WO2022204202A1

WO2022204202A1 - Antibodies that bind to multiple sarbecoviruses

Info

Publication number: WO2022204202A1
Application number: PCT/US2022/021415
Authority: WO
Inventors: Davide Corti; Anna De Marco; Dora PINTO; Matteo Samuele PIZZUTO
Original assignee: Vir Biotechnology, Inc.
Priority date: 2021-03-23
Filing date: 2022-03-22
Publication date: 2022-09-29

Abstract

The instant disclosure provides antibodies and antigen-binding fragments thereof that can bind to S proteins of sarbecoviruses (including, in some embodiments, multiple sarbecoviruses) and, in certain embodiments, are capable of neutralizing infection by multiple sarbecoviruses.

Description

ANTIBODIES THAT BIND TO MULTIPLE SARBECOVIRUSES

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is 930585_423WO_SEQUENCE_LISTING.txt. The text file is 208 KB, was created on March 14, 2022, and is being submitted electronically via EFS-Web.

BACKGROUND

A novel sarbecovirus emerged in Wuhan, China, in late 2019. As of March 202022, approximately 6.07 million cases of infection by this virus (termed, among other names, SARS- CoV-2), were confirmed worldwide, and had resulted in approximately 470 million deaths. SARS-CoV-2 and SARS-CoV are members of the sarbecovirus lineage. Sarbecoviruses are further divided into four clades: la, lb, 2, and 3. SARS-CoV is a member of clade la, while SARS-CoV-2 is a member of clade lb. Therapies for preventing or treating sarbecovirus infections, and diagnostic reagents for diagnosing sarbecovirus infections are needed.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

Figure 1 shows a phylogenetic tree of sarbecoviruses, divided into clade la (also referred to as clade 1), clade lb (also referred to as clade 1/2), clade 2, and clade 3.

Figure 2 shows differences in receptor binding domain (RBD) amino acid sequences among various sarbecoviruses and the amino acids that are likely significant for S2K146 interaction with viral antigens (boxes).

Figure 3 shows inhibitory effects of various antibodies on the ability of the SARS-CoV and the SARS-CoV-2 RBDs to bind the ACE2 receptor in a competitive ELISA.

Figure 4 shows effects of antibodies S2E12, S309, S2X259, and S2M11 on SI staining (which reflects SI shedding) over time. S2E12 was used as positive control whereas S2M11 and S309 were used as negative controls.

Figure 5 shows binding results for binding of antibodies S2X259 and S2K146 to the RDB of SARS-CoV, WIV-1, SARS-CoV-2, RatG13, PangGD, PangGX, Anlongl 12, YN2013, SC2018, SC2011, ZC45, BtKY72, and BGR2008, and the SARS-CoV-2 variants N501Y,

Y453F, N439K, K417V, E484K, N501Y-K417N-E484K, Californian variant, Brazilian variant, and Swiss variant, representing sarbecovirus Clades la, lb, 2 and 3, as well as several variants of SARS-CoV-2, as measured by ELISA.

Figure 6 shows binding results for binding of antibodies S2X259-v2, S2X59-vl5,

S2H90, S2K146, S2X259-v5, S2X259-v6, and S2X259-v7 to the sarbecoviruses SARS-Co-V, WIV1, SARS-CoV-2, RatG13, PangGD, PangGX, Anlongll2, YN2013, SC2018, SX2011, ZC45, BtKY72, and BRG2008, and the SARS-CoV-2 variants N501Y, Y453F, N439K, K417V, E484K, B.1.351 (beta), B.1.429 (epsilon), P.l (delta) and B.1.1.222, as measured by ELISA.

IC50 values (ng/mL) are shown and are color-coordinated in accordance with the scale at bottom.

Figure 7 shows EC50 values of S2K146 and comparator antibody S2E12 for binding to the RDB of SARS-CoV, WIV-1, SARS-CoV-2, RatG13, PangGD, PangGX, Anlongll2, YN2013, SC2018, SC2011, ZC45, BtKY72, and BGR2008, and the SARS-CoV-2 variants K417V, E484K, B.l.1.7, B1.298, B.1.1.258, B.1.351, B.1.429, P.l, and B.1.1.519, representing sarbecovirus Clades la, lb, 2 and 3, as well as several variants of SARS-CoV-2. EC50 values from at least two independent experiments are shown.

Figure 8 shows binding results for binding of antibody S2K146 to various sarbecoviruses, including SARS-Co-V, WIV1, SARS-CoV-2, RatG13, PangGD, and PangGX, and the SARS-CoV-2 variants alpha (B.l.1.7), beta (B.1.351), gamma (P.l), epsilon (B.1.427 and B.1.429), B.1.1.519, Bl.1.298, B.1.258, K417V, and E484K, as measured by ELISA.

Figure 9 shows ELISA binding by wild type antibody S2K146 (left) and antibody S2K146 UCA (the Unmutated Common Ancestor of Antibody S2K146) (right) against SARS- CoV, SARS-CoV-2, WIV-1, RatG13, PangGD (PG-GD), and PangGX (PG-GX) RBDs.

Figure 10 summarizes flow cytometry results for S2K146 and comparator antibody S2E12 cross-reactivity with a panel of twelve spike glycoproteins representative of sarbecovirus clades la and lb transiently expressed on the surface of mammalian cells.

Figure 11 shows results of a biolayer interference assay (BLI) for SARS-CoV-2 RBD using S2K146 in the presence of site I-targeting antibody S2E12, site IV-targeting antibody S309, or site Il-targeting antibody S2X259.

Figure 12 biolayer interferometry results for antibody S2K146 and for antibody S2K146 UCA binding to SARS-CoV and SARS-CoV-2 prefusion-stabilized S ectodomain trimers.

Figure 13 shows neutralization of SARS-CoV and SARS-CoV-2 infection by S2K146 and S2K146 UCA using a VSV-based pseudovirus system. Figure 14 shows neutralization of YSV pseudotypes harboring Wuhan-Hu-1 SARS-CoV- 2 S, and variants B.1.351 (beta), B.l.1.7 (alpha), P.l (gamma), B.1.429 (epsilon), C.37 (lambda), AY.2 (delta+) SARS-CoV-2 S or SARS-CoV S by S2K146.

Figure 15 shows neutralization of SARS-CoV-2 MLV-based pseudotypes by antibody S2K146 and comparator antibody S2E12.

Figure 16 shows neutralization of SARS-CoV-2 infection by antibody S2K146 and comparator antibodies S2H90 and S2E12 using a VSV-based pseudovirus system in Vero E6 cells.

Figure 17 shows neutralization of SARS-CoV-2 infection by antibody S2K146 and comparator antibodies S2H90 and S2E12 using a VSV-based pseudovirus system in Vero- TMPRRS2 cells.

Figure 18 shows neutralization of SARS-CoV-2 infection by antibody S2K146 and comparator antibodies S2H90 and S2E12 using a MLV-based pseudovirus system in Vero E6 cells.

Figure 19 shows fold change in neutralization of SARS-CoV-2 infection by antibody S2K146 (VSV-based pseudovirus neutralization assay) using SARS-CoV-2 wild type (WT; Wuhan-Hu-1) or certain variants of concern: B.l.1.7 (alpha), B.1.351 (Beta), P.l (Gamma),

B.1.429 (Epsilon), C.37 (Lambda), AY.1 (Delta Plus) and AY.2 (Delta Plus).

Figure 20 shows neutralization of SARS-CoV-2 by antibody S2K146 and comparator antibody S2E12 in a VSV-based pseudovirus neutralization assay for WT SARS-CoV-2 and certain variants of concern.

Figure 21 shows neutralization of replication-competent nanoluciferase SARS-CoV-2 Wuhan-Hu-1 and SARS-CoV-2 variant of concern viruses by antibody S2K146 or comparator antibody S2E12.

Figure 22 shows shows inhibitory effects of antibody S2K146 and comparator antibody S2E12 in an ACE2 receptor competitive ELISA.

Figure 23 shows NFAT-driven luciferase signal induced in Jurkat cells stably expressing FcyRIIaH131 by S2K146 or comparator antibodies S309, S2E12, or S309-GRLRby binding to full-length wild type SARS-CoV-2 spike protein on CHO target cells.

Figure 24 shows NFAT-driven luciferase signal induced in Jurkat cells stably expressing FcyRIIIa V148 by S2K146 or comparator antibodies S309, S2E12, or S309-GRLRby binding to full-length wild type SARS-CoV-2 spike protein on CHO target cells. Figure 25 shows NFAT-driven luciferase signal induced in Jurkat cells stably expressing FcyRIIaH131 by S2K146 or comparator antibodies S309, S2E12, or S309-GRLRby binding to full-length wild type SARS-CoV-2 uncleavable spike protein on CHO target cells.

Figure 26 shows NFAT-driven luciferase signal induced in Jurkat cells stably expressing FcyRIIIa V148 by S2K146 or comparator antibodies S309, S2E12, or S309-GRLRby binding to full-length wild type SARS-CoV-2 uncleavable spike protein on CHO target cells.

Figure 27 shows effects of antibody S2K146 and comparator antibodies S2M11 and S2E12 on SI staining (which reflects SI shedding) overtime.

Figure 28 shows viral RNA titers in the lungs of Syrian hamsters intranasally infected with the B.1.351 variant of SARS-CoV-2 and treated with the indicated amounts of S2K146 or comparator antibody S2E12 at 24 hours after infection. 4 days post-infection, animals were euthanized and lungs were collected. n=6/5 animal for each group for S2K146, n=6 animals for S2E12. Isotype control was administered at 10 mg/kg (n=6 animals).

Figure 29 shows replicating virus titers in the lungs of Syrian hamsters treated and collected as described with respect to Figure 28.

Figure 30 shows overall serum mAb concentration measured at day 4 post-infection correlated with viral RNA loads in the lungs of Syrian hamsters treated and collected as described with respect to Figure 28.

Figure 31 shows overall serum mAb concentration measured at day 4 post-infection correlated with replicating virus titers in the lungs of Syrian hamsters treated and collected as described with respect to Figure 28.

Figure 32 shows SARS-Co-2 S protein trimer in fully open conformation (PDB: 7K.4N) with positions of mutated residues in the Omicron variant in and outside the ACE footprint.

Figure 33 shows Omicron mutations in a primary structure of SARS-CoV-2 S with domains and cleavage sites highlighted.

Figure 34 shows single-cycle kinetics surface plasmon resonance (SPR) analysis of ACE2 binding to five SARS-CoV-2 RBDs. ACE2 is injected successively 11, 33, 100, and 300 nM (human) or 22, 100, 300, and 900 nM (mouse). Monomeric and dimeric ACE2 were tested. Curves show fits to a 1 : 1 binding model. Vertical dashed lines indicate transitions between association and dissociation phases.

Figure 35 shows quantification of human ACE2 binding data shown in Figure 34.

Figure 36 shows neutralization of Omicron SARS-CoV-2 VSV pseudovirus by plasma from COVID-19 convalescent and vaccinated individuals. Specifically, Figure 36 shows plasma neutralizing activity in COVID-19 convalescent vaccinated (BNT162b2 mRNA vaccine) individuals collected at the indicated timepoints for pairwise neutralizing antibody titers (ID50).

Figure 37 also shows neutralization of Omicron SARS-CoV-2 VSV pseudovirus by plasma from COVID-19 convalescent and vaccinated individuals. Specifically, Figure 37 shows fold change loss in ID50 values between Omicron versus Wuhan-Hu-1. Data are an average of n=2 replicates.

Figure 38 shows the RBD sequence of SARS-CoV-2 Wuhan-Hu-1 with highlighted footprints of ACE2 (cross-hatching) and mAbs (slashes).

Figure 39 shows neutralization of SARS-CoV-2 VSV pseudoviruses carrying Wuhan- Hu-1 (WT) or Omicron S proteins by clinical-stage mAbs. Date are representative of n=2 replicates.

Figure 40 shows mean IC50 values for Omicron (white circles) and Wuhan-Hu-1 (black circles) in the top panel and the mean fold change in the bottom panel for clinical-stage mAbs. Non-neutralizing IC50 titers and fold change were set to 10⁴ and 10³, respectively.

Figure 41 shows mean IC50 values for Omicron (white circles) and Wuhan-Hu-1 (black circles) in the top panel and mean fold change in the bottom panel for 4 N-terminal domain (NTD) mAbs and 32 RBD mAbs. Non-neutralizing IC50 titers and fold change were set to 10⁴ and 10³, respectively.

Figure 42 illustrates RBD sites targeted by 4 mABs that cross-neutralize Omicron and representative antibody Fv regions bound to S. Shaded surfaces on the RBD depict the epitopes and the receptor binding motif (RBM) is shown as a black outline.

Figure 43 shows mutations shared by Omicron with other sarbecoviruses and SARS- CoV-2 variants of concern (VOCs).

Figure 44 shows that since the beginning of the SARS-CoV-2 pandemic there is a progressive coalescence of Omicron-defming mutations into non-Omicron haplotypes that may carry as many as 10 of the Omicron-defming mutations.

Figure 45 shows that Pango lineages (dots) assessed to-date rarely carry more than 10-15 lineage-defining mutations.

Figures 46A-46D show that some non-Omicron haplotypes may carry up to a maximum of 19 Omicron-defming mutations. Selected exceptional haplotypes are shown. Spike G142D and Y145del may also be noted as G142del and Y145D.

Figure 47 shows single-cycle kinetics SPR analysis of ACE2 binding to five RBD variants. Dimeric mink or pangolin ACE2 was injected successively at 33, 100, 300, and 90 nM. Vertical dashed lines indicate transitions between association and dissociation phases. Monomeric human ACE2 binding to Wuhan-Hu-1 RBD (ACE2 concentrations of 11, 33, 100, and 300 nM) are shown for comparison.

Figure 48 shows neutralization of SARS-CoV-2 pseudotyped VSV carrying WT or Omicron S protein by plasma from COVID-19 convalescent individuals 2-4 weeks after infection by WT SARS-CoV-2.

Figure 49 shows neutralization of SARS-CoV-2 pseudotyped VSV carrying WT or Omicron S protein by plasma from individuals previously infected by WT SARS-Co-V-22-4 weeks after receiving a second dose of Pfizer/BioNtech BNT162b2 mRNA vaccine.

Figure 50 shows neutralization of SARS-CoV-2 pseudotyped VSV carrying WT or Omicron S protein by plasma from naive individuals not known to have been infected by WT SARS-CoV-2, 2-4 weeks after receiving a second dose of Pfizer/BioNtech BNT162b2 mRNA vaccine.

Figure 51 shows neutralization of SARS-CoV-2 pseudotyped VSV carrying WT or Omicron S protein by plasma from naive individuals not known to have been infected by WT SARS-CoV-2, 7-10 months after receiving a second dose of Pfizer/BioNtech BNT162b2 mRNA vaccine.

Figure 52 shows neutralization of SARS-CoV-2 pseudotyped VSV carrying wild-type D614 virus (black circles) or Omicron (white circles) S protein by NTD-targeting mAbs. Date are representative of one independent experiment out of two. Mean +/- s.d. of 2 technical replicates is shown.

Figure 53 shows neutralization of SARS-CoV-2 pseudotyped VSV carrying wild-type D614 virus (black circles) or Omicron (white circles) S protein by RBM-targeting mAbs. Date are representative of one independent experiment out of two. Mean +/- s.d. of 2 technical replicates is shown.

Figure 54 shows neutralization of SARS-CoV-2 pseudotyped VSV carrying wild-type D614 virus (black circles) or Omicron (white circles) S protein by core RBD-targeting mAbs. Date are representative of one independent experiment out of two. Mean +/- s.d. of 2 technical replicates is shown.

DETAILED DESCRIPTION

Provided herein are antibodies and antigen-binding fragments that are capable of binding to a sarbecovirus (e.g. SARS-CoV-2). In some embodiments, an antibody or antigen-binding fragment is 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 sarbecoviruses, optionally comprised on a virion and/or expressed on the surface of a cell infected by two or more sarbecoviruses). In certain embodiments, presently disclosed antibodies and antigen-binding fragments can neutralize infection by one or more sarbecovirus (e.g. , one, two, three, four, 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 one or more sarbecovirus (e.g. one, two, three, four, 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, an “anti-sarbecovirus antibody or antigen-binding fragment” specifically binds at least one sarbecovirus and may bind two or more, three or more, four or more, or five or more sarbecoviruses.

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, RatG13, Pangolin-Guanxi-2017 (PANG/GX) and Pangolin-Guangdon-2019 (PANG/GD). Examples of clade lb also include SARS-CoV-2 variants, for example N501Y, Y453F, N439K, K417V, E484K, N501Y-K417N-E484K, B.E1.7 , B.1.351, B.1.429, P.1, B.1.1.222, C.37, AY.l, AY.2, a California variant, a Brazilian variant, or a Swiss variant. Examples of clade 2 sarbecoviruses are Bat ZC45 (ZC45), Bat ZXC21 (ZXC21), YN2013, RmYN02, Anlongll2, SC2018, SX2011. Examples of clade 3 sarbecoviruses are BtkY72 and BGR2008. Sarbecovirus clades are also illustrated in Figure 1 and differences in RBDs among sarbecoviruses are illustrated in Figure 2 and Figure 38.

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 (e.g. Bat ZC45 (ZC45), Bat ZXC21 (ZXC21), YN2013, RmYN02, Anlongll2, SC2018, SX2011, or any combination thereof); and/or a sarbecovirus of clade 3 (e.g. BtkY72, BGR2008, or both). In certain further embodiments, an antibody or antigen-binding fragment thereof is capable of binding to a SARS-CoV-2 variant; e.g., aN501Y variant; a Y453F variant; aN439K variant; a K417V variant; a N501Y-K417N-E484K variant; a E484K variant; a California variant; a Brazilian variant; a Swiss variant; BEE7; B.1.351; B.E429, P.l; B.1.1.222; C.37;

AY.1 AY.2, or any combination thereof. In some embodiments, a SARS-Cov-2 variant is an Omicron variant, also refered-to as B.1.1.529.1.

In some embodiments, an antibody or antigen-binding fragment thereof is capable of inhibiting a binding interaction between human ACE2 and a sarbecovirus (e.g., SARS-CoV-2) receptor binding domain (RBD) with an IC50 of about 12 ng/mL, about 12.5 ng/mL, or about 13 ng/mL.

As used herein, "SARS-CoV-2", also originally referred to as "Wuhan coronavirus", "Wuhan seafood market pneumonia virus", or "Wuhan CoV", "novel CoV", or "nCoV", or "2019 nCoV", or "Wuhan nCoV", or a variant thereof, is a betacoronavirus 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. SARS CoV-2 infection can result in a disease known as COVID-19; symptoms of COVID-19 include fever or chills, dry cough, dyspnea, fatigue, body aches, headache, new loss of taste or smell, sore throat, congestions or runny nose, nausea or vomiting, diarrhea, persistent pressure or pain in the chest, new confusion, inability to wake or stay awake, and bluish lips or face.

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), 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 Wuhan-Hu-1 surface glycoprotein is provided in SEQ ID NO.:3. The amino acid sequence of the Wuhan-Hu-1 RBD is provided in SEQ ID NO.:4. Wuhan-Hu-1 S protein has approximately 73% amino acid sequence identity with SARS-CoV. The amino acid sequence of Wuhan-Hu-1 RBM is provided in SEQ ID NO.:5.

There have been a number of emerging SARS-CoV-2 variants, which may differ in genomic and amino acid sequences, particularly of the surface glycoprotein or the RBD. For example the SARS-CoV, Urbani strain, surface glycoprotein has a sequence provided in SEQ ID NO:2. 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 N501Y mutation, which is associated with increased transmissibility, including the lineages B.1.1.7 (also known as 201/501 Y. VI and VOC 202012/01) and B.1.351 (also known as 20H 501Y.V2), which were discovered in the United Kingdom and South Africa, respectively (Tegally, H., 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 N501Y 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, H., 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.1, 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 https://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 B 1.1.207; 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.l.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), and Gamma (P.1, 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). SARS-CoV-2 Omicron variant (B.1.1.529.1) comprises the mutations shown in Figure 32 and Figure 33. Treating a SARS CoV-2 infection in accordance with the present disclosure includes treating infection by any one or more of the aforementioned SARS-CoV-2 viruses. In certain embodiments, treating a SARS-CoV-2 infection comprises treating any one or more of: SARS CoV-2 Wuhan-Hu-1; a SARS-CoV-2 variant comprising a N439K mutation; a SARS-CoV-2 variant comprising a N501Y mutation; a SARS-CoV-2 variant comprising a K417N mutation and/or a E484K mutation; a SARS-CoV-2 comprising a L452R mutation; B.1.1.28; B.1.1.7 (also referred-to as the "alpha" variant); B.1.351 (also referred-to as the "beta" variant); P.l (also referred-to as the "gamma" variant); B.1.617.1 (also referred-to as the "kappa" variant); B.1.429 (also referred-to as the "epsilon" variant); B.1.525 (also referred-to as the "eta" variant); B.1.526 (also referred-to as the "iota" variant); B.1.258; a variant of Wuhan-Hu-1 comprising aN440K mutation;

B.1.243.1; B.1.258 with a K417N mutation; A.27.1; R.l; P.2; R.2; B.1.1.519; A.23.1; B.1.318;

B.1.619; A.VOI.V2; B.1.618; a variant of Wuhan-Hu-1 comprising N440K and E484K mutations; B.1.617.2 (also referred-to as the "delta" variant); B.1.1.298; B.1.617.2-AY.1; B.1.617.2-AY.2; C.37 (also referred-to as the "lambda" variant); a SARS CoV-2 of clade 19A; SARS CoV-2 of clade 19B; a SARS CoV-2 of clade 20 A; 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 a SARS CoV-2 omicron variant.

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 of¹ 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 (i.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, g- carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.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 (lie or I), Leucine (Leu or L), Methionine (Met or M), Valine (Val 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, Val, Leu, and lie. 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, Val, 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.

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.

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 (i.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. 8: 108, 2003: Mates etal, Nat. Genet. 41:153, 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 (i.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 g-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-BLY group, lentivirus, spumavirus (Coffin, J. M., Retroviridae: The viruses and their replication, In Fundamental Virology, Third Edition, B. N. Fields et ah, 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 eta/., PLoS One 6: 327930, 2011; Zhao etal., J. Immunol. 777:4415, 2005; Engels et al., Hum. Gene Ther. 77:1155, 2003; Frecha etal., Mol. Ther. 75:1748, 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 multicistronic 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 fragment-encoding 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 sarbecovirus infection, a "host" refers to a cell or a subject infected with a 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 cytotoxicity, 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., a surface glycoprotein or portion thereof), such as present in a virion, or expressed or presented on the surface of a cell infected by a sarbecovirus.

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 isolated antibody, or an antigen-binding fragment thereof, that is capable of binding to a surface glycoprotein of two or more sarbecoviruses, three or more sarbecoviruses, four or more sarbeco viruses, or five or more sarbecoviruses. In some embodiments, the antibody or antigen-binding fragment comprises a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3.

In some embodiments, the two or more, three or more, four or more, or five or more sarbecoviruses are selected from: clade la sarbecoviruses and/or clade lb sarbecoviruses; clade 2 sarbecoviruses; clade 3 sarbecoviruses; or naturally occurring 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, three or more, four or more, or five or more sarbecoviruses; e.g. capable of binding when a sarbecovirus surface glycoprotein is expressed on a cell surface of a host cell and/or on a sarbecovirus virion. In certain embodiments, the two or more, three or more, four or more, or five or more sarbecoviruses are selected from SARS-CoV, WIY1, SARS-CoV2, PANG/GD, PANG/GX, RatG13, ZXC21,

ZC45, RmYN02, BGR2008, BtkY72, Anlongll2, YN2013, SC2018, SX2011, and naturally occurring variants thereof. In some embodiments, the two or more, three or more, four or more, or five or more sarbecoviruses include one or more of SARS-CoV-2 variants P.1, B.l.1.7,

B.1.429, B.1.351, Bl.1.222, B.l.1.529, C.37, AY.l, and AY.2.

In some embodiments, the two or more, three or more, four or more, or five or more sarbecoviruses include one or more SARS-CoV-2 variants having S protein mutations D614G, Q493R, G496S, Q498R, N501Y, Y453F, N439K, K417V, E484K, or any combination thereof.

In certain embodiments, two or more sarbecoviruses include one or more SARS-CoV-2 variants having S protein mutations K417N, Q493K, G496S, or any combination thereof.

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 some embodiments, the epitope is a conformational epitope or a linear epitope.

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. 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 (i.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_0ff] 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 (Kd) of a particular binding interaction with units of M (e.g., lO 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 10¹⁰ M ¹, at least 10^u M ¹, at least 10¹²M^_1, or at least 10¹³ M ¹. "Low-affinity" antibodies refer to those antibodies having a K_a of up to 10⁷ M 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 VI (e.g., 10⁵ M to 10 ¹³ M).

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 embodiments, an antibody or antigen-binding fragment of the present disclosure binds to a spike (S) protein RBD from each of two or more sarbecoviruses with an EC50 in a range from about 15 to about 100 ng/mL. In certain embodiments, an antibody or antigen-binding fragment of the present disclosure binds to each of two or more sarbecoviruses with a EC50 in a range from about 15 to about 50 ng/mL, or of about 20, 25, 30, 35, or 40 ng/mL, as determined by ELISA. In some embodiments, the antibody or antigen-binding fragment is capable of binding to two or more sarbecoviruses selected from Clade la (including, for example, SARS-CoV-2 variants comprising N501Y, Y453F, N439K, K417V, N501Y- K417N-E484K, B.l.1.7 , B.1.351, B.1.429, P.1, B.1.1.222, B.l.1.529, C.37, AY.l, AY.2, Californian, Brazilian, and/or Swiss) and/or Clade lb with an EC50 of about 15, about 17, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 ng/mL.

In some embodiments, the antibody or antigen-binding fragment is capable of binding to a first and a second sarbecovirus each independently selected from Clade la and/or Clade lb, wherein the antibody or antigen-binding fragment and is capable of binding to the first sarbecovirus with an EC50 of about 15, about 17, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 ng/mL, and is capable of binding to the second sarbecovirus with an EC50 of about 15, about 17, about 20, about 25, about 30, about 35, about 40, about 45, or about 50 ng/mL.

In some embodiments, an antibody or antigen-binding fragment is capable of binding to a SARS-CoV-2 RBD with a KD of about 5 x 10 ^u, or about 5.5 x 10 ¹¹, or 5.7 x 10 ¹¹, or 5.8 x 10 ^u, and/or to a SARS-CoV RBD with a KD of about 1.2 x 10 ⁹; e.g., as determined using biolayer interferometry (e.g., using Octet).

In certain examples, binding can be determined by recombinantly expressing a sarbecovirus 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 cytometry ( 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 sarbecovirus-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, three or more, four or more, or five 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, as measured using biolayer interferometry.

In certain embodiments, an antibody of the present disclosure is capable of neutralizing infection by two or more sarbecoviruses. As used herein, a "neutralizing antibody" is one that can neutralize, i.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 infection by two or more sarbecoviruses 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 of the present disclosure is capable of neutralizing infection by SARS-CoV-2 in a pseudovirus system (e.g., MLV-pp- based or VSV-pp-based ) with an IC50 in a range from about 0.001 pg/mL to about 0.5 pg/mL, in a range from about 0.01 pg/mL to about 0.5 pg/mL, in a range from about 0.1 pg/mL to about 0.5 pg/mL, in a range from about 0.001 pg/mL to about 0.25 pg/mL, in a range from about 0.001 pg/mL to about 0.1 pg/mL, or in a range from about 0.001 pg/mL to about 0.05 pg/mL. In some embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing infection by SARS-CoV in a pseudovirus system (e.g. MLV-pp-based) with an IC50 in a range of about 0.02 pg/mL to about 0.25 pg/mL. In some embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing infection by SARS- CoV in a pseudovirus system (e.g., MLV-pp-based) with an IC50 in a range from about 0.01 pg/mL to about 10 pg/mL. In some embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing infection by a coronavirus Pangolin-Guangdong- 2019 (PANG/GD19 or PANG/GD) in a pseudovirus system (e.g., VSV-pp-based) with an IC50 in a range from about 0.03 pg/mL to about 0.3 pg/mL. In some embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing infection by coronavirus Pangolin-Guanzi-2017 (PANG/GX17 or PANG/GX) in a pseudovirus system (e.g., VSV-pp-based) with an IC50 in a range from about 0.06 pg/mL to about 11 pg/mL. In some embodiments, an antibody or antigen-binding fragment of the present disclosure is capable of neutralizing a sarbecovirus infection in a VSV-based pseudovirus system with an IC50 in a range from about 0.001 pg/mL to about 0.15 pg/mL.

In certain embodiments, the antibody or antigen-binding fragment (i) recognizes an epitope in the Spike protein of two or more sarbecoviruses; (ii) is capable of blocking an interaction between the Spike protein of one or more sarbecoviruses and a cell surface receptor;

(iii) recognizes an epitope that is conserved in the Spike protein of two or more sarbecoviruses;

(iv) is cross-reactive against two or more sarbecoviruses; or (v) any combination of (i)-(iv).

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., bispecific 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, 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 (l) 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/or 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 non-conservative substitutions), deletions, or combinations thereof.

Numbering of CDR and framework regions may be according to any known method or scheme, such as the Rabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Rabat 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. 196901-917 (1987)); Lefranc et al. , Dev. Comp. Immunol. 27:55, 2003; Honegger and Pliickthun, J. Mol. Bio. 309:657-670 (2001)). 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). Accordingly, identification of CDRs of an exemplary 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 certain embodiments, an antibody or antigen-binding fragment is provided that comprises CDRs identified in i) a VH sequence according to any one of SEQ ID NOs.: 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, and 169 and in a VL sequence according to any one of SEQ ID NOs.: 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, and 173, ii) in a VH sequence according to SEQ ID NO.: 143 and a VL sequence according to SEQ ID NO.: 144, iii) in a VH sequence according to SEQ ID NO.: 143 and a VL sequence according to SEQ ID NO.: 173, iv) in a VH sequence according to SEQ ID NO.: 169 and a VL sequence according to SEQ ID NO.: 144, or v) in a VH sequence according to SEQ ID NO. : 169 and a VL sequence according to SEQ ID NO.: 173, as determined using any known CDR numbering method, including the Rabat, Chothia, EU, IMGT, Martin (Enhanced Chothia), Contact, and AHo numbering methods. In certain embodiments, CDRs are according to the antibody numbering method developed by the Chemical Computing Group (CCG); e.g., using Molecular Operating Environment (MOE) software (www.chemcomp.com).

In certain embodiments, an antibody or an antigen-binding fragment is provided that comprises a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein: (i) the CDRH1 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, 144, or 170 or a sequence variant thereof comprising one, two, or three acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (ii) the CDRH2 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, 145, or 171 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (iii) the CDRH3 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, 146, or 172 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid; (iv) the CDRL1 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, 148, or 174 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid; (v) the CDRL2 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, 149, or 175 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid; and/or (vi) the CDRL3 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or 176 or a sequence variant thereof comprising having one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid, wherein the antibody or antigen binding fragment is capable of binding to a surface glycoprotein of two or more sarbecoviruses expressed on a cell surface of a host cell. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1. In certain embodiments, an antibody or an antigen-binding fragment is provided that comprises a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein: (i) the CDRH1 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 144 or 170 or a sequence variant thereof comprising one, two, or three acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (ii) the CDRH2 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 145 or 171 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid; (iii) the CDRH3 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 146 or 172 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (iv) the CDRL1 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 148 or 174 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (v) the CDRL2 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 149 or 175 or a sequence variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid; and/or (vi) the CDRL3 comprises or consists of the amino acid sequence according to any one of SEQ ID NOs.: 150 or 176 or a sequence variant thereof comprising having one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid, wherein the antibody or antigen binding fragment is capable of binding to a surface glycoprotein of two or more sarbecoviruses expressed on a cell surface of a host cell. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1.

In any of the presently disclosed embodiments, the antibody or antigen-binding fragment is capable of preventing and/or neutralizing infection by two or more sarbecoviruses in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.

In any of the presently disclosed embodiments, the antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs.: (i) 24-26 and 28-30, respectively; (ii) 34-36 and 38-40, respectively; (iii) 44-46 and 48-50, respectively; (iv) 54-56 and 58-60, respectively; (v) 64-66 and 68-70, respectively; (vi) 74-76 and 78-80, respectively; (vii) 84-86 and 88-90, respectively; (viii) 94-96 and 98-100, respectively; (ix) 104-106 and 108-110, respectively; (x) 114-116 and 118-120, respectively; (xi) 124-126 and 128-130, respectively; (xii) 134-136 and 138-140, respectively; (xiii) 144-146 and 148-150, or (xiv) 170-172 and 174-176 respectively. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1.

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 a CDRL3, wherein each CDR is independently selected from a corresponding CDR of antibody S2E22, antibody S2H97, antibody S2L17, antibody S2L23, antibody S2L37, antibody S2M18, antibody S2H90, antibody S2H94, antibody S2N27, antibody S2K15, antibody S2K21, antibody S2K23, antibody S2K146, or antibody S2K146 UCA as provided in Table 2. That is, all combinations of CDRs from the sarbecovirus antibodies and the variant sequences thereof provided in Table 2 are contemplated. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1.

The term "CL" refers to an "immunoglobulin light chain constant region" or a "light chain constant region," i.e., a constant region from an antibody light chain. The term "CH" refers to an "immunoglobulin heavy chain constant region" or a "heavy chain constant region," which is further divisible, depending on the antibody isotype into CHI, CH2, and CH3 (IgA, IgD, IgG), or CHI, CH2, CH3, and CH4 domains (IgE, IgM). The Fc region of an antibody heavy chain is described further herein. In any of the presently disclosed embodiments, an antibody or antigen binding fragment of the present disclosure comprises any one or more of CL, a CHI, a CH2, and a CH3. In certain embodiments, a CL comprises an amino acid sequence having 90%, 91%,

92%, 93%, 94%, 95%, 96%, 975, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.:6 or SEQ ID NO.: 7. In certain embodiments, a CH1-CH2-CH3 comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 975, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO.:6 or SEQ ID NO.:7.

It will be understood that, for example, production in a mammalian cell line can remove one or more C-terminal lysine 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 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, and embodiments where a lysine is the C-terminal residue. 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 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 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, i.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 "antigen-binding 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 (i.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 ah, Gene 40:3946 (1985); Murphy et ah, 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 ah, 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 ah, 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. Variable region and CDR amino acid sequences of exemplary anti-sarbecovirus antibodies of the present disclosure are provided in Table 2 herein.

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, deamination, 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 ("parent") antibody, wherein the variant antibody is capable of binding to a SARS- CoV-2 antigen.

In certain embodiments, the VH comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence according to any one of SEQ ID NOs.: 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, and 169, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline- encoded amino acid; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence according to any one of SEQ ID NOs.: 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, and 173, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline- encoded amino acid. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1.

In certain embodiments, the VH comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence according to any one of SEQ ID NOs.: 143 or 169, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence according to any one of SEQ ID NOs.: 147 or 173, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1.

In certain embodiments, the VH comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence according to any one of SEQ ID NO.: 143, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence according to any one of SEQ ID NO.: 147, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1.

In certain embodiments, the VH comprises or consists of any VH amino acid sequence set forth in Table 2, and the VL comprises or consists of any VL amino acid sequence set forth in Table 2. In particular embodiments, the VH and the VL comprise or consist of the amino acid sequences according to SEQ ID NOs : (i) 23 and 27, respectively; (ii) 33 and 37, respectively;

(iii) 43 and 47, respectively; (iv) 53 and 57, respectively; (v) 63 and 67, respectively; (vi) 73 and 77, respectively; (vii) 83 and 87, respectively; (viii) 93 and 97, respectively; (ix) 103 and 107, respectively; (x) 113 and 117, respectively; (xi) 123 and 127, respectively; (xii) 133 and 137, respectively; (xiii) 143 and 147, or (xiv) 169 and 173, respectively. The antibody may further comprise an Fc region, particularly an Fc region containing a mutation, particularly a mutation of Table 1.

In some embodiments, the antibody or antigen-binding fragment is an IgG, IgA, IgM,

IgE, or IgD isotype. In some embodiments, the antibody or antigen-binding fragment is human, humanized, or chimeric.

In some embodiments, 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.

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, kl-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 DVI-IgG (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 (IEI-Ig; 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 (i.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 epitopes 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 multi specific 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 antigen-binding fragment may, in some embodiments, comprise one, two, or more antigen-binding 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: (i) a first VH and a first VL; and (ii) a second VH and a second VL, wherein the first VH and the second VH are different and each independently comprise an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, and 143, and wherein the first VL and the second VL are different and each independently comprise an amino acid sequence having at least 85% (i.e.,

85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs.: 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, and 147, and wherein the first VH and the first VL together form a first antigen-binding site, and wherein the second VH and the second VL together form a second antigen-binding site.

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/P331 S, E333A, S239D/A330L/I332E, P257EQ311, K326W/E333S,

S239D/I332E/G236 A, 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 ofGlu318, 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 FceR, 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., JLab. 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 moieties providing cross-linking 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 homologous; 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 ah, 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 ah, 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 ah, 2008: Inhibition of B cell receptor-mediated activation of primary human B cells by coengagement of CD 19 and FcgammaRIIb with Fc-engineered antibodies. Molecular Immunology 45, 3926-3933. Thereby, the clearance of immune complexes can be enhanced (Chu, S., et ah, 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 ah, 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 "F158" variant, which binds to IgGl Fc with low affinity, and the "V158" 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., J. Immunol. 2000; 164: 5313 - 5318). Moreover, FcyRI appears to bind to the same site on IgGFc, 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., J. 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 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 IgGFc and a half-life-extending mutation comprises any one or more of: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I Q311I; D376V; T307A; E380A (EU numbering). In certain embodiments, a half-life-extending mutation comprises M428L/N434S (also referred to herein as "MLNS"). 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. 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 and G236A/A330L/I332E, and optionally does not comprise S239D. In certain embodiments, an antibody or antigen-binding fragment includes aFc polypeptide or fragment thereof that comprises the substitution mutations: M428L/N434S and G236 A/S239D/A330L/I332E.

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 etal. Clin. Cancer Res. 73(6):1875-82 (2007); Huang etal. MAbs 6:1-12 (2018)).

In certain embodiments, an antibody or antigen-binding fragment has one or more altered characteristics (e.g., increased binding to a human FcyRa, decreased binding to a human FcyRIIb, binding to a human FcyRa that is increased relative to the binding to a FcyRIIb, increased binding to a human Clq, increased binding to a human FcRn, an increased Tm, increased binding to a FcyRIIIa, or any combination thereof), as compared to a reference antibody or antigen binding fragment that comprises a variant Fc containing the following mutation(s): G236A; G236S; G236A/A330L/I332E; G236A/A330L/I332E/M428L/N434S;

G236 A/S239D/A330L/I332E; or A330L/I332E.

Certain embodiments of antibodies or antigen-binding fragments comprise a variant Fc comprising substitution mutation(s) and properties as shown in Table 1.

Table 1. Certain Fc variants and properties thereof

In some embodiments, the antibody or antigen-binding fragment comprises at least a portion of a human IgGl heavy chain comprising the amino acid mutation(s) set forth in any one of (i)-(xvii): (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, wherein the numbering of amino acid residues is according to the EU index as set forth in Rabat.

In some embodiments, any of the presently disclosed antibodies or antigen-binding fragments can comprise an IgGl isotype (optionally comprising an IgGlm3 allotype, an IgGlm3,l allotype, an IgGlml7 allotype, or an IgGlml7,l 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. 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 antigen-binding fragment thereof comprises an IgGlml7,l allotype.

In some embodiments, the polypeptide or antibody further comprises one or more mutation that enhances binding to a human FcRn, such as M428L and N434S mutations (EU numbering).

Any antibody or antigen-binding fragment of the present disclosure can be fucosylated ( e.g ., comprising one or more fucosyl moiety, and typically comprising a native (wild-type) fucosylation pattern or a fucosylation pattern that includes one or more additional, or fewer, fucosyl moieties as compared to native), or can be afucosylated. 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 polypeptide in a host cell which has been genetically engineered to lack the ability (or have an inhibited or compromised ability) to fucosylate the polypeptide; by expressing the polypeptide under conditions in which a host cell is impaired in its ability to fucosylate the polypeptide (e.g., in the presence of 2-fluoro- L-fucose (2FF)), or the like. An afucosylated polypeptide can comprise no fucosyl moieties, or substantially no fucosyl moieties, and/or can be expressed by a host cell that is genetically engineered to lack the ability (or have an inhibited or compromised ability) to fucosylate the polypeptide and/or can be expressed under conditions in which a host cell is impaired in its ability to fucosylate the polypeptide (e.g., in the presence of 2-fluoro-L-fucose (2FF)).

In any of the presently disclosed embodiments, an antibody or antigen-binding fragment can comprise only the specified or recited amino acid mutations (e.g. substitutions), and 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 polypeptide may comprise one or more further amino acid mutations ( e.g . substitutions), which can be specified (e.g., M428L_N434S).

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 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, i.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 etal., J. Mol. Biol., 222:581-597 (1991), for example. Monoclonal antibodies may also be obtained using methods disclosed in PCT Publication No. WO 2004/076677 A2.

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 etal, Proc. Natl. Acad. Sci. USA, §7:6851-6855 (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 etal, 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 etal, Nature, 332:323-327 (1988); Verhoeyen etal., 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.

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 etal., Clin. Immunol. 779:135, 2006). Codon-optimized sequences include sequences that are partially codon- optimized (i.e., one or more codon is optimized for expression in the host cell) and those that are fully codon-optimized.

It will also be appreciated that polynucleotides encoding antibodies and antigen-binding 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.

In certain embodiments, the polynucleotide comprises a polynucleotide having at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the polynucleotide sequence according to any one or more of SEQ ID NOs.:31, 32, 41, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 151, 152, 177, and 178. In certain embodiments the polynucleotide comprises a polynucleotide having at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the polynucleotide sequence according to any one or more of SEQ ID NOs.: 151, 152, 177, and 178. In certain embodiments, the polynucleotide comprises a polynucleotide having at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99%, or 100%) identity to the polynucleotide sequence according to i) SEQ ID NOs.: 151 and 152, ii) SEQ ID NOs.: 151 and 178, iii) SEQ ID NOs.: 177 and 152, or iv) SEQ ID NOs.:

177 and 178. In certain embodiments, any of the polynucleotides may further comprise a polynucleotide encoding an Fc region, particularly an Fc region comprising a mutation, particularly a mutation as set forth in Table 1. In certain embodiments, the polynucleotide comprises a polynucleotide having at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the polynucleotide sequence according to i) SEQ ID NOs.: 151 and 152, ii) SEQ ID NOs.: 151 and 178, iii) SEQ ID NOs.: 177 and 152, or iv) SEQ ID NOs.: 177 and 178 and a polynucleotide encoding an Fc region, particularly an Fc region comprising a mutation, particularly a mutation as set forth in Table 1.

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).

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 two or more sarbecoviruses). 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 etal, J Infect Dis. 2/-/(3 ):369-378 (2016); Muthumani et al., Hum Vaccin Immunother 9:2253-2262 (2013)); Flingai etal, 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 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. In some embodiments, the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide comprised in a single plasmid. In other embodiments, the substituent components of the antibody or antigen-binding fragment are encoded by a polynucleotide 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 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 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 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 if. 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 etal, PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0 cells. NS0 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 SY40 (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 3A); 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 in BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWTOl “Mimic™” cells. See, e.g., Palmberger etal., 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 Gemgross, Nat. Biotech. 22:1409-1414 (2004); Li etal., 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 two or more different antibodies or antigen-binding 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 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 antibody or antigen-binding fragment, comprising a VH comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NOs: 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, and 169, and a VL comprising or consisting of the amino acid sequence as set forth in any one of SEQ ID NOs: 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, and 173; and a second antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence as set forth in SEQ ID NOs: 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, 143, and 169, and a VL comprising of consisting of the amino acid sequence as set forth in SEQ ID NOs: 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, 147, and 173. In certain embodiments, a composition comprises a first antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: (i) 24-26, respectively, (ii) 34-36, respectively; (iii) 44-46, respectively; (iv) 54-56, respectively; (v) 64-66, respectively, (vi) 74-76, respectively; (vii) 84-86, respectively; (viii) 94-96, respectively; (ix) 104-106, respectively; (x) 114-116, respectively; (xi) 124-126, respectively; (xii) 134-136, respectively; (xiii) 144-146, or (xiv) 170-172, respectively; and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: (i) 28-30, respectively; (ii) 38-40, respectively; (iii) 48-50, respectively; (iv) 58-60, respectively; (v) 68-70, respectively; (vi) 78-80, respectively; (vii) 88-90, respectively; (viii) 98-100, respectively; (ix) 108-110, respectively; (x) 118-120, respectively; (xi) 128-130, respectively; (xii) 138-140, respectively; (xiii) 148-150, or (xiv) 174-176, respectively; and a second antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: (i) 24-26, respectively, (ii) 34-36, respectively; (iii) 44-46, respectively; (iv) 54-56, respectively; (v) 64-66, respectively, (vi) 74-76, respectively; (vii) 84-86, respectively; (viii) 94- 96, respectively; (ix) 104-106, respectively; (x) 114-116, respectively; (xi) 124-126, respectively; (xii) 134-136, respectively; (xiii) 144-146, or (xiv) 170-172, respectively; and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: (i) 28-30, respectively; (ii) 38-40, respectively; (iii) 48-50, respectively; (iv) 58-60, respectively; (v) 68-70, respectively; (vi) 78-80, respectively; (vii) 88-90, respectively; (viii) 98-100, respectively; (ix) 108-110, respectively; (x) 118-120, respectively; (xi) 128-130, respectively; (xii) 138-140, respectively; (xiii) 148-150, or (xiv) 174-176, respectively.

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. 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 etal. Wilery Inter discip Rev. Nanomed Nanobiotechnol. I /(2):e 1530 (2019)). Principles, reagents, and techniques for designing appropriate mRNA and formulating mRNA-LNP and delivering the same are described in, for example, Pardi etal. (J Control Release 277345-351 (2015)); Thess etal. (Mol Ther 23: 1456- 1464 (2015)); Thran etal. (EMBO Mol Med 9(10): 1434-1448 (2017); Kose et al. (Sci. Immunol.

4 eaaw6647 (2019); and Sabnis etal. (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 (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 sarbecovirus. "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 (i.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 (i.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 late-stage 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 sarbecovirus antigens, which in certain embodiments, may be the same or different sarbecovirus antigens, and/or can comprise the same or different epitopes.

Accordingly, in certain embodiments, methods are provided for treating a sarbecovirus 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 infant, juvenile, adolescent, adult, and geriatric subjects.

A number of criteria are believed to contribute to high risk for severe symptoms or death associated with a sarbecovirus 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 sarbecovirus 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 a sarbecovirus 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, intrasternal 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, antigen-binding 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, com 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 sarbecovirus 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, following 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 (i.e., 0.07 mg) to about 100 mg/kg (i.e., 7.0 g); preferably a therapeutically effective dose is (for a 70 kg mammal) from about 0.01 mg/kg (i.e., 0.7 mg) to about 50 mg/kg (i.e., 3.5 g); more preferably a therapeutically effective dose is (for a 70 kg mammal) from about 1 mg/kg (i.e., 70 mg) to about 25 mg/kg (i.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. 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 antigen-binding 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, i.e., concurrently, or at separately staggered times, i.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 (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-Ib, IL-7, IL-8, IL-9, IL-10, FGF, G-CSF, GM-CSF, IFN-g, IP-10, MCP-1, MIP-1A, 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 nucleotide 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. 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.

The present disclosure further provides a kit compriaing one or more of any antibodies, antigen-binding fragments, polynucleotides, nucleic acids, vectors, or other compositions disclosed herein. The kit may further include one or more of a container, such as a tube, vial, or syringe, an activator, a valve, a subcontainer, or instructions for use, such as for administering to a subject.

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

The disclosure further provides the following embodiments:

Embodiment 1. An antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein: (i) the CDRH1 comprises or consists of the amino acid sequence according to SEQ ID NO: 144, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, or 170, or a functional variant thereof comprising one, two, or three acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (ii) the CDRH2 comprises or consists of the amino acid sequence according to SEQ ID NO: 145, 25, 35, 45, 55, 65, 75, 85,

95, 105, 115, 125, 135, or 171, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (iii)the CDRH3 comprises or consists of the amino acid sequence according to SEQ ID NO: 146, 26, 36, 46, 56, 66, 76, 86,

96, 106, 116, 126, 136, or 172, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (iv) the CDRL1 comprises or consists of the amino acid sequence according to SEQ ID NO: 148, 28, 38, 48, 58, 68, 78, 88,

98, 108, 118, 128, 138, or 174, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; (v) the CDRL2 comprises or consists of the amino acid sequence according to SEQ ID NO: 149, 29, 39, 49, 59, 69, 79, 89,

99, 109, 119, 129, 139, or 175, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid; and/or (vi) the CDRL3 comprises or consists of the amino acid sequence according to SEQ ID NO: 150, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 176, or a functional variant thereof comprising having one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid, wherein the antibody or antigen-binding fragment is capable of binding to the surface glycoprotein of a sarbecovirus.

Embodiment 2. The antibody or antigen-binding fragment of Embodiment 1, wherein the antibody or antigen-binding fragment is capable of binding to the surface glycoprotein when the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 3. The antibody or antigen-binding fragment of Embodiment 1 or 2, which is capable of binding to a surface glycoprotein from two or more ( e.g ., two, three, four, five, or more) sarbecoviruses.

Embodiment 4. The antibody or antigen-binding fragment of any one of Embodiments 1- 3, which is capable of neutralizing an infection by one or more sarbecoviruses in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human. Embodiment 5. The antibody or antigen-binding fragment of any one of Embodiments 1-

4, which is capable of neutralizing an infection by two or more sarbecoviruses in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.

Embodiment 6. The antibody or antigen-binding fragment of any one of Embodiments 1-

5, comprising CDRH1, CDRH2, CDRH3, CDRE1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs.: (i) 144-146 and 148-150, respectively; (ii) 24-26 and 28-30, respectively; (iii) 34-36 and 38-40, respectively; (iv) 44-46 and 48-50, respectively; (v) 54-56 and 58-60, respectively; (vi) 64-66 and 68-70, respectively; (vii) 74-76 and 78-80, respectively; (viii) 84-86 and 88-90, respectively; (vix) 94-96 and 98-100, respectively; (x) 104-106 and 108- 110, respectively; (xi) 114-116 and 118-120, respectively; (xii) 124-126 and 128-130, respectively; (xiii) 134-136 and 138-140, respectively; or (xiv) 170-172 and 174-176, respectively.

Embodiment 7. The antibody or antigen-binding fragment of any one of Embodiments 1-

6, wherein: (i) the VH comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO: 143, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, and 169, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid; and/or (ii) the VL comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO: 147, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, and 173, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid.

Embodiment 8. The antibody or antigen-binding fragment of any one of Embodiment 1-6, wherein: (i) the VH comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO:

143 and 169, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid; and/or (ii) the VL comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO: 147 and 173, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid.

Embodiment 9. The antibody or antigen-binding fragment of any one of Embodiments 1- 6, wherein: (i) the VH comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO: 143, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid; and/or (ii) the VL comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO: 147, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid.

Embodiment 10. The antibody or antigen-binding fragment of any one of claims 1-9, wherein the VH and the VL comprise or consist of amino acid sequences having 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 to, or comprising or consisting of, the amino acid sequences according to SEQ ID NOs.: (i) 143 and 147, respectively; (ii) 23 and 27, respectively; (iii) 33 and 37, respectively; (iv) 43 and 47, respectively; (v) 53 and 57, respectively; (vi) 63 and 67, respectively; (vii) 73 and 77, respectively; (viii) 83 and 87, respectively; (vix) 93 and 97, respectively; (x) 103 and 107, respectively; (xi) 113 and 117, respectively; (xii) 123 and 127, respectively; (xiii) 133 and 137, respectively; or (xiv) 169 and 173, respectively.

Embodiment 11. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 143 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 147.

Embodiment 12. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 24-26, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 28-30, respectively.

Embodiment 13. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 11 or Embodiment 12, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more ( e.g ., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 14. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 33 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 37.

Embodiment 15. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 34-36, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 38-40, respectively.

Embodiment 16. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 14 or Embodiment 15, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more (e.g., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 17. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 43 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 47.

Embodiment 18. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRLl, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 44-46, respectively, and the CDRLl, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 48-50, respectively. Embodiment 19. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 17 or Embodiment 18, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more ( e.g ., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 20. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 53 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 57.

Embodiment 21. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 54-56, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 58-60, respectively.

Embodiment 22. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 20 or Embodiment 21, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more (e.g., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 23. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 63 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 67.

Embodiment 24. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRLl, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 64-66, respectively, and the CDRLl, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 68-70, respectively.

Embodiment 25. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 23 or Embodiment 24, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more (e.g., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 26. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 73 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 77.

Embodiment 27. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRE3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 74-76, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 78-80, respectively.

Embodiment 28. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 26 or Embodiment 27, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more ( e.g ., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 29. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 83 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 87.

Embodiment 30. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 84-86, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 88-90, respectively.

Embodiment 31. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 29 or Embodiment 30, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more (e.g., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 32. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 93 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 97.

Embodiment 33. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRE3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 94-96, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 98-100, respectively.

Embodiment 34. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 32 or Embodiment 33, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more ( e.g ., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 35. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 103 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 107.

Embodiment 36. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 104-106, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 108-110, respectively.

Embodiment 37. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 35 or Embodiment 36, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more (e.g., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 38. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 113 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 117.

Embodiment 39. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 114-116, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 118-120, respectively.

Embodiment 40. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 38 or Embodiment 39, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more ( e.g ., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 4E An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 133 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 137.

Embodiment 42. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 134-136, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 138-140, respectively.

Embodiment 43. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 41 or Embodiment 42, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more (e.g., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 44. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 143 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 147.

Embodiment 45. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRLl, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 144-146, respectively, and the CDRLl, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 148-150, respectively. Embodiment 46. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 44 or Embodiment 45, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more ( e.g ., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 47. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein the VH comprises or consists of the amino acid sequence set forth in SEQ ID NO: 169 and the VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 173.

Embodiment 48. An anti-sarbecovirus antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein the CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 170-172, respectively, and the CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 174-176, respectively.

Embodiment 49. The anti-sarbecovirus antibody or antigen-binding fragment thereof of Embodiment 47 or Embodiment 48, wherein the antibody or antigen binding fragment is capable of binding to the surface glycoprotein of two or more (e.g., two, three, four, five, or more) sarbecoviruses, optionally wherein the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

Embodiment 50. The antibody or antigen-binding fragment of any one of Embodiments 1-49, which: (i) recognizes an epitope in the Spike protein of two or more, three or more, four or more, or five or more sarbecoviruses; (ii) is capable of blocking an interaction between the Spike protein of two or more, , three or more, four or more, or five or more sarbecoviruses and their respective cell surface receptor(s), wherein, optionally, a cell surface receptor comprises a human ACE2; (iii) recognizes an epitope that is conserved in the Spike protein of two or more, , three or more, four or more, or five or more sarbecoviruses; (iv) is cross-reactive against two or more, , three or more, four or more, or five or more sarbecoviruses; or (v) any combination of (i)-(iv).

Embodiment 51. The antibody or antigen-binding fragment of any one of Embodiments 1-

50, which is a IgG, IgA, IgM, IgE, or IgD isotype.

Embodiment 52. The antibody or antigen-binding fragment of any one of Embodiments 1-

51, which is an IgG isotype selected from IgGl, IgG2, IgG3, and IgG4.

Embodiment 53. The antibody or antigen-binding fragment of any one of Embodiments 1-52, which is human, humanized, or chimeric. Embodiment 54. The antibody or antigen-binding fragment of any one of Embodiments 1-53, 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.

Embodiment 55. The antibody or antigen-binding fragment of Embodiment 54, wherein the scFv comprises more than one VH domain and more than one VL domain.

Embodiment 56. The antibody or antigen-binding fragment of any one of Embodiments 1-55, wherein the antibody or antigen-binding fragment is a multi-specific antibody or antigen binding fragment.

Embodiment 57. The antibody or antigen-binding fragment of Embodiment 56, wherein the antibody or antigen binding fragment is a bispecific antibody or antigen-binding fragment.

Embodiment 58. The antibody or antigen-binding fragment of Embodiment 56 or 57, comprising: (i) a first VH and a first VL; and (ii) a second VH and a second VL, wherein the first VH and the second VH are different and each independently comprise an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO: 143, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, and 169, and wherein the first VL and the second VL are different and each independently comprise an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO: 147, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, and 173; and wherein the first VH and the first VL together form a first antigen-binding site, and wherein the second VH and the second VL together form a second antigen-binding site.

Embodiment 59. The antibody or antigen-binding fragment of Embodiment 56 or 57, comprising: (i) a first VH and a first VL; and (ii) a second VH and a second VL, wherein the first VH and the second VH are different and each independently comprise an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO: 143, and wherein the first VL and the second VL are different and each independently comprise an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO: 147; and wherein the first VH and the first VL together form a first antigen-binding site, and wherein the second VH and the second VL together form a second antigen-binding site.

Embodiment 60. The antibody or antigen-binding fragment of any one of Embodiments 1-59, wherein the antibody or antigen-binding fragment further comprises a Fc polypeptide or a fragment thereof.

Embodiment 6E The antibody or antigen-binding fragment of Embodiment 60, 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; (ii) a mutation that enhances binding to a FcyR as compared to a reference Fc polypeptide that does not comprise the mutation; (iii) a mutation that enhances binding to human FcyRIIa and/or decreases binding to a human FcyRIIb as compared to a reference Fc polypeptide that does not comprise the mutation; and/or (iv) a mutation that enhances binding to a human Clq compared to a reference Fc polypeptide that does not comprise the mutation.

Embodiment 62. The antibody or antigen-binding fragment of Embodiment 60, 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.

Embodiment 63. The antibody or antigen-binding fragment of Embodiment 61 or 62, wherein the mutation that enhances binding to FcRn comprises: (i) M428L N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; or (viii) any combination of (i)-(vii).

Embodiment 64. The antibody or antigen-binding fragment of any one of Embodiments 61-63, wherein the mutation that enhances binding to FcRn comprises M428L/N434S.

Embodiment 65. The antibody or antigen-binding fragment of any one of Embodiments 61-64, wherein the mutation that enhances binding to a FcyR comprises S239D; I332E; A330L; G236A; or any combination thereof.

Embodiment 66. The antibody or antigen-binding fragment of any one of Embodiments 61-64, 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 67. The antibody or antigen-binding fragment of any one of Embodiments 1-66, 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 69. The antibody or antigen-binding fragment of any one of Embodiments 1-67, comprising a Fc polypeptide or fragment thereof that comprises: (i) A at position 236, V at position 328, and E at position 295; (ii) A at position 236, A at position 230, and E at position 295; (iii) A at position 236, P at position 292, and N at position 377; (iv) A at position 236, A at position 334, and E at position 295; or (v) S at position 236, P at position 292, and L at position 300.

Embodiment 70. The antibody or antigen-binding fragment of any one of Embodiments 1-68, comprising a Fc polypeptide or fragment thereof that comprises: (i) L at position 300; (ii) K at position 345, S at position 236, Y at position 235, and E at position 267; (iii) R at position 272, T at position 309, Y at position 219, and E at position 267; (iv) Y or W at position 236; (v) A at position 236, wherein the antibody or antigen-binding fragment is afucosylated; (vi) A at position 236, L at position 330, and E at position 332, wherein the antibody or antigen-binding fragment is afucosylated; (vii) A at position 236, L at position 330, and E at position 332, and does not comprise D at position 239, wherein the antibody or antigen-binding fragment is afucosylated; (viii) A at position 236, L at position 330, and E at position 332, S at position 239, wherein the antibody or antigen-binding fragment is afucosylated; (ix) L at position 243, E at position 446, L at position 396, and E at position 267; or (x) A at position 236, D at position 239, E at position 332, L at position 428, and S at position 434.

Embodiment 71. An isolated polynucleotide encoding the antibody or antigen-binding fragment of any one of Embodiments 1-70 or 84-87, or encoding a VH, a heavy chain, a VL, and/or a light chain of the antibody or the antigen-binding fragment.

Embodiment 72. The polynucleotide of Embodiment 71, wherein the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), wherein the RNA optionally comprises messenger RNA (mRNA).

Embodiment 73. The polynucleotide of Embodiment 71 or 72, which is codon-optimized for expression in a host cell.

Embodiment 74. The polynucleotide of any one of Embodiments 71-73, comprising a 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 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 to, or comprising or consisting of, the polynucleotide sequence according to any one of SEQ ID NOs.: 151, 152, 31, 32, 41, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 177, and 178.

Embodiment 75. A recombinant vector comprising the polynucleotide of any one of Embodiments 71-74.

Embodiment 76. A host cell comprising the polynucleotide of any one of Embodiments 71-74 and/or the vector of Embodiment 75, wherein the polynucleotide is heterologous to the host cell.

Embodiment 77. A human B cell comprising the polynucleotide of any one of Embodiments 71-74, wherein polynucleotide is heterologous to the human B cell and/or wherein the human B cell is immortalized.

Embodiment 78. A composition comprising: (i) the antibody or antigen-binding fragment of any one of Embodiments 1-70 or 84-87; (ii) the polynucleotide of any one of Embodiments 71-74; (iii) the recombinant vector of Embodiment 75; (iv) the host cell of Embodiment 76; and/or (v) the human B cell of Embodiment 77, and a pharmaceutically acceptable excipient, carrier, or diluent.

Embodiment 79. The composition of Embodiment 78, comprising two or more antibodies or antigen-binding fragments of any one of Embodiments 1-70 or 84-87.

Embodiment 80. A composition comprising the polynucleotide of any one of Embodiments 71-74 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.

Embodiment 8E A method of treating a sarbecovirus infection in a subject, the method comprising administering to the subject an effective amount of (i) the antibody or antigen binding fragment of any one of Embodiments 1-70 or 84-87; (ii) the polynucleotide of any one of Embodiments 71-74; (iii) the recombinant vector of Embodiment 75; (iv) the host cell of Embodiment 76; (v) the human B cell of Embodiment 77; and/or (vi) the composition of any one of Embodiments 78-80.

Embodiment 82. The antibody or antigen-binding fragment of any one of Embodiments 1-70 or 84-87, the polynucleotide of any one of Embodiments 71-74, the recombinant vector of Embodiment 75, the host cell of Embodiment 76, the human B cell of Embodiment 77, and/or the composition of any one of Embodiments 78-80 for use in a method of treating a sarbecovirus infection in a subject. Embodiment 83. The antibody or antigen-binding fragment of any one of Embodiments 1-70 or 84-87, the polynucleotide of any one of Embodiments 71-74, the recombinant vector of Embodiment 75, the host cell of Embodiment 76, the human B cell of Embodiment 77, and/or the composition of any one of Embodiments 78-80 for use in the preparation of a medicament for the treatment of a sarbecovirus infection in a subject.

Embodiment 84. The antibody or antigen-binding fragment of any one of Embodiments 61-69, wherein the Fc polypeptide comprises a L234A mutation and a L235A mutