment thereof, wherein the variant comprises an alanine (A) at EU position 236, an aspartic acid (D) at EU position 239, a glutamic acid (E) and EU position 332, a leucine (L) at EU position 428, and a serine (S) or an alanine (A) at EU position 434. In some embodiments, the IgG Fc polypeptide or fragment thereof comprises an (e.g., otherwise wild-type) IgGl Fc polypeptide or fragment thereof (“GASDIEMLNS” or “GASDIEMLNA”).

In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of an (e.g. IgGl) IgG Fc polypeptide, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434S, G236A, L328V, and Q295E; (ii) M428L, N434S, G236A, R292P, and I377N; (iii) M428L, N434S, G236A, and Y300L; (iv) M428L, N434S, G236A, R292P, and Y300L; (v) M428L, N434S, G236A, L328V, and Q295E, wherein the antibody or antigenbinding fragment or antigen-binding fragment is afucosylated; (vi) M428L, N434S, G236A, R292P, and I377N, wherein the antibody or antigen-binding fragment is afucosylated; (vii) M428L, N434S, G236A, and Y300L, wherein the antibody or antigen-binding fragment is afucosylated; or (viii) M428L, N434S, G236A, R292P, and Y300L, wherein the antibody or antigen-binding fragment is afucosylated. In some embodiments, the variant of an (e.g., IgGl) IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody or antigen-binding fragment comprises a kappa light chain.

In some embodiments, an antibody or antigen-binding fragment of the present disclosure comprises a variant of an (e.g. IgGl) IgG Fc polypeptide, wherein the variant comprises the following mutations, according to EU numbering: (i) M428L, N434A, G236A, L328V, and Q295E; (ii) M428L, N434A, G236A, R292P, and I377N; (iii) M428L, N434A, G236A, and Y300L; (iv) M428L, N434A, G236A, R292P, and Y300L; (v) M428L, N434A, G236A, L328V, and Q295E, wherein the antibody or antigen-binding fragment is afucosylated; (vi) M428L, N434A, G236A, R292P, and I377N, wherein the antibody or antigen-binding fragment is afucosylated; (vii) M428L, N434A, G236A, and Y300L, wherein the antibody or antigen-binding fragment is afucosylated; or (viii) M428L, N434A, G236A, R292P, and Y300L, wherein the antibody or antigen-binding fragment is afucosylated. In some embodiments, the variant of an IgG Fc polypeptide comprises amino acid substitutions that consist essentially of the substitution mutations in (i), (ii), (iii), (iv), (v), (vi), (vii), or (viii) above. In some embodiments, the antibody or antigen-binding fragment comprises a kappa light chain. In certain embodiments, the antibody or antigen-binding fragment is capable of eliciting continued protection in vivo in a subject even once no detectable levels of the antibody or antigen-binding fragment can be found in the subject (z.e., when the antibody has been cleared from the subject following administration). Such protection is referred to herein as a vaccinal effect. Without wishing to be bound by theory, it is believed that dendritic cells can internalize complexes of antibody (or antigen-binding fragment) and antigen and thereafter induce or contribute to an endogenous immune response against antigen. In certain embodiments, an antibody or antigen-binding fragment comprises one or more modifications, such as, for example, mutations in the Fc comprising G236A, A330L, and I332E, that can activate dendritic cells that may induce, e.g., T cell immunity to the antigen. In any of the presently disclosed embodiments, the antibody or antigen-binding fragment comprises a Fc polypeptide or a fragment thereof, including a CH2 (or a fragment thereof, a CH3 (or a fragment thereof), or a CH2 and a CH3, wherein the CH2, the CH3, or both can be of any isotype and may contain amino acid substitutions or other modifications as compared to a corresponding wild-type CH2 or CH3, respectively. In certain embodiments, a Fc of the present disclosure comprises two CH2-CH3 polypeptides that associate to form a dimer.

In some embodiments, any of the presently disclosed antibodies or antigenbinding fragments can comprise an IgGl isotype (optionally comprising an IgGlm3 allotype, an IgGl m3, 1 allotype, an IgGlml7 allotype, or an IgGl ml 7,1 allotype) comprising (according to EU numbering): (i) M428L and N434S mutations; (ii) G236A, L328V, and Q295E mutations; (iii) G236A, L328V, Q259E, M428L, and N434S mutations; (iv) G236A, L328V, Q295E, M428L, and N434S mutations, wherein the antibody or antigen-binding fragment is afucosylated; (v) G236A, R292P, and Y300L mutations; (vi) G236A, R292P, Y300L, M428L, and N434S mutations; (vii) G236A, A330L, I332E, M428L, and N434S mutations; (viii) a G236A mutation, optionally wherein the antibody or antigen-binding fragment is afucosylated; (ix) G236A, M428L, and N434S mutations, optionally wherein the antibody or antigenbinding fragment is afucosylated; (x) G236R and L328R mutations; or (xi) G236R, L328R, M428L, and N434S mutations. Alternatively in any of these embodiments, N434A may be used instead of N434S. In some embodiments, the antibody or antigenbinding fragment comprises an IgGl isotype and, in a heavy chain, G236A, R292P, Y300L, M428L, and N434S mutations. In some embodiments, the antibody or antigenbinding fragment comprises an IgGl isotype and, in a heavy chain, G236A, Y300L, M428L, and N434S mutations.

In certain further embodiments, the antibody or antigen-binding fragment does not comprise any other mutations in the Fc. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgGlm3 allotype. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgGlml7 allotype. In some embodiments, the antibody or antigen-binding fragment thereof comprises an IgGlm3,l allotype. In some embodiments, the antibody or antigenbinding fragment thereof comprises an IgGlml7,l allotype.

In any of the presently disclosed embodiments, the antibody or antigen-binding fragment comprises a Fc polypeptide or a fragment thereof, including a CH2 (or a fragment thereof, a CH3 (or a fragment thereof), or a CH2 and a CH3, wherein the CH2, the CH3, or both can be of any isotype and may contain amino acid substitutions or other modifications as compared to a corresponding wild-type CH2 or CH3, respectively. In certain embodiments, a Fc polypeptide of the present disclosure comprises two CH2-CH3 polypeptides that associate to form a dimer.

In any of the presently disclosed embodiments, the antibody or antigen-binding fragment can be monoclonal. The term “monoclonal antibody” (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, z.e., individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present, in some cases in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to polyclonal antibody preparations that include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of the antigen. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The term "monoclonal" is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology first described by Kohler et al., Nature 256 :495 (1975), or may be made using recombinant DNA methods in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Pat. No. 4,816,567). Monoclonal antibodies may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. Monoclonal antibodies may also be obtained using methods disclosed in PCT Publication No. WO 2004/076677A2.

Antibodies and antigen-binding fragments of the present disclosure include "chimeric antibodies" in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, U.S. Pat. Nos. 4,816,567; 5,530,101 and 7,498,415; and Morrison et al., Proc. Natl. Acad. Sci. USA, 57:6851-6855 (1984)). For example, chimeric antibodies may comprise human and non-human residues. Furthermore, chimeric antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. For further details, see Jones et al., Nature 321 :522-525 (1986); Riechmann et al., Nature 332:323- 329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). Chimeric antibodies also include primatized and humanized antibodies. A "humanized antibody" is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are typically taken from a variable domain. Humanization may be performed following the method of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by substituting non-human variable sequences for the corresponding sequences of a human antibody. Accordingly, such “humanized” antibodies are chimeric antibodies (U.S. Pat. Nos. 4,816,567; 5,530,101 and 7,498,415) wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In some instances, a "humanized" antibody is one which is produced by a non-human cell or animal and comprises human sequences, e.g., He domains.

A "human antibody" is an antibody containing only sequences that are present in an antibody that is produced by a human. However, as used herein, human antibodies may comprise residues or modifications not found in a naturally occurring human antibody (e.g., an antibody that is isolated from a human), including those modifications and variant sequences described herein. These are typically made to further refine or enhance antibody performance. In some instances, human antibodies are produced by transgenic animals. For example, see U.S. Pat. Nos. 5,770,429; 6,596,541 and 7,049,426.

In certain embodiments, an antibody or antigen-binding fragment of the present disclosure is chimeric, humanized, or human.

In certain embodiments, an antibody is provided that comprises (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:402 (or SEQ ID NO.:402 without the C-terminal lysine) and (ii) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:403.

In certain embodiments, an antibody is provided that comprises (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:402 (or SEQ ID NO.:402 without the C-terminal lysine) and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:403. In certain embodiments, an antibody is provided that comprises (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:404 (or SEQ ID NO.:404 without the C-terminal lysine or SEQ ID NO.:404 without the C- terminal glycine-lysine) and (ii) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:405.

In certain embodiments, an antibody is provided that comprises (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:404 (or SEQ ID NO.:404 without the C-terminal lysine or SEQ ID NO.:404 without the C-terminal glycine-lysine) and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:405.

In certain embodiments, an antibody is provided that comprises (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:406 and (ii) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:407.

In certain embodiments, an antibody is provided that comprises (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:406 and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:407.

Polynucleotides, Vectors, and Host cells

In another aspect, the present disclosure provides isolated polynucleotides that encode any of the presently disclosed antibodies or an antigen-binding fragment thereof, or a portion thereof (e.g., a CDR, a VH, a VL, a heavy chain, or a light chain). In certain embodiments, the polynucleotide is codon-optimized for expression in a host cell. Once a coding sequence is known or identified, codon optimization can be performed using known techniques and tools, e.g., using the GenScript® OptimiumGene™ tool; see also Scholten et al., Clin. Immunol. 119 : 135, 2006). Codon-optimized sequences include sequences that are partially codon-optimized (ie., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.

Table 4 shows variable domain polynucleotide sequences of certain antibodies of the present disclosure; in some embodiments, a polynucleotide comprises a VH- encoding polynucleotide and a VL-encoding polynucleotide having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity to, or comprising or consisting of, a VH and VL amino acid sequence according to Table 4. Table 4. Example Variable Domain Polynucleotide SEQ ID NOs. of Certain Antibodies of the Present Disclosure.

It will also be appreciated that polynucleotides encoding antibodies and antigenbinding fragments of the present disclosure may possess different nucleotide sequences while still encoding a same antibody or antigen-binding fragment due to, for example, the degeneracy of the genetic code, splicing, and the like.

It will be appreciated that in certain embodiments, a polynucleotide encoding an antibody or antigen-binding fragment is comprised in a polynucleotide that includes other sequences and/or features for, e.g., expression of the antibody or antigen-binding fragment in a host cell. Exemplary features include a promoter sequence, a polyadenylation sequence, a sequence that encodes a signal peptide (e.g., located at the N-terminus of a expressed antibody heavy chain or light chain), or the like.

In any of the presently disclosed embodiments, the polynucleotide can comprise deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). In some embodiments, the RNA comprises messenger RNA (mRNA).

In some embodiments, the polynucleotide comprises a modified nucleoside, a cap-1 structure, a cap-2 structure, or any combination thereof. In certain embodiments, the polynucleotide comprises a pseudouridine, a N6-methyladenonsine, a 5- methylcytidine, a 2-thiouridine, or any combination thereof. In some embodiments, the pseudouridine comprises N1 -methylpseudouridine.

Vectors are also provided, wherein the vectors comprise or contain a polynucleotide as disclosed herein (e.g., a polynucleotide that encodes an antibody or antigen-binding fragment that binds to SARS-CoV-2). A vector can comprise any one or more of the vectors disclosed herein. In particular embodiments, a vector is provided that comprises a DNA plasmid construct encoding the antibody or antigen-binding fragment, or a portion thereof (e.g., so-called "DMAb"; see, e.g., Muthumani et al., J Infect Dis. 277(3):369-378 (2016); Muthumani et al., Hum Vaccin Immunother 9:2253- 2262 (2013)); Flingai et al., Sci Rep. 5: 12616 (2015); and Elliott et al., NPJ Vaccines 18 (2017), which antibody-coding DNA constructs and related methods of use, including administration of the same, are incorporated herein by reference). In certain embodiments, a DNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigenbinding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide. In certain embodiments, a mRNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigenbinding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide. In some embodiments, the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide (e.g. DNA or mRNA) comprised in a single plasmid. In other embodiments, the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide e.g. DNA or mRNA) comprised in two or more plasmids (e.g., a first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL). In certain embodiments, a single plasmid comprises a polynucleotide e.g. DNA or mRNA) encoding a heavy chain and/or a light chain from two or more antibodies or antigen-binding fragments of the present disclosure. An exemplary expression vector is pVaxl, available from Invitrogen®. A DNA or mRNA plasmid of the present disclosure can be delivered to a subject by, for example, electroporation (e.g., intramuscular electroporation), or with an appropriate formulation (e.g., hyaluronidase). In some embodiments, a vector of the present disclosure comprises a nucleotide sequence encoding a signal peptide. The signal peptide may or may not be present (e.g., can be enzymatically cleaved from) on the mature antibody or antigen-binding fragment. In some embodiments, a vector of the present disclosure comprises a polyadenylation signal sequence.

In some embodiments, a vector of the present disclosure comprises a CMV promoter.

In some embodiments, a method is provided that comprises administering to a subject a first polynucleotide (e.g., DNA or mRNA) encoding an antibody heavy chain, a VH, or a Fd (VH + CHI), and administering to the subject a second polynucleotide (e.g., DNA or mRNA) encoding the cognate antibody light chain, VL, or VL+CL. In some embodiments, the first polynucleotide and the second polynucleotide comprise or consist of DNA. In some embodiments, the first polynucleotide and the second polynucleotide comprise or consist of mRNA.

In some embodiments, a polynucleotide (e.g., mRNA) is provided that encodes a heavy chain and a light chain of an antibody or antigen binding fragment thereof. In some embodiments, a polynucleotide (e.g., mRNA) is provided that encodes two heavy chains and two light chains of an antibody or antigen binding fragment thereof. See, e.g. Li, JQ., Zhang, ZR., Zhang, HQ. et al. Intranasal delivery of replicating mRNA encoding neutralizing antibody against SARS-CoV-2 infection in mice. Sig Transduct Target Ther 6, 369 (2021). https://doi.org/10.1038/s41392-021-00783-l, the antibodyencoding mRNA constructs, vectors, and related techniques of which are incorporated herein by reference. In some embodiments, a polynucleotide is delivered to a subject via an alphavirus replicon particle (VRP) delivery system. In some embodiments, a replicon comprises a modified VEEV replicon comprising two subgenomic promoters. In some embodiments, a polynucleotide or replicon can translate simultaneously the heavy chain (or VH, or VH+1) and the light chain (or VL, or VL+CL) of an antibody or antigen binding fragment thereof. In some embodiments, a method is provided that comprises delivering to a subject such a polynucleotide or replicon. In a further aspect, the present disclosure also provides a host cell expressing an antibody or antigen-binding fragment according to the present disclosure; or comprising or containing a vector or polynucleotide according the present disclosure.

Examples of such cells include but are not limited to, eukaryotic cells, e.g., yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In certain such embodiments, the cells are a mammalian cell line such as CHO cells (e.g., DHFR- CHO cells (Urlaub et al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells. NSO cells, human liver cells, e.g., Hepa RG cells, myeloma cells or hybridoma cells. Other examples of mammalian host cell lines include mouse sertoli cells (e.g, TM4 cells); monkey kidney CV1 line transformed by SV40 (COS-7); baby hamster kidney cells (BHK); African green monkey kidney cells (VERO-76); monkey kidney cells (CV1); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3 A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Mammalian host cell lines suitable for antibody production also include those described in, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255- 268 (2003).

In certain embodiments, a host cell is a prokaryotic cell, such as an E. coli. The expression of peptides in prokaryotic cells such as E. coli is well established (see, e.g., Pluckthun, A. Bio/Technology 9:545-551 (1991). 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.

In particular embodiments, the cell may be transfected with a vector according to the present description with an expression vector. The term “transfection” refers to the introduction of nucleic acid molecules, such as DNA or RNA (e.g., mRNA) molecules, into cells, such as into eukaryotic cells. In the context of the present description, the term “transfection” encompasses any method known to the skilled person for introducing nucleic acid molecules into cells, such as into eukaryotic cells, including into mammalian cells. Such methods encompass, for example, electroporation, lipofection, e.g., based on cationic lipids and/or liposomes, calcium phosphate precipitation, nanoparticle based transfection, virus based transfection, or transfection based on cationic polymers, such as DEAE-dextran or polyethylenimine, etc. In certain embodiments, the introduction is non-viral.

Moreover, host cells of the present disclosure may be transfected stably or transiently with a vector according to the present disclosure, e.g., for expressing an antibody, or an antigen-binding fragment thereof, according to the present disclosure. In such embodiments, the cells may be stably transfected with the vector as described herein. Alternatively, cells may be transiently transfected with a vector according to the present disclosure encoding an antibody or antigen-binding fragment as disclosed herein. In any of the presently disclosed embodiments, a polynucleotide may be heterologous to the host cell.

Accordingly, the present disclosure also provides recombinant host cells that heterologously express an antibody or antigen-binding fragment of the present disclosure. For example, the cell may be of a species that is different to the species from which the antibody was fully or partially obtained (e.g., CHO cells expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not express the antibody or antigen-binding fragment in nature. Moreover, the host cell may impart a post-translational modification (PTM; e.g., glycosylation or fucosylation) on the antibody or antigen-binding fragment that is not present in a native state of the antibody or antigen-binding fragment (or in a native state of a parent antibody from which the antibody or antigen binding fragment was engineered or derived). Such a PTM may result in a functional difference (e.g., reduced immunogenicity). Accordingly, an antibody or antigen-binding fragment of the present disclosure that is produced by a host cell as disclosed herein may include one or more post-translational modification that is distinct from the antibody (or parent antibody) in its native state (e.g., a human antibody produced by a CHO cell can comprise a more post-translational modification that is distinct from the antibody when isolated from the human and/or produced by the native human B cell or plasma cell).

Insect cells useful expressing a binding protein of the present disclosure are known in the art and include, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWTOl “Mimic™” cells. See, e.g., Palmberger et al., J. Biotechnol. 753(3-4): 160-166 (2011). Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Eukaryotic microbes such as filamentous fungi or yeast are also suitable hosts for cloning or expressing protein-encoding vectors, and include fungi and yeast strains with “humanized” glycosylation pathways, resulting in the production of an antibody with a partially or fully human glycosylation pattern. See Gerngross, Nat.

Biotech. 22: 1409-1414 (2004); Li et al., Nat. Biotech. 24:210-215 (2006).

Plant cells can also be utilized as hosts for expressing a binding protein of the present disclosure. For example, PLANTIBODIES™ technology (described in, for example, U.S. Pat. Nos. 5,959,177; 6,040,498; 6,420,548; 7,125,978; and 6,417,429) employs transgenic plants to produce antibodies.

In certain embodiments, the host cell comprises a mammalian cell. In particular embodiments, the host cell is a CHO cell, a HEK293 cell, a PER.C6 cell, a Y0 cell, a Sp2/0 cell, a NS0 cell, a human liver cell, a myeloma cell, or a hybridoma cell.

In a related aspect, the present disclosure provides methods for producing an antibody, or antigen-binding fragment, wherein the methods comprise culturing a host cell of the present disclosure under conditions and for a time sufficient to produce the antibody, or the antigen-binding fragment. Methods useful for isolating and purifying recombinantly produced antibodies, by way of example, may include obtaining supernatants from suitable host cell/vector systems that secrete the recombinant antibody into culture media and then concentrating the media using a commercially available filter. Following concentration, the concentrate may be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reverse phase HPLC steps may be employed to further purify a recombinant polypeptide. These purification methods may also be employed when isolating an immunogen from its natural environment. Methods for large scale production of one or more of the isolated/recombinant antibody described herein include batch cell culture, which is monitored and controlled to maintain appropriate culture conditions. Purification of soluble antibodies may be performed according to methods described herein and known in the art and that comport with laws and guidelines of domestic and foreign regulatory agencies.

Compositions

Also provided herein are compositions that comprise any one or more of the presently disclosed antibodies, antigen-binding fragments, polynucleotides, vectors, or host cells, singly or in any combination, and can further comprise a pharmaceutically acceptable carrier, excipient, or diluent. Carriers, excipients, and diluents are discussed in further detail herein.

In certain embodiments, a composition comprises a plurality of an antibody and/or an antigen-binding fragment of the present disclosure, wherein one or more antibody or antigen-binding fragment does not comprise a lysine residue (or glycinelysine motif) at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein one or more antibody or antigen-binding fragment comprises a lysine residue (or glycine-lysine motif) at the C-terminal end of the heavy chain, CH1-CH3, or Fc polypeptide.

In certain embodiments, a composition comprises two or more different antibodies or antigen-binding fragments, which are optionally antibodies or antigen- bindign fragments according to the present disclosure. In certain embodiments, antibodies or antigen-binding fragments to be used in a combination each independently have one or more of the following characteristics: neutralize naturally occurring SARS-CoV-2 variants; do not compete with one another for Spike protein binding; bind distinct Spike protein epitopes; have a reduced formation of resistance to SARS-CoV-2; when in a combination, have a reduced formation of resistance to SARS- CoV-2; potently neutralize live SARS-CoV-2 virus; exhibit additive or synergistic effects on neutralization of live SARS-CoV-2 virus when used in combination; exhibit effector functions; are protective in relevant animal model(s) of infection; are capable of being produced in sufficient quantities for large-scale production.

In certain embodiments, antibodies or antigen-binding fragments to be used in a combination each independently have one or more of the following characteristics: neutralize one, two, three, four, five, or more naturally occurring sarbecovirus variants; do not compete with one another for Spike protein binding; bind distinct sarbecovirus Spike protein epitopes; have a reduced formation of resistance to sarbecovirus; when in a combination, have a reduced formation of resistance to sarbecovirus; potently neutralize one, two, three, four, five or more live sarbecoviruses; exhibit additive or synergistic effects on neutralization of one, two, three, four, five or more or more live sarbecoviruses when used in combination; exhibit effector functions; are protective in relevant animal model(s) of infection; are capable of being produced in sufficient quantities for large-scale production.

In certain embodiments, a composition comprises two or more different antibodies or antigen-binding fragments according to the present disclosure.

In certain embodiments, a composition comprises a first vector comprising a first plasmid, and a second vector comprising a second plasmid, wherein the first plasmid comprises a polynucleotide encoding a heavy chain, VH, or VH+CH, and a second plasmid comprises a polynucleotide encoding the cognate light chain, VL, or VL+CL of the antibody or antigen-binding fragment thereof. In certain embodiments, a composition comprises a polynucleotide (e.g., mRNA) coupled to a suitable delivery vehicle or carrier. In certain embodiments, a composition comprises a first polynucleotide (e.g., mRNA) encoding an antibody heavy chain, a VH, or a Fd (VH + CHI), and a second polynucleotide (e.g., mRNA) encoding the cognate antibody light chain or VL. Exemplary vehicles or carriers for administration to a human subject include a lipid or lipid-derived delivery vehicle, such as a liposome, solid lipid nanoparticle, oily suspension, submicron lipid emulsion, lipid microbubble, inverse lipid micelle, cochlear liposome, lipid microtubule, lipid microcylinder, or lipid nanoparticle (LNP) or a nanoscale platform (see, e.g., Li el al. Wilery Interdiscip Rev. Nanomed Nanobiotechnol. 77(2):el530 (2019)). Principles, reagents, and techniques for designing appropriate mRNA and and formulating mRNA-LNP and delivering the same are described in, for example, Pardi et al. (J Control Release 277345-351 (2015)); Thess et al. Mol Ther 23: 1456-1464 (2015)); Thran et al. (EMBO Mol Med 9(10): 1434-1448 (2017); Kose et al. (Set. Immunol. 4 eaaw6647 (2019); and Sabnis et al. (Mol. Ther. 26: 1509-1519 (2018)), which techniques, include capping, codon optimization, nucleoside modification, purification of mRNA, incorporation of the mRNA into stable lipid nanoparticles (e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG-lipid; ionizable lipid:distearoyl PC:cholesterol:polyethylene glycol lipid), and subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intratracheal administration of the same, are incorporated herein by reference.

Methods and Uses

Also provided herein are methods for use of an antibody or antigen-binding fragment, nucleic acid, vector, cell, or composition of the present disclosure in the diagnosis of a sarbecovirus infection, such as a SARS-CoV-2 infection (e.g., in a human subject, or in a sample obtained from a human subject).

Methods of diagnosis (e.g., in vitro, ex vivo) may include contacting an antibody, antibody fragment (e.g., antigen binding fragment) with a sample. Such samples may be isolated from a subject, for example an isolated tissue sample taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain, skin or blood. The methods of diagnosis may also include the detection of an antigen/antibody complex, in particular following the contacting of an antibody or antibody fragment with a sample. Such a detection step can be performed at the bench, i.e., without any contact to the human or animal body. Examples of detection methods are well-known to the person skilled in the art and include, e.g., ELISA (enzyme-linked immunosorbent assay), including direct, indirect, and sandwich ELISA. Also provided herein are methods of treating a subject using an antibody or antigen-binding fragment of the present disclosure, or a composition comprising the same, wherein the subject has, is believed to have, or is at risk for having an infection by a sarbecorvirus, such as SARS-CoV-2. "Treat," "treatment," or "ameliorate" refers to medical management of a disease, disorder, or condition of a subject (e.g., a human or non-human mammal, such as a primate, horse, cat, dog, goat, mouse, or rat). In general, an appropriate dose or treatment regimen comprising an antibody or composition of the present disclosure is administered in an amount sufficient to elicit a therapeutic or prophylactic benefit. Therapeutic or prophylactic/preventive benefit includes improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay or prevention of disease progression; remission; survival; prolonged survival; or any combination thereof. In certain embodiments, therapeutic or prophylactic/preventive benefit includes reduction or prevention of hospitalization for treatment of a sarbecovirus infection, such as a SARS-CoV-2 infection (z.e., in a statistically significant manner). In certain embodiments, therapeutic or prophylactic/preventive benefit includes a reduced duration of hospitalization for treatment of a sarbecovirus infection, such as a SARS-CoV-2 infection (z.e., in a statistically significant manner). In certain embodiments, therapeutic or prophylactic/preventive benefit includes a reduced or abrogated need for respiratory intervention, such as intubation and/or the use of a respirator device. In certain embodiments, therapeutic or prophylactic/preventive benefit includes reversing a latestage disease pathology and/or reducing mortality.

A "therapeutically effective amount" or "effective amount" of an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition of this disclosure refers to an amount of the composition or molecule sufficient to result in a therapeutic effect, including improved clinical outcome; lessening or alleviation of symptoms associated with a disease; decreased occurrence of symptoms; improved quality of life; longer disease-free status; diminishment of extent of disease, stabilization of disease state; delay of disease progression; remission; survival; or prolonged survival in a statistically significant manner. When referring to an individual active ingredient, administered alone, a therapeutically effective amount refers to the effects of that ingredient or cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of active ingredients or combined adjunctive active ingredient with a cell expressing an active ingredient that results in a therapeutic effect, whether administered serially, sequentially, or simultaneously. A combination may comprise, for example, two different antibodies that specifically bind a SARS-CoV-2 antigen, which in certain embodiments, may be the same or different SARS-CoV-2 antigen, and/or can comprise the same or different epitopes.

Accordingly, in certain embodiments, methods are provided for treating a sarbecovirus infection, such as a SARS-CoV-2 infection, in a subject, wherein the methods comprise administering to the subject an effective amount of an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition as disclosed herein.

Subjects that can be treated by the present disclosure are, in general, human and other primate subjects, such as monkeys and apes for veterinary medicine purposes. Other model organisms, such as mice and rats, may also be treated according to the present disclosure. In any of the aforementioned embodiments, the subject may be a human subject. The subjects can be male or female and can be any suitable age, including infantjuvenile, adolescent, adult, and geriatric subjects.

A number of criteria are believed to contribute to high risk for severe symptoms or death associated with a SARS CoV-2 infection. These include, but are not limited to, age, occupation, general health, pre-existing health conditions, and lifestyle habits. In some embodiments, a subject treated according to the present disclosure comprises one or more risk factors.

In certain embodiments, a human subject treated according to the present disclosure is an infant, a child, a young adult, an adult of middle age, or an elderly person. In certain embodiments, a human subject treated according to the present disclosure is less than 1 year old, or is 1 to 5 years old, or is between 5 and 125 years old (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 125 years old, including any and all ages therein or therebetween). In certain embodiments, a human subject treated according to the present disclosure is 0- 19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older. Persons of middle, and especially of elderly age are believed to be at particular risk. In particular embodiments, the human subject is 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years old, or older. In some embodiments, the human subject is male. In some embodiments, the human subject is female.

In certain embodiments, a human subject treated according to the present disclosure is a resident of a nursing home or a long-term care facility, is a hospice care worker, is a healthcare provider or healthcare worker, is a first responder, is a family member or other close contact of a subject diagnosed with or suspected of having a SARS-CoV-2 infection, is overweight or clinically obese, is or has been a smoker, has or had chronic obstructive pulmonary disease (COPD), is asthmatic (e.g., having moderate to severe asthma), has an autoimmune disease or condition (e.g., diabetes), and/or has a compromised or depleted immune system (e.g., due to AIDS/HIV infection, a cancer such as a blood cancer, a lymphodepleting therapy such as a chemotherapy, a bone marrow or organ transplantation, or a genetic immune condition), has chronic liver disease, has cardiovascular disease, has a pulmonary or heart defect, works or otherwise spends time in close proximity with others, such as in a factory, shipping center, hospital setting, or the like.

In certain embodiments, a subject treated according to the present disclosure has received a vaccine for SARS-CoV-2 and the vaccine is determined to be ineffective, e.g., by post-vaccine infection or symptoms in the subject, by clinical diagnosis or scientific or regulatory criteria.

In certain embodiments, treatment is administered as peri-exposure prophylaxis. In certain embodiments, treatment is administered to a subject with mild- to-moderate disease, which may be in an outpatient setting. In certain embodiments, treatment is administered to a subject with moderate-to-severe disease, such as requiring hospitalization.

Typical routes of administering the presently disclosed compositions thus include, without limitation, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term "parenteral", as used herein, includes subcutaneous injections, intravenous, intramuscular, intrastemal injection or infusion techniques. In certain embodiments, administering comprises administering by a route that is selected from oral, intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, topical, transdermal, intracisternal, intrathecal, intranasal, and intramuscular. In particular embodiments, a method comprises orally administering the antibody, antigenbinding fragment, polynucleotide, vector, host cell, or composition to the subject.

Pharmaceutical compositions according to certain embodiments of the present invention are formulated so as to allow the active ingredients contained therein to be bioavailable upon administration of the composition to a patient. Compositions that will be administered to a subject or patient may take the form of one or more dosage units, where for example, a tablet may be a single dosage unit, and a container of a herein described an antibody or antigen-binding in aerosol form may hold a plurality of dosage units. Actual methods of preparing such dosage forms are known, or will be apparent, to those skilled in this art; for example, see Remington: The Science and Practice of Pharmacy, 20th Edition (Philadelphia College of Pharmacy and Science, 2000). The composition to be administered will, in any event, contain an effective amount of an antibody or antigen-binding fragment, polynucleotide, vector, host cell, , or composition of the present disclosure, for treatment of a disease or condition of interest in accordance with teachings herein.

A composition may be in the form of a solid or liquid. In some embodiments, the carrier(s) are particulate, so that the compositions are, for example, in tablet or powder form. The carrier(s) may be liquid, with the compositions being, for example, an oral oil, injectable liquid or an aerosol, which is useful in, for example, inhalatory administration. When intended for oral administration, the pharmaceutical composition is preferably in either solid or liquid form, where semi solid, semi liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

As a solid composition for oral administration, the pharmaceutical composition may be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like. Such a solid composition will typically contain one or more inert diluents or edible carriers. In addition, one or more of the following may be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, gum tragacanth or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, Primogel, corn starch and the like; lubricants such as magnesium stearate or Sterotex; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule, for example, a gelatin capsule, it may contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol or oil.

The composition may be in the form of a liquid, for example, an elixir, syrup, solution, emulsion or suspension. The liquid may be for oral administration or for delivery by injection, as two examples. When intended for oral administration, preferred compositions contain, in addition to the present compounds, one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition intended to be administered by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent may be included.

Liquid pharmaceutical compositions, whether they be solutions, suspensions or other like form, may include one or more of the following adjuvants: sterile diluents such as water for injection, saline solution, preferably physiological saline, Ringer’s solution, isotonic sodium chloride, fixed oils such as synthetic mono or diglycerides which may serve as the solvent or suspending medium, polyethylene glycols, glycerin, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methyl paraben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic. Physiological saline is a preferred adjuvant. An injectable pharmaceutical composition is preferably sterile.

A liquid composition intended for either parenteral or oral administration should contain an amount of an antibody or antigen-binding fragment as herein disclosed such that a suitable dosage will be obtained. Typically, this amount is at least 0.01% of the antibody or antigen-binding fragment in the composition. When intended for oral administration, this amount may be varied to be between 0.1 and about 70% of the weight of the composition. Certain oral pharmaceutical compositions contain between about 4% and about 75% of the antibody or antigen-binding fragment. In certain embodiments, pharmaceutical compositions and preparations according to the present invention are prepared so that a parenteral dosage unit contains between 0.01 to 10% by weight of antibody or antigen-binding fragment prior to dilution.

The composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base, for example, may comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, bee wax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in a composition for topical administration. If intended for transdermal administration, the composition may include a transdermal patch or iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form, for example, of a suppository, which will melt in the rectum and release the drug. The composition for rectal administration may contain an oleaginous base as a suitable nonirritating excipient. Such bases include, without limitation, lanolin, cocoa butter and polyethylene glycol.

A composition may include various materials which modify the physical form of a solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The materials that form the coating shell are typically inert, and may be selected from, for example, sugar, shellac, and other enteric coating agents. Alternatively, the active ingredients may be encased in a gelatin capsule. The composition in solid or liquid form may include an agent that binds to the antibody or antigen-binding fragment of the disclosure and thereby assists in the delivery of the compound. Suitable agents that may act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome. The composition may consist essentially of dosage units that can be administered as an aerosol. The term aerosol is used to denote a variety of systems ranging from those of colloidal nature to systems consisting of pressurized packages. Delivery may be by a liquefied or compressed gas or by a suitable pump system that dispenses the active ingredients. Aerosols may be delivered in single phase, bi phasic, or tri phasic systems in order to deliver the active ingredient(s). Delivery of the aerosol includes the necessary container, activators, valves, subcontainers, and the like, which together may form a kit. One of ordinary skill in the art, without undue experimentation, may determine preferred aerosols.

It will be understood that compositions of the present disclosure also encompass carrier molecules for polynucleotides, as described herein (e.g., lipid nanoparticles, nanoscale delivery platforms, and the like).

The pharmaceutical compositions may be prepared by methodology well known in the pharmaceutical art. For example, a composition intended to be administered by injection can be prepared by combining a composition that comprises an antibody, antigen-binding fragment thereof, or antibody conjugate as described herein and optionally, one or more of salts, buffers and/or stabilizers, with sterile, distilled water so as to form a solution. A surfactant may be added to facilitate the formation of a homogeneous solution or suspension. Surfactants are compounds that non-covalently interact with the peptide composition so as to facilitate dissolution or homogeneous suspension of the antibody or antigen-binding fragment thereof in the aqueous delivery system.

In general, an appropriate dose and treatment regimen provide the composition(s) in an amount sufficient to provide therapeutic and/or prophylactic benefit (such as described herein, including an improved clinical outcome (e.g., a decrease in frequency, duration, or severity of diarrhea or associated dehydration, or inflammation, or longer disease-free and/or overall survival, or a lessening of symptom severity). For prophylactic use, a dose should be sufficient to prevent, delay the onset of, or diminish the severity of a disease associated with disease or disorder.

Prophylactic benefit of the compositions administered according to the methods described herein can be determined by performing pre-clinical (including in vitro and in vivo animal studies) and clinical studies and analyzing data obtained therefrom by appropriate statistical, biological, and clinical methods and techniques, all of which can readily be practiced by a person skilled in the art.

Compositions are administered in an effective amount (e.g., to treat a SARS- CoV-2 infection), which will vary depending upon a variety of factors including the activity of the specific compound employed; the metabolic stability and length of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of excretion; the drug combination; the severity of the particular disorder or condition; and the subject undergoing therapy. In certain embodiments, tollowing administration of therapies according to the formulations and methods of this disclosure, test subjects will exhibit about a 10% up to about a 99% reduction in one or more symptoms associated with the disease or disorder being treated as compared to placebo-treated or other suitable control subjects.

Generally, a therapeutically effective daily dose of an antibody or antigen binding fragment is (for a 70 kg mammal) from about 0.001 mg/kg (z.e., 0.07 mg) to about 100 mg/kg (z.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (z.e., 0.7 mg) to about 50 mg/kg (z.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (z.e., 70 mg) to about 25 mg/kg (z.e., 1.75 g). For polynucleotides, vectors, host cells, and related compositions of the present disclosure, a therapeutically effective dose may be different than for an antibody or antigen-binding fragment.

In certain embodiments, a method comprises administering the antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition to the subject at 2, 3, 4, 5, 6, 7, 8, 9, 10 times, or more. In certain embodiments, a method comprises administering the antibody, antigen-binding fragment, or composition to the subject a plurality of times, wherein a second or successive administration is performed at about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 24, about 48, about 74, about 96 hours, or more, following a first or prior administration, respectively.

In certain embodiments, a method comprises administering the antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition at least one time prior to the subject being infected by a sarbecovirus, such as SARS-CoV-2.

Compositions comprising an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition of the present disclosure may also be administered simultaneously with, prior to, or after administration of one or more other therapeutic agents. Such combination therapy may include administration of a single pharmaceutical dosage formulation which contains a compound of the invention and one or more additional active agents, as well as administration of compositions comprising an antibody or antigen-binding fragment of the disclosure and each active agent in its own separate dosage formulation. For example, an antibody or antigenbinding fragment thereof as described herein and the other active agent can be administered to the patient together in a single oral dosage composition such as a tablet or capsule, or each agent administered in separate oral dosage formulations. Similarly, an antibody or antigen-binding fragment as described herein and the other active agent can be administered to the subject together in a single parenteral dosage composition such as in a saline solution or other physiologically acceptable solution, or each agent administered in separate parenteral dosage formulations. Where separate dosage formulations are used, the compositions comprising an antibody or antigen-binding fragment and one or more additional active agents can be administered at essentially the same time, z.e., concurrently, or at separately staggered times, z.e., sequentially and in any order; combination therapy is understood to include all these regimens.

In certain embodiments, a combination therapy is provided that comprises one or more anti-sarbecovirus antibody, such as an anti-SARS-CoV-2 antibody, (or one or more nucleic acid, host cell, vector, or composition) of the present disclosure and one or more anti-inflammatory agent and/or one or more anti-viral agent. In particular embodiments, the one or more anti-inflammatory agent comprises a corticosteroid such as, for example, dexamethasone, prednisone, or the like. In some embodiments, the one or more anti-inflammatory agents comprise a cytokine antagonist such as, for example, an antibody that binds to IL6 (such as siltuximab), or to IL-6R (such as tocilizumab), or to IL-ip, IL-7, IL-8, IL-9, IL-10, FGF, G-CSF, GM-CSF, IFN-y, IP-10, MCP-1, MIP- 1 A, MIP1-B, PDGR, TNF-a, or VEGF. In some embodiments, anti-inflammatory agents such as leronlimab, ruxolitinib and/or anakinra are used. In some embodiments, the one or more anti-viral agents comprise nucleotide analogs or nucelotide analog prodrugs such as, for example, remdesivir, sofosbuvir, acyclovir, and zidovudine. In particular embodiments, an anti-viral agent comprises lopinavir, ritonavir, favipiravir, or any combination thereof. Other anti-inflammatory agents for use in a combination therapy of the present disclosure include non-steroidal anti-inflammatory drugs (NSAIDS). It will be appreciated that in such a combination therapy, the one or more antibody (or one or more nucleic acid, host cell, vector, or composition) and the one or more anti-inflammatory agent and/or one or the more antiviral agent can be administered in any order and any sequence, or together.

In some embodiments, an antibody (or one or more nucleic acid, host cell, vector, or composition) is administered to a subject who has previously received one or more anti-inflammatory agent and/or one or more antiviral agent. In some embodiments, one or more anti-inflammatory agent and/or one or more antiviral agent is administered to a subject who has previously received an antibody (or one or more nucleic acid, host cell, vector, or composition).

In certain embodiments, a combination therapy is provided that comprises two or more anti-sarbecovirus antibodies of the present disclosure, such as two or more anti- SARS-CoV-2 antibodies. A method can comprise administering a first antibody to a subject who has received a second antibody, or can comprise administering two or more antibodies together. For example, in particular embodiments, a method is provided that comprises administering to the subject (a) a first antibody or antigen-binding fragment, when the subject has received a second antibody or antigen-binding fragment; (b) the second antibody or antigen-binding fragment, when the subject has received the first antibody or antigen-binding fragment; or (c) the first antibody or antigen-binding fragment, and the second antibody or antigen-binding fragment.

In a related aspect, uses of the presently disclosed antibodies, antigen-binding fragments, vectors, host cells, and compositions are provided.

In certain embodiments, an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition is provided for use in a method of treating a SARS- CoV-2 infection in a subject.

In certain embodiments, an antibody, antigen-binding fragment, or composition is provided for use in a method of manufacturing or preparing a medicament for treating a sarbecovirus infection, such as a SARS-CoV-2 infection, in a subject.

Table 5. Sequences

The present disclosure also provides the following non-limiting, numbered Embodiments, which may be referenced by number in other Embodiments:

Embodiment 1. An anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising: (i) the CDRH1 amino acid sequence of any one of SEQ ID NOs.:39, 49, 59, 69, 79, 89, 99, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 361, 371, 381, and 391; (ii) the CDRH2 amino acid sequence of any one of SEQ ID NOs.:40, 50, 60, 70, 80, 90, 100, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232,

242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 362, 372, 382, and 392; (iii) the CDRH3 amino acid sequence of any one of SEQ ID NOs.:41, 51, 61, 71, 81, 91, 101, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263,

273, 283, 293, 303, 313, 323, 333, 343, 353, 363, 373, 383, and 393; (iv) the CDRL1 amino acid sequence of any one of SEQ ID NOs.: 44, 54, 64, 74, 84, 94, 104, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 366, 376, 386, and 396; (v) the CDRL2 amino acid sequence of any one of SEQ ID NOs. :45, 55, 65, 75, 85, 95, 105, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 367, 377, 387, and 397; and (vi) the CDRL3 amino acid sequence of any one of SEQ ID NOs.:46, 56, 66, 76, 86, 96, 106, 118, 128, 138, 148, 158, 168, 178, 198, 208, 218, 228, 238, 248, 258, 268, 278, 288, 298, 308, 318, 328, 338, 348, 358, 368, 378, 388, and 398.

Embodiment 2. An anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of SEQ ID NOs.: (i) 39, 40, 41, 44, 45, and 46, respectively; (ii) 49, 50, 51, 54, 55, and 56, respectively; (iii) 59, 60, 61, 64, 65, and 66, respectively; (iv) 69, 70, 71, 74, 75, and 76, respectively; (v) 79, 80, 81, 84, 85, and 86, respectively; (vi) 89, 90, 91, 94, 95, and 96, respectively; (vii) 99, 100, 101, 104, 105, and 106, respectively; (viii) 111, 112, 113, 116, 117, and 118, respectively; (ix) 121, 122, 123, 126, 127, and 128, respectively; (x) 131, 132, 133, 136, 137, and 138, respectively; (xi) 141, 142, 143, 146, 147, and 148, respectively; (xi) 151, 152, 153, 156, 157, and 158, respectively; (xii) 161, 162, 163, 166, 167, and 168, respectively; (xiii) 171, 172, 173, 176, 177, and 178, respectively; (xiv) 181, 182, 183, 186, 187, and 188, respectively; (xv) 191, 192, 193, 196, 197, and 198, respectively; (xvi) 201, 202, 203, 206, 207, and 208, respectively; (xvii) 211, 212, 213, 216, 217, and 218, respectively; (xviii) 221, 222, 223, 226, 227, and 228, respectively; (xix) 231, 232, 233, 236, 237, and 238, respectively; (xx) 241, 242, 243, 246, 247, and 248, respectively; (xxi) 251, 252, 253, 256, 257, and 258, respectively; (xxii) 261, 262, 263, 266, 267, and 268, respectively; (xxiii) 271, 272, 273, 276, 277, and 278, respectively; (xxiv) 281, 282, 283, 286, 287, and 288, respectively; (xxiv) 291, 292, 293, 296, 297, and 298, respectively; (xxv) 301, 302, 303, 306, 307, and 308, respectively; (xxvi) 311, 312, 313, 316, 317, and 318, respectively; (xxvii) 321, 322, 323, 326, 327, and 328, respectively; (xxviii) 331, 332, 333, 336, 337, and 338, respectively, (xxix) 341, 342, 343, 346, 347, and 348, respectively, (xxx) 351, 352, 353, 356, 357, and 358, respectively, (xxxi) 361, 362, 363, 366, 367, and 368, respectively, (xxxii) 371, 372, 373, 376, 377, and 378, respectively, (xxxiii) 381, 382, 383, 386, 387, and 388, respectively, or (xxxiv) 391, 392, 393, 396, 397, and 398, respectively.

Embodiment 3. An isolated anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, comprising: (i) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 39, 40, and 41, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 44, 45, and 46; (ii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 49, 50, and 51, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 54, 55, and 56; (iii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 59, 60, and 61, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 64, 65, and 66; (iv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 69, 70, and 71, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 74, 75, and 76; (v) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 79, 80, and 81, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 84, 85, and 86; (vi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 89, 90, and 91, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 94, 95, and 96; (vii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 99, 100, and 101, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 104, 105, and 106; (viii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: I l l, 112, and 113, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 116, 117, and 118; (ix) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 121, 122, and 123, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 126, 127, and 128; (x) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 131, 132, and 133, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 136, 137, and 138; (xi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 141, 142, and 143, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 146, 147, and 148; (xi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 151, 152, and 153, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 156, 157, and 158; (xii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 161, 162, and 163, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 166, 167, and 168; (xiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 171, 172, and 173, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 176, 177, and 178; (xiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 181, 182, and 183, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 186, 187, and 188; (xv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 191, 192, and 193, and in a VL, the amino acid sequences set forth in SEQ ID NOs : 196, 197, and 198; (xvi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:201, 202, and 203, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:206, 207, and 208; (xvii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:211, 212, and 213, and in a VL, the amino acid sequences set forth in SEQ ID NOs. :216, 217, and 218; (xviii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:221, 222, and 223, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:226, 227, and 228; (xix) in a VH, the amino acid sequences set forth in SEQ ID NOs.:231, 232, and 233, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:236, 237, and 238; (xx) in a VH, the amino acid sequences set forth in SEQ ID NOs.:241, 242, and 243, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:246, 247, and 248; (xxi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:251, 252, and 253, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:256, 257, and 258; (xxii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:261, 262, and 263, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:266, 267, and 268; (xxiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:271, 272, and 273, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:276, 277, and 278; (xxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:281, 282, and 283, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:286, 287, and 288; (xxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:291, 292, and 293, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:296, 297, and 298; (xxv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:301, 302, and 303, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 306, 307, and 308; (xxvi) in a VH, the amino acid sequences set forth in SEQ ID NOs. :311, 312, and 313, and in a VL, the amino acid sequences set forth in SEQ ID NOs. :316, 317, and 318; (xxvii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:321, 322, and 323, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:326, 327, and 328; (xxviii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:331, 332, and 333, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:336, 337, and 338, (xxix) in a VH, the amino acid sequences set forth in SEQ ID NOs.:341, 342, and 343, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:346, 347, and 348, (xxx) in a VH, the amino acid sequences set forth in SEQ ID NOs.:351, 352, and 353, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:356, 357, and 358, (xxxi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:361, 362, and 363, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:366, 367, and 368, (xxxii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:371, 372, and 373, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:376, 377, and 378, (xxxiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:381, 382, and 383, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:386, 387, and 388, or (xxxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:391, 392, and 393, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:396, 397, and 398.

Embodiment 4. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-3, comprising a VH having at least 85% identity (z.e., at least 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to a VH amino acid sequence provided in Table 3, and/or a VL having at least 85% identity (z.e., at least 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to a VL amino acid sequence provided in Table 3.

Embodiment 5. An anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising:

(i) the VH amino acid sequence of SEQ ID NO.:38 and the VL amino acid sequence of SEQ ID NO.:43;

(ii) the VH amino acid sequence of SEQ ID NO. :48 and the VL amino acid sequence of SEQ ID NO.: 53;

(iii) the VH amino acid sequence of SEQ ID NO. :58 and the VL amino acid sequence of SEQ ID NO.:63;

(iv) the VH amino acid sequence of SEQ ID NO.:68 and the VL amino acid sequence of SEQ ID NO.:73;

(v) the VH amino acid sequence of SEQ ID NO.:78 and the VL amino acid sequence of SEQ ID NO.: 83; (vi) the VH amino acid sequence of SEQ ID NO.: 88 and the VL amino acid sequence of SEQ ID NO.:93;

(vii) the VH amino acid sequence of SEQ ID NO.:98 and the VL amino acid sequence of SEQ ID NO.: 103;

(viii) the VH amino acid sequence of SEQ ID NO. : 110 and the VL amino acid sequence of SEQ ID NO.: 115;

(ix) the VH amino acid sequence of SEQ ID NO.: 120 and the VL amino acid sequence of SEQ ID NO.: 125;

(x) the VH amino acid sequence of SEQ ID NO.: 130 and the VL amino acid sequence of SEQ ID NO.: 135;

(xi) the VH amino acid sequence of SEQ ID NO.: 140 and the VL amino acid sequence of SEQ ID NO.: 145;

(xii) the VH amino acid sequence of SEQ ID NO.: 150 and the VL amino acid sequence of SEQ ID NO.: 155;

(xiii) the VH amino acid sequence of SEQ ID NO. : 160 and the VL amino acid sequence of SEQ ID NO.: 165;

(xiv) the VH amino acid sequence of SEQ ID NO.: 170 and the VL amino acid sequence of SEQ ID NO.: 175;

(xv) the VH amino acid sequence of SEQ ID NO. : 180 and the VL amino acid sequence of SEQ ID NO.: 185;

(xvi) the VH amino acid sequence of SEQ ID NO.: 190 and the VL amino acid sequence of SEQ ID NO.: 195;

(xvii) the VH amino acid sequence of SEQ ID NO. :200 and the VL amino acid sequence of SEQ ID NO.:205;

(xviii) the VH amino acid sequence of SEQ ID NO.:210 and the VL amino acid sequence of SEQ ID NO.:215;

(xix) the VH amino acid sequence of SEQ ID NO.:220 and the VL amino acid sequence of SEQ ID NO.:225;

(xx) the VH amino acid sequence of SEQ ID NO. :230 and the VL amino acid sequence of SEQ ID NO.:235; (xxi) the VH amino acid sequence of SEQ ID NO.:240 and the VL amino acid sequence of SEQ ID NO.:245;

(xxii) the VH amino acid sequence of SEQ ID NO. :250 and the VL amino acid sequence of SEQ ID NO.:255;

(xxiii) the VH amino acid sequence of SEQ ID NO.:260 and the VL amino acid sequence of SEQ ID NO.:265;

(xxiv) the VH amino acid sequence of SEQ ID NO.:270 and the VL amino acid sequence of SEQ ID NO.:275;

(xxv) the VH amino acid sequence of SEQ ID NO. :280 and the VL amino acid sequence of SEQ ID NO.:285;

(xxvi) the VH amino acid sequence of SEQ ID NO.:290 and the VL amino acid sequence of SEQ ID NO.:295;

(xxvii) the VH amino acid sequence of SEQ ID NO.:300 and the VL amino acid sequence of SEQ ID NO.:305;

(xxviii)the VH amino acid sequence of SEQ ID NO.:310 and the VL amino acid sequence of SEQ ID NO.:315;

(xxix) the VH amino acid sequence of SEQ ID NO.:320 and the VL amino acid sequence of SEQ ID NO.:325;

(xxx) the VH amino acid sequence of SEQ ID NO. :330 and the VL amino acid sequence of SEQ ID NO.:335;

(xxxi) the VH amino acid sequence of SEQ ID NO.:340 and the VL amino acid sequence of SEQ ID NO.:345;

(xxxii) the VH amino acid sequence of SEQ ID NO.:350 and the VL amino acid sequence of SEQ ID NO.:355;

(xxxiii)the VH amino acid sequence of SEQ ID NO.:360 and the VL amino acid sequence of SEQ ID NO.:365;

(xxxiv)the VH amino acid sequence of SEQ ID NO.:370 and the VL amino acid sequence of SEQ ID NO.:375;

(xxxv) the VH amino acid sequence of SEQ ID NO.:380 and the VL amino acid sequence of SEQ ID NO.:385; or (xxxvi)the VH amino acid sequence of SEQ ID NO.:390 and the VL amino acid sequence of SEQ ID NO.:395.

Embodiment 6. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-5, which is capable of neutralizing a SARS- CoV-2 infection in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human, wherein, optionally, the SARS-CoV-2 infection comprises a SARS-CoV-2 comprising the amino acid sequence according to SEQ ID NO.:3, or comprises a SARS-CoV-2 Omicron variant or subvariant.

Embodiment 7. The anti-SARS-CoV-2 antibody antibody or antigenbinding fragment of any one of Embodiments 1-6, wherein the antibody, or the antigenbinding fragment, comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, a Fab', a F(ab')2, a Fv, a scFv, or a scFab.

Embodiment 8. The anti-SARS-CoV-2 antibody or antigen-binding fragment of Embodiment 7, wherein the antibody or antigen-binding fragment comprises a scFv comprising more than one VH domain and more than one VL domain.

Embodiment 9. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-8, wherein the antibody or antigen-binding fragment is a multi-specific antibody or antigen binding fragment.

Embodiment 10. The anti-SARS-CoV-2 antibody or antigen-binding fragment of Embodiment 9, wherein the antibody or antigen binding fragment is a bispecific antibody or antigen-binding fragment.

Embodiment 11. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-10, wherein the antibody or antigen-binding fragment comprises a Fc polypeptide or a fragment thereof, wherein, optionally, (1) the antibody or antigen-binding fragment comprises a CH1-CH3 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:6, 7, 108, or 109, and/or (2) the antibody or antigenbinding fragment comprises a CL having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:9 or 8, and preferably comprising a CH1-CH3 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:6, 7, 108, or 109 and a CL having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO. :9.

Embodiment 12. The antibody or antigen-binding fragment of any one of Embodiments 1-11, which is human, humanized, or chimeric.

Embodiment 13. The antibody or antigen-binding fragment of any one of Embodiments 1-12, which is a IgG, IgA, IgM, IgE, or IgD isotype.

Embodiment 14. The antibody or antigen-binding fragment of any one of Embodiments 1-13, which is an IgG isotype selected from IgGl, IgG2, IgG3, and IgG4, optionally with a C-terminal lysine or a C-terminal glycine-lysine removed, and is optionally an IgGl glm,17 allotype or an IgGl glm3 allotype.

Embodiment 15. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 11-14, wherein the Fc polypeptide or fragment thereof comprises:

(i) a mutation that enhances binding to a FcRn as compared to a reference Fc polypeptide that does not comprise the mutation; and/or

(ii) a mutation that enhances binding to a FcyR as compared to a reference Fc polypeptide that does not comprise the mutation.

Embodiment 16. The anti-SARS-CoV-2 antibody or antigen-binding fragment of Embodiment 15, wherein the mutation that enhances binding to a FcRn comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q31 II; D376V; T307A; E380A; or any combination thereof. Embodiment 17. The anti-SARS-CoV-2 antibody or antigen-binding fragment of Embodiment 15 or 16, wherein the mutation that enhances binding to FcRn comprises:

(i) M428L/N434S;

(ii) M428L/N434A;

(iii) M252Y/S254T/T256E;

(iv) T250Q/M428L;

(v) P257VQ311I;

(vi) P257I/N434H;

(vii) D376V/N434H;

(viii) T307A/E380A/N434A; or

(ix) any combination of (i)-(viii).

Embodiment 18. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 15-17, wherein the mutation that enhances binding to FcRn comprises M428L/N434S or M428L/N434A.

Embodiment 19. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 15-17, wherein the mutation that enhances binding to a FcyR comprises S239D; I332E; A330L; G236A; or any combination thereof.

Embodiment 20. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 15-19, wherein the mutation that enhances binding to a FcyR comprises:

(i) S239D/I332E;

(ii) S239D/A330L/I332E;

(iii) G236A/S239D/I332E; or

(iv) G236A/A330L/I332E.

Embodiment 21. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-20, comprising an IgGl isotype, optionally an IgGlm3 allotype or an IgGlml7 allotype, comprising (according to EU numbering): (i) M428L and N434S mutations; (ii) G236A, L328V, and Q295E mutations; (iii) G236A, L328V, Q259E, M428L, and N434S mutations; (iv) G236A, L328V, Q295E, M428L, and N434S mutations, wherein the antibody or antigen-binding fragment is afucosylated; (v) G236A, R292P, and Y300L mutations; (vi) G236A, R292P, Y300L, M428L, and N434S mutations; (vii) G236A, A330L, I332E, M428L, and N434S mutations; (viii) a G236A mutation, optionally wherein the antibody or antigen-binding fragment is afucosylated; (ix) G236A, M428L, and N434S mutations, optionally wherein the antibody or antigen-binding fragment is afucosylated; (x) G236R and L328R mutations; or (xi) G236R, L328R, M428L, and N434S mutations, wherein, preferably, the antibody or antigen-binding fragment comprises: (1) M428L and N434S mutations; (2) G236A, R292P, Y300L, M428L, and N434S mutations; or (3) G236A, Y300L, M428L, and N434S mutations.

Embodiment 22. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-21, which comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or which is aglycosylated and/or afucosylated.

Embodiment 23. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-22, which is capable of activating a human FcyRIIa, a human FcyRIIIa, or both, when bound to a SARS-CoV-2 S protein expressed on a surface of a target cell, wherein, optionally:

(i) the target cell comprises an EpiCHO cell;

(ii) the human FcyRIIa comprises a Hl 31 allele;

(iii) the human FcyRIIIa comprises a VI 58 allele; and/or

(iv) the human FcyRIIIa and/or the human FcyRIIa is expressed by a host cell, such as a Jurkat cell or a Natural Killer cell, and activation is determined using a NFAT-driven luciferase signal in the host cell.

Embodiment 24. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 1-23, wherein the antibody or antigen-binding fragment is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody dependent cellular phagocytosis (ADCP) against a target cell infected by SARS-CoV-2. Embodiment 25. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments 11-24, wherein the Fc polypeptide or fragment thereof comprises a L234A mutation and a L235A mutation.

Embodiment 25a. An anti-SARS-CoV-2 antibody comprising:

(1) (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:402 (or SEQ ID NO.:402 without the C-terminal lysine) and (ii) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:403; or

(2) (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:402 (or SEQ ID NO.:402 without the C-terminal lysine) and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:403; or

(3) (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:404 (or SEQ ID NO.:404 without the C-terminal lysine or SEQ ID NO.:404 without the C-terminal glycine-lysine) and a (ii) light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:405;

(4) (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:404 (or SEQ ID NO.:404 without the C-terminal lysine or SEQ ID NO.:404 without the C-terminal glycine-lysine) and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:405; or

(5) (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:406 and (ii) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:407; or

(6) (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:406 and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:407.

Embodiment 26. An isolated polynucleotide encoding the anti-SARS-CoV- 2 antibody or antigen-binding fragment of any one of Embodiments l-25a, or encoding a VH, a Fd, a heavy chain, a VL, and/or a light chain of the antibody or the antigenbinding fragment, wherein, optionally, the polynucleotide comprises mRNA and/or comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigen-binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide.

Embodiment 27. The polynucleotide of Embodiment 26, wherein the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), wherein the RNA optionally comprises messenger RNA (mRNA).

Embodiment 28. The polynucleotide of Embodiment 26 or 27, which is codon-optimized for expression in a host cell.

Embodiment 29. A recombinant vector comprising the polynucleotide of any one of Embodiments 26-28.

30. A host cell comprising the polynucleotide of any one of Embodiments 26-28 and/or the vector of Embodiment 29, wherein the polynucleotide is heterologous to the host cell, wherein, optionally, the host cell is a mammalian cell, an insect cell, or a plant cell.

Embodiment 31. A human B cell comprising the polynucleotide of any one of Embodiments 26-28, wherein polynucleotide is heterologous to the human B cell and/or wherein the human B cell is immortalized.

Embodiment 32. A composition comprising:

(i) the anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments l-25a;

(ii) the polynucleotide of any one of Embodiments 26-28;

(iii) the recombinant vector of Embodiment 29;

(iv) the host cell of Embodiment 30; and/or

(v) the human B cell of Embodiment 31 ; and a pharmaceutically acceptable excipient, carrier, or diluent. Embodiment 33. A composition comprising the polynucleotide of any one of Embodiments 26-28, encapsulated in a carrier molecule, wherein the carrier molecule optionally comprises a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), or a nanoscale platform, and/or wherein the polynucleotide comprises mRNA.

Embodiment 34. A method of treating a sarbecovirus (e.g. SARS-CoV-2) infection in a subject, the method comprising administering to the subject an effective amount of

(ii) the polynucleotide of any one of Embodiments 26-28;

(iii) the recombinant vector of Embodiment 29;

(iv) the host cell of Embodiment 30;

(v) the human B cell of Embodiment 31; and/or

(vi) the composition of Embodiment 32.

Embodiment 35. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments l-25a, the polynucleotide of any one of Embodiments 26-28, the recombinant vector of Embodiment 29, the host cell of Embodiment 30, the human B cell of Embodiment 31, and/or the composition of Embodiment 32, for use in a method of treating a sarbecovirus (e.g. SARS-CoV-2) infection in a subject.

Embodiment 36. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of Embodiments l-25a, the polynucleotide of any one of Embodiments 26-28, the recombinant vector of Embodiment 29, the host cell of Embodiment 30, the human B cell of Embodiment 31, and/or the composition of Embodiment 32, for use in the preparation of a medicament for the treatment of a sarbecovirus (e.g. SARS-CoV-2) infection in a subject. Embodiment 37. The method of Embodiment 34 or the anti-SARS-CoV-2 antibody, antigen-binding fragment, polynucleotide, recombinant vector, host cell, human B cell, and/or composition for use of Embodiment 35 or 36, wherein the sarbecovirus comprises a sarbecovirus of Clade la, a sarbecovirus of clade lb, a sarbecovirus of clade 2, and/or a sarbecovirus of clade 3, and optionally comprises a SARS-CoV-2.

Embodiment 38. A method for in vitro diagnosis of a sarbecovirus (e.g. SARS-CoV-2) infection, the method comprising:

Embodiment (i) contacting a sample from a subject with an anti-SARS- CoV-2 antibody or antigen-binding fragment of any one of Embodiments l-25a; and (ii) detecting a complex comprising an antigen and the antibody, or comprising an antigen and the antigen-binding fragment.

Embodiment 39. The method of Embodiment 38, wherein the sample comprises blood isolated from the subject.

EXAMPLES

EXAMPLE 1 ENGINEERING IN A SARS-CoV-2 ANTIBODY

Sotrovimab is an engineered variant of S309, a human IgGl kappa monoclonal antibody isolated from B cells of a SARS-CoV survivor (Pinto et al. Nature 583:590- 295 (2020)). Sotrovimab has the VH amino acid sequence of SEQ ID NO.:30 and the VL amino acid sequence of SEQ ID NO.:34. Sotrovimab further comprises M428L and N434S mutations in the Fc and may also be referred herein to as S309 N55Q (referring to a N^Q substitution mutation in VH) or S309 N55Q MLNS or VIR-7831. Sotrovimab neutralizes some SARS-CoV-2 infections in vivo and has received limited authorizations to treat COVID-19.

Engineering in the Sotrovimab variable domains was performed to potentially improve binding affinity for SARS-CoV-2 and other features. Yeast display libraries were constructed, containing l-3x!0⁸ variant sequences. Binding to SARS-CoV-2 BA.2 was assessed by FACS. The 56 top-performing engineered variants were produced in yeast cells, and in vitro neutralization of BA.5 VSV pseudovirus was assessed. For in vitro neutralization experiments, VSV-pseudovirus and antibody were incubated for 1 hour. Antibodies were used at an equal final dilution of 1/10 (absolute IgG concentrations were adjusted during data analysis, after BLI-based quantification) and virus was diluted to reach MOI of 0.1. Vero E6 cells were incubated with virus/antibody mix for 1 hour. A luminescence-based read out was obtained after 24 hours. Briefly, neutralization potency is inversely proportional to the luminescence signal. Percentages of neutralization are calculated referencing to lurnmax/lurnmincontrols, and dose-response curves are plotted in Prism an IC50 values were interpolated.

The top approximately two-thirds of the engineered variants (32 variants) were tested for in vitro neutralization against a panel of SARS-CoV-2 or SARS-CoV VSV pseudoviruses (Wuhan-Hu-1, BA.2, BA.5, BA4.6, SARS-CoV), and fold-change in neutralization IC50 versus that of the parental S309 N55Q was assessed. Binding by the top thirteen variant antibodies to a panel of SARS-CoV-2 RBDs was assessed using SPR. A second round of engineering was performed, yielding 301 further variants. These, and the four best-performing variant antibodies from the first round, were produced in CHO cells.

In vitro neutralization of infection of SARS-CoV-2 Wuhan-Hu-1 (with D614G mutation) and Omicron haplotype BQ.1.1 was tested using S309 N55Q and several variant antibodies. The BQ.1.1 haplotype contains the following set of mutations in the spike protein: T19I / L24- / P25- / P26- / A27S / A69-70 / G142D / V213G / G339D / R346T / S371F / S373P / S375F / T376A / D405N / R408S / K417N / N440K / K444T / L452R / N460K / S477N / T478K / E484A / F486V / Q498R / N501 Y / Y505H / D614G / H655Y / N679K / P681H / N764K / D796Y / Q954H / N969K.

Antibodies were produced in parallel in Expi-CHO cells by transient transfection. IgG quantification was performed by BLI (Octet, using Protein A pins). Neutralization results are summarized in Figure 1. Several engineered variants had improved neutralization IC50 (ng/mL) values against Wuhan-Hu-1 D614G as compared to the parental S309 N55Q antibody, and all tested engineered variants had at least a 3- fold (up to a 14.4-fold) improvement in neutralization of BQ.1.1 as compared to the parental S309 N55Q antibody. The rightmost column in the table in Figure 1 shows the fold-change in neutralization potency for each tested antibody against BQ.1.1 versus Wuhan-Hu- 1 D614G. All data referred to a single experiment performed on cell culture supernatants.

Variable domain amino acid sequence alignments of S309 N55Q and the engineered variant antibodies tested in Figure 1 are shown in Figures 2A and 2B (see also Figures 3A-3G). The engineered variants contained one or more substitution mutation within regions identified as follows:

• IMGT CDRH1 plus the two C-terminal flanking amino acids

• IMGT CDRH2 plus the one N-terminal flanking amino acid and the eight C-terminal flanking amino acids

• IMGT CDRH3

• IMGT CDRL1 plus the three N-terminal flanking amino acids plus the two C-terminal flanking amino acids

• IMGT CDRL2 plus the four N-terminal flanking amino acids

• IMGT CDRL3.

Additional engineered variant antibodies were generated. Variable domain amino acid and corresponding exemplary polynucleotide sequences of certain of these antibodies are shown in Figures 4A-4X.

Figures 5A-7 show additional characterization of engineered variant antibodies. Figure 5A shows (left) neutralization IC50 values (ng/mL) by twenty-seven variants vs. a panel of SARS or SARS-CoV-2 VSV pseudoviruses and (right) fold-change in neutralization IC50 of the parental antibody versus the variant. The parental antibody (S309-N55Q) was included as a comparator. Variants were selected for further studies based on: improved or not reduced neutralization potency against circulating SARS- CoV-2 variants as compared to the parental antibody; and retained neutralization activity against SARS-CoV-2 prototypic virus and SARS-CoV. Figures 5B-5D show neutralization curves of variant antibodies. Figures 6A and 6B show binding affinities of twelve engineered variant antibodies and the parental antibody to a panel of Receptor Binding Domains (assessed by SPR). Briefly, the surface used anti-human Fc capture and the ligands were CHO cell supernatants. A 4-fold dilution series with 3 concentrations (50, 12.5, and 3.125 nM) was used. Single-cycle kinetics and a 1 : 1 bindin model were used. Affinity measurement revealed an improved binding profile for all of the variants tested. Figure 7 provides a table summarizing binding affinity and neutralization potency fold-improvement (versus the parental S309-N55Q) of variant antibodies, along with the mutations in the (IMGT + CCG) CDRs as compared to the parental antibody. Increased binding affinity did not correlate in all instances with the same magnitude in neutralization potency improvement against a match VSV pseudotype (or vice versa; e.g. S309-887 vs. Wuhan-Hu-1 and BA.5).

Figure 8 shows binding of antibodies to SARS-CoV-2 bearing escape mutations. Figure 9 shows binding affinity correlations between SARS and SARS- CoV-2 strains based on studies using antibodies of the present disclosure. Figure 10 shows binding (SPR) of certain high-performing engineered variant antibodies (selected based on neutralization studies) versus a panel of RBDs. Figure 11 shows fold-change in binding of antibodies (S309-N55Q and engineered variants) compared to purified S309 IgG, for the indicated SARS-CoV-2 antigens. Figure 12 summarizes neutralization IC50-values of parental S309-N55Q and certain engineered variants against a panel of SARS or SARS-CoV-2 VSV pseudoviruses. Figures 13A-13B show IC50 fold change graphs of parental S309-N55Q and 40 variants against BQ.1.1 VSV. Figures 14A-14G show engineered variants that were further tested versus 12 VSV viruses. Red boxes highlight the clones selected for a second NT test against the following VSV viruses: Wuhan-Hu-1 D614G, BQ.1.1, BA.5, BA.5 S8, BA.5 E340A, BA.5 P337T, BA.2.75.2 S8, BA.2.75-R346T, BA.4.6, BF.7, XBB.l, SARS-CoV. Figure 15A-15B show graphs of S309 neutralization vs. binding against SARS-CoV-2 and SARS-CoV-1 pseudoviruses for a first cycle (la) and a second cycle (lb) of engineered variant antibodies. Cycle lb improved neutralization and binding to BA 4.6 and BA.5. Compared to cycle la, cycle lb variants have a weaker binding to SARS1 and Wuhan-WT (n=l), though neutralization potency is similar. Figure 16A-16B show graphs of S309 fold change improvement compared to parent antibody against SARS- CoV-2 and SARS-CoV-1 pseudoviruses for the first cycle la and the second cycle lb. Figure 17 shows sensorgram data of weak binders vs non-binders to RBDs with escape mutations. Figures 18A-18H show graphs of S309 neutralization vs. binding against SARS-CoV-2 and SARS-CoV-1 pseudoviruses for step 2 of cycle lb. Blue indicates parental S309 N55Q (supernatant). Figures 19A-19B show neutralizing activity of 56 engineered variant antibodies as fold improvement to the parental S309-N55Q against BA.5. There was a gain in potency up to 5-fold compared to the parental. Maximal neutralization plateau is improved for some variants. 32 variants were selected for a second screening step (shown by red dots) based on fold-change of BA.5 vs Wuhan/SARSl for each mAb (not vs parental mAb). Figure 20 shows neutralizing activity of S309-N55Q versus a panel of SARS-CoV-2 and SARS-CoV-1 pseudoviruses (n=32). IC50 was improved as well as the level of maximal neutralization achieved (e.g. BA.4.6). Variants -828, -846, and -838 were selected for recombinant expression in mammalan cells. None of the variants acquired neutralization ability vs P337T and E340A inserted in the BA.5 S backbone. Figure 21A shows neutralization IC50 values (ng/mL) by engineered variant antibodies vs. a panel of SARS or SARS-CoV-2 VSV pseudoviruses. Figure 21B shows the same neutralization data sorted based on BA.5. Figure 22 shows correlation of neutralizing potency across different SARS-CoV-2 strains for S309-N55Q (VIR-7831). Figure 23 shows IC50 values and the top 13 engineered variant antibodies from cycle la neutralization.

Figure 24 shows SPR experimental setup for 15 engineered variant antibodies x 8 RBDs.

Figure 25 shows a graph of S309 neutralization vs binding against SARS-CoV- 2 and SARS-CoV-1 pseudoviruses. The plateau for neutralization potency appears to be different for Omicron vs Wuhan/SARS 1. The best binders for Omicron RBDs reach the same affinity as for Wuhan/SARSl, but not the same neutralization potency.

Figure 26 shows a graph of correlation between neutralization and affinity for S309 plotted as fold-change improvement to parental antibody. Figure 27 shows binding affinity correlations between SARS and SARS-CoV-2 strains based on studies using engineered variant antibodies.

Figure 28 shows S309 neutralization vs. k-off, neutralization vs. k-on, and KD correlation.

Figure 29 shows neutralization curves of engineered variant antibodies. The parental antibody was included for comparison. VSV pseudovirus neutralization assay was performed using Vero E6 cells. Vero E6 cells were grown in DMEM supplemented with 10% FBS and seeded into clear bottom white 96 well plates (PerkinElmer, 6005688) at a density of 20,000 cells per well. The next day, monoclonal antibodies were serially diluted in pre-warmed complete medium, mixed with pseudoviruses and incubated for 1 h at 37 °C in round bottom polypropylene plates. Medium from cells was aspirated and 50 pl of virus-monoclonal antibody complexes was added to cells, which were then incubated for 1 h at 37 °C. An additional 100 pl of prewarmed complete medium was then added on top of complexes and cells were incubated for an additional 16-24 h. Conditions were tested in duplicate wells on each plate and four wells per plate contained untreated infected cells (defining the 0% of neutralization, ‘MAX RLU’ value) and uninfected cells (defining the 100% of neutralization, ‘MIN RLU’ value). Virus-monoclonal antibody-containing medium was then aspirated from cells and 100 pl of a 1 :2 dilution of SteadyLite Plus (PerkinElmer, 6066759) in PBS with Ca2+ and Mg2+ was added to cells. Plates were incubated for 15 min at room temperature and then were analyzed on the Synergy-Hl (Biotek). The average relative light units (RLUs) of untreated infected wells (MAX RLUave) was subtracted by the average of MIN RLU (MIN RLUave) and used to normalize percentage of neutralization of individual RLU values of experimental data according to the following formula: (1 - (RLUx - MIN RLUave)/ (MAX RLUave - MIN RLUave)) x 100. Data were analyzed and visualized with Prism. IC50 values were calculated from the interpolated value from the log(inhibitor) versus response, using variable slope (four parameters) nonlinear regression with an upper constraint of <100.

Figure 30 shows neutralization data including IC50 values and fold change improvement to parent S309 summarized in table format for S309 variants. All variants have been purified and quantified by BLI in parallel with all other tested mAbs. Absolute IC50s remain higher than usual. This difference is also seen in other experiments done by different operators. Fold improve to parental S309 still points to S309.887-vl9.1 and vl5.1 as the most interesting. Although SBD variants display a good behavior too (better than what we have seen with supernatants). All variants have higher potency than S309 and S309.887 on CH.1.1 and BR.2. Figure 31 summarizes IC50 values for engineered variant antibodies. Figure 32 summarizes IC50 values for engineered variant antibodies without BN.1.

Figures 33A-39 are as shown and described in the Brief Description of the Drawings.

The various embodiments described above can be combined to provide further embodiments. All of the U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications and non-patent publications referred to in this specification and/or listed in the Application Data Sheet, including U.S. Provisional Application No. 63/384,765, filed on November 22, 2023, U.S. Provisional Application No. 63/386,477, filed on December 7, 2022 and U.S. Provisional Application No. 63/488,763, filed on March 6, 2023, are incorporated herein by reference, in their entirety. Aspects of the embodiments can be modified, if necessary to employ concepts of the various patents, applications and publications to provide yet further embodiments.

These and other changes can be made to the embodiments in light of the abovedetailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by the disclosure.

Claims

CLAIMS What is claimed is:

1. An anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising: (i) the CDRH1 amino acid sequence of any one of SEQ ID NOs.:361, 39,

49, 59, 69, 79, 89, 99, 111, 121, 131, 141, 151, 161, 171, 181, 191, 201, 211, 221, 231, 241, 251, 261, 271, 281, 291, 301, 311, 321, 331, 341, 351, 371, 381, and 391; (ii) the CDRH2 amino acid sequence of any one of SEQ ID NOs.:362, 40, 50, 60, 70, 80, 90, 100, 112, 122, 132, 142, 152, 162, 172, 182, 192, 202, 212, 222, 232, 242, 252, 262, 272, 282, 292, 302, 312, 322, 332, 342, 352, 372, 382, and 392; (iii) the CDRH3 amino acid sequence of any one of SEQ ID NOs.:363, 41, 51, 61, 71, 81, 91, 101, 113, 123, 133, 143, 153, 163, 173, 183, 193, 203, 213, 223, 233, 243, 253, 263, 273, 283, 293, 303, 313, 323, 333, 343, 353, 373, 383, and 393; (iv) the CDRL1 amino acid sequence of any one of SEQ ID NOs.:366, 44, 54, 64, 74, 84, 94, 104, 116, 126, 136, 146, 156, 166, 176, 186, 196, 206, 216, 226, 236, 246, 256, 266, 276, 286, 296, 306, 316, 326, 336, 346, 356, 376, 386, and 396; (v) the CDRL2 amino acid sequence of any one of SEQ ID NOs.:367, 45, 55, 65, 75, 85, 95, 105, 117, 127, 137, 147, 157, 167, 177, 187, 197, 207, 217, 227, 237, 247, 257, 267, 277, 287, 297, 307, 317, 327, 337, 347, 357, 377, 387, and 397; and (vi) the CDRL3 amino acid sequence of any one of SEQ ID NOs.:368, 46, 56, 66, 76, 86, 96, 106, 118, 128, 138, 148, 158, 168, 178, 198, 208, 218, 228, 238, 248, 258, 268, 278, 288, 298, 308, 318, 328, 338, 348, 358, 378, 388, and 398.

2. An anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising the CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences of SEQ ID NOs.: (i) 361, 362, 363, 366, 367, and 368, respectively; (ii) 49,

50, 51, 54, 55, and 56, respectively; (iii) 59, 60, 61, 64, 65, and 66, respectively; (iv) 69, 70, 71, 74, 75, and 76, respectively; (v) 79, 80, 81, 84, 85, and 86, respectively; (vi) 89, 90, 91, 94, 95, and 96, respectively; (vii) 99, 100, 101, 104, 105, and 106, respectively; (viii) 111, 112, 113, 116, 117, and 118, respectively; (ix) 121, 122, 123, 126, 127, and 128, respectively; (x) 131, 132, 133, 136, 137, and 138, respectively; (xi) 141, 142, 143, 146, 147, and 148, respectively; (xi) 151, 152, 153, 156, 157, and 158, respectively;

(xii) 161, 162, 163, 166, 167, and 168, respectively; (xiii) 171, 172, 173, 176, 177, and 178, respectively; (xiv) 181, 182, 183, 186, 187, and 188, respectively; (xv) 191, 192, 193, 196, 197, and 198, respectively; (xvi) 201, 202, 203, 206, 207, and 208, respectively; (xvii) 211, 212, 213, 216, 217, and 218, respectively; (xviii) 221, 222, 223, 226, 227, and 228, respectively; (xix) 231, 232, 233, 236, 237, and 238, respectively; (xx) 241, 242, 243, 246, 247, and 248, respectively; (xxi) 251, 252, 253, 256, 257, and 258, respectively; (xxii) 261, 262, 263, 266, 267, and 268, respectively; (xxiii) 271, 272, 273, 276, 277, and 278, respectively; (xxiv) 281, 282, 283, 286, 287, and 288, respectively; (xxiv) 291, 292, 293, 296, 297, and 298, respectively; (xxv) 301, 302, 303, 306, 307, and 308, respectively; (xxvi) 311, 312, 313, 316, 317, and 318, respectively; (xxvii) 321, 322, 323, 326, 327, and 328, respectively; (xxviii) 331, 332, 333, 336, 337, and 338, respectively; (xxix) 341, 342, 343, 346, 347, and 348, respectively; (xxx) 351, 352, 353, 356, 357, and 358, respectively; (xxxi) 39, 40, 41, 44, 45, and 46, respectively; (xxxii) 371, 372, 373, 376, 377, and 378, respectively; (xxxiii) 381, 382, 383, 386, 387, and 388, respectively; or (xxxiv) 391, 392, 393, 396, 397, and 398, respectively.

3. An isolated anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising: (i) in a VH, the amino acid sequences set forth in SEQ ID NOs.:361, 362, and 363, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:366, 367, and 368; (ii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 49, 50, and 51, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 54, 55, and 56; (iii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 59, 60, and 61, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 64, 65, and 66; (iv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 69, 70, and 71, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 74, 75, and 76; (v) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 79, 80, and 81, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 84, 85, and 86; (vi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 89, 90, and 91, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 94, 95, and 96; (vii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 99, 100, and 101, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 104, 105, and 106; (viii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: I l l, 112, and 113, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 116, 117, and 118; (ix) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 121, 122, and 123, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 126, 127, and 128; (x) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 131, 132, and 133, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 136, 137, and 138; (xi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 141, 142, and 143, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 146, 147, and 148; (xi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 151, 152, and 153, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 156, 157, and 158; (xii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 161, 162, and 163, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 166, 167, and 168; (xiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 171, 172, and 173, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 176, 177, and 178; (xiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 181, 182, and 183, and in a VL, the amino acid sequences set forth in SEQ ID NOs. : 186, 187, and 188; (xv) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 191, 192, and 193, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 196, 197, and 198; (xvi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:201, 202, and 203, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:206, 207, and 208; (xvii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:211, 212, and 213, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:216, 217, and 218; (xviii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:221, 222, and 223, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:226, 227, and 228; (xix) in a VH, the amino acid sequences set forth in SEQ ID NOs.:231, 232, and 233, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:236, 237, and 238; (xx) in a VH, the amino acid sequences set forth in SEQ ID NOs.:241, 242, and 243, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:246, 247, and 248; (xxi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:251, 252, and 253, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:256, 257, and 258; (xxii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:261, 262, and 263, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:266, 267, and 268; (xxiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:271, 272, and 273, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:276, 277, and 278; (xxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:281, 282, and 283, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:286, 287, and 288; (xxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:291, 292, and 293, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:296, 297, and 298; (xxv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:301, 302, and 303, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:306, 307, and 308; (xxvi) in a VH, the amino acid sequences set forth in SEQ ID NOs.:311, 312, and 313, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:316, 317, and 318; (xxvii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:321, 322, and 323, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:326, 327, and 328; (xxviii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:331, 332, and 333, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:336, 337, and 338; (xxix) in a VH, the amino acid sequences set forth in SEQ ID NOs.:341, 342, and 343, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:346, 347, and 348; (xxx) in a VH, the amino acid sequences set forth in SEQ ID NOs.:351, 352, and 353, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:356, 357, and 358; (xxxi) in a VH, the amino acid sequences set forth in SEQ ID NOs.: 39, 40, and 41, and in a VL, the amino acid sequences set forth in SEQ ID NOs.: 44, 45, and 46; (xxxii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:371, 372, and 373, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:376, 377, and 378; (xxxiii) in a VH, the amino acid sequences set forth in SEQ ID NOs.:381, 382, and 383, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:386, 387, and 388; or (xxxiv) in a VH, the amino acid sequences set forth in SEQ ID NOs.:391, 392, and 393, and in a VL, the amino acid sequences set forth in SEQ ID NOs.:396, 397, and 398.

4. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-3, comprising a VH having at least 85% identity (z.e., at least 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to a VH amino acid sequence provided in Table 3, and/or a VL having at least 85% identity (z.e., at least 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to a VL amino acid sequence provided in Table 3.

5. An anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising:

(i) the VH amino acid sequence of SEQ ID NO.:360 and the VL amino acid sequence of SEQ ID NO.:365;

(v) the VH amino acid sequence of SEQ ID NO.:78 and the VL amino acid sequence of SEQ ID NO.: 83;

(vi) the VH amino acid sequence of SEQ ID NO.: 88 and the VL amino acid sequence of SEQ ID NO.:93;

(viii) the VH amino acid sequence of SEQ ID NO. : 110 and the VL amino acid sequence of SEQ ID NO.: 115; (ix) the VH amino acid sequence of SEQ ID NO.: 120 and the VL amino acid sequence of SEQ ID NO.: 125;

(xx) the VH amino acid sequence of SEQ ID NO. :230 and the VL amino acid sequence of SEQ ID NO.:235;

(xxi) the VH amino acid sequence of SEQ ID NO.:240 and the VL amino acid sequence of SEQ ID NO.:245;

(xxiii) the VH amino acid sequence of SEQ ID NO.:260 and the VL amino acid sequence of SEQ ID NO.:265; (xxiv) the VH amino acid sequence of SEQ ID NO.:270 and the VL amino acid sequence of SEQ ID NO.:275;

(xxxiii) the VH amino acid sequence of SEQ ID NO.:38 and the VL amino acid sequence of SEQ ID NO.:43;

(xxxv) the VH amino acid sequence of SEQ ID NO.:380 and the VL amino acid sequence of SEQ ID NO.:385; or

(xxxvi)the VH amino acid sequence of SEQ ID NO.:390 and the VL amino acid sequence of SEQ ID NO.:395.

6. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-5, which is capable of neutralizing a SARS-CoV-2 infection in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human, wherein, optionally, the SARS-CoV-2 infection comprises a SARS-CoV-2 comprising the amino acid sequence according to SEQ ID NO.:3, or comprises a SARS-CoV-2 Omicron variant or subvariant.

7. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-6, wherein the antibody, or the antigen-binding fragment, comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, a Fab', a F(ab')2, a Fv, a scFv, or a scFab.

8. The anti-SARS-CoV-2 antibody or antigen-binding fragment of claim 7, wherein the antibody or antigen-binding fragment comprises a scFv comprising more than one VH domain and more than one VL domain.

9. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-8, wherein the antibody or antigen-binding fragment is a multi-specific antibody or antigen binding fragment.

10. The anti-SARS-CoV-2 antibody or antigen-binding fragment of claim 9, wherein the antibody or antigen binding fragment is a bispecific antibody or antigenbinding fragment.

11. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-10, wherein the antibody or antigen-binding fragment comprises a Fc polypeptide or a fragment thereof, wherein, optionally, (1) the antibody or antigenbinding fragment comprises a CH1-CH3 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:6, 7, 108, or 109, and/or (2) the antibody or antigen-binding fragment comprises a CL having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:9 or 8, and preferably comprising a CH1-CH3 having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:6, 7, 108, or 109 and a CL having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or 99% identity to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO.:9.

12. The antibody or antigen-binding fragment of any one of claims 1-11, which is human, humanized, or chimeric.

13. The antibody or antigen-binding fragment of any one of claims 1-12, which is a IgG, IgA, IgM, IgE, or IgD isotype.

14. The antibody or antigen-binding fragment of any one of claims 1-13, which is an IgG isotype selected from IgGl, IgG2, IgG3, and IgG4, optionally with a C-terminal lysine or a C-terminal glycine-lysine removed, and is optionally an IgGl glm,17 allotype or an IgGl glm3 allotype.

15. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 11-14, wherein the Fc polypeptide or fragment thereof comprises:

16. The anti-SARS-CoV-2 antibody or antigen-binding fragment of claim 15, wherein the mutation that enhances binding to a FcRn comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V;

T307A; E380A; or any combination thereof.

17. The anti-SARS-CoV-2 antibody or antigen-binding fragment of claim 15 or 16, wherein the mutation that enhances binding to FcRn comprises:

(i) M428L/N434S;

(ii) M428L/N434A;

(iii) M252Y/S254T/T256E;

(iv) T250Q/M428L;

(v) P257VQ311I;

(vi) P257I/N434H;

(vii) D376V/N434H;

(viii) T307A/E380A/N434A; or

(ix) any combination of (i)-(viii).

18. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 15-17, wherein the mutation that enhances binding to FcRn comprises M428L/N434S or M428L/N434A.

19. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 15-17, wherein the mutation that enhances binding to a FcyR comprises S239D; I332E; A330L; G236A; or any combination thereof.

20. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 15-19, wherein the mutation that enhances binding to a FcyR comprises:

(i) S239D/I332E;

(ii) S239D/A330L/I332E;

(iii) G236A/S239D/I332E; or

(iv) G236A/A330L/I332E.

21. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-20, comprising an IgGl isotype, optionally an IgGlm3 allotype or an IgGlml7 allotype, comprising (according to EU numbering): (i) M428L and N434S mutations; (ii) G236A, L328V, and Q295E mutations; (iii) G236A, L328V, Q259E, M428L, and N434S mutations; (iv) G236A, L328V, Q295E, M428L, and N434S mutations, wherein the antibody or antigen-binding fragment is afucosylated; (v) G236A, R292P, and Y300L mutations; (vi) G236A, R292P, Y300L, M428L, and N434S mutations; (vii) G236A, A330L, I332E, M428L, and N434S mutations; (viii) a G236A mutation, optionally wherein the antibody or antigen-binding fragment is afucosylated; (ix) G236A, M428L, and N434S mutations, optionally wherein the antibody or antigen-binding fragment is afucosylated; (x) G236R and L328R mutations; or (xi) G236R, L328R, M428L, and N434S mutations, wherein, preferably, the antibody or antigen-binding fragment comprises: (1) M428L and N434S mutations; (2) G236A, R292P, Y300L, M428L, and N434S mutations; or (3) G236A, Y300L, M428L, and N434S mutations.

22. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-21, which comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or which is aglycosylated and/or afucosylated.

23. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-22, which is capable of activating a human FcyRIIa, a human FcyRIIIa, or both, when bound to a SARS-CoV-2 S protein expressed on a surface of a target cell, wherein, optionally:

(i) the target cell comprises an EpiCHO cell;

(ii) the human FcyRIIa comprises a Hl 31 allele;

(iii) the human FcyRIIIa comprises a VI 58 allele; and/or (iv) the human FcyRIIIa and/or the human FcyRIIa is expressed by a host cell, such as a Jurkat cell or a Natural Killer cell, and activation is determined using a NFAT-driven luciferase signal in the host cell.

24. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-23, wherein the antibody or antigen-binding fragment is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody dependent cellular phagocytosis (ADCP) against a target cell infected by SARS-CoV-2.

25. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 11-24, wherein the Fc polypeptide or fragment thereof comprises a L234A mutation and a L235A mutation.

26. An anti-SARS-CoV-2 antibody comprising:

(1) (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:404 (or SEQ ID NO.:404 without the C-terminal lysine or SEQ ID NO.:404 without the C-terminal glycine-lysine) and a (ii) light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:405; or

(2) (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:404 (or SEQ ID NO.:404 without the C-terminal lysine or SEQ ID NO.:404 without the C-terminal glycine-lysine) and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:405; or

(3) (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:406 and (ii) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:407; or

(4) (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:406 and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:407; or (5) (i) a heavy chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:402 (or SEQ ID NO.:402 without the C-terminal lysine) and (ii) a light chain comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:403; or

(6) (i) two heavy chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:402 (or SEQ ID NO.:402 without the C-terminal lysine) and (ii) two light chains, each comprising or consisting of the amino acid sequence set forth in SEQ ID NO.:403.

27. An isolated polynucleotide encoding the anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-26, or encoding a VH, a Fd, a heavy chain, a VL, and/or a light chain of the antibody or the antigen-binding fragment, wherein, optionally, the polynucleotide comprises mRNA and/or comprises a single open reading frame encoding a heavy chain and a light chain (or a VH and a VL) of the antibody or antigen-binding fragment, wherein the sequence encoding the heavy chain and the sequence encoding the light chain are optionally separated by polynucleotide encoding a protease cleavage site and/or by a polynucleotide encoding a self-cleaving peptide.

28. The polynucleotide of claim 27, wherein the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), wherein the RNA optionally comprises messenger RNA (mRNA).

29. The polynucleotide of claim 27 or 28, which is codon-optimized for expression in a host cell.

30. A recombinant vector comprising the polynucleotide of any one of claims 27-29.

31. A host cell comprising the polynucleotide of any one of claims 27-29 and/or the vector of claim 30, wherein the polynucleotide is heterologous to the host cell, wherein, optionally, the host cell is a mammalian cell, an insect cell, or a plant cell.

32. A human B cell comprising the polynucleotide of any one of claims 27- 29, wherein polynucleotide is heterologous to the human B cell and/or wherein the human B cell is immortalized.

33. A composition comprising:

(i) the anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-26;

(ii) the polynucleotide of any one of claims 27-29;

(iii) the recombinant vector of claim 30;

(iv) the host cell of claim 31; and/or

(v) the human B cell of claim 32; and a pharmaceutically acceptable excipient, carrier, or diluent.

34. A composition comprising the polynucleotide of any one of claims 27-29 encapsulated in a carrier molecule, wherein the carrier molecule optionally comprises a lipid, a lipid-derived delivery vehicle, such as a liposome, a solid lipid nanoparticle, an oily suspension, a submicron lipid emulsion, a lipid microbubble, an inverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, lipid nanoparticle (LNP), or a nanoscale platform, and/or wherein the polynucleotide comprises mRNA.

35. A method of treating a sarbecovirus (e.g. SARS-CoV-2) infection in a subject, the method comprising administering to the subject an effective amount of:

(ii) the polynucleotide of any one of claims 27-29; (iii) the recombinant vector of claim 30;

(iv) the host cell of claim 31 ;

(v) the human B cell of claim 32; and/or

(vi) the composition of claim 33 or 34.

36. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-26, the polynucleotide of any one of claims 27-29, the recombinant vector of claim 30, the host cell of claim 31, the human B cell of claim 32, and/or the composition of claim 33 or 34, for use in a method of treating a sarbecovirus (e.g. SARS-CoV-2) infection in a subject.

37. The anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-26, the polynucleotide of any one of claims 27-29, the recombinant vector of claim 30, the host cell of claim 31, the human B cell of claim 32, and/or the composition of claim 33 or 34, for use in the preparation of a medicament for the treatment of a sarbecovirus (e.g. SARS-CoV-2) infection in a subject.

38. The method of claim 35 or the anti-SARS-CoV-2 antibody, antigenbinding fragment, polynucleotide, recombinant vector, host cell, human B cell, and/or composition for use of claim 36 or 37, wherein the sarbecovirus comprises a sarbecovirus of Clade la, a sarbecovirus of clade lb, a sarbecovirus of clade 2, and/or a sarbecovirus of clade 3, and optionally comprises a SARS-CoV-2.

39. A method for in vitro diagnosis of a sarbecovirus (e.g. SARS-CoV-2) infection, the method comprising:

(i) contacting a sample from a subject with an anti-SARS-CoV-2 antibody or antigen-binding fragment of any one of claims 1-26; and

(ii) detecting a complex comprising an antigen and the antibody, or comprising an antigen and the antigen-binding fragment.

40. The method of claim 39, wherein the sample comprises blood isolated from the subject.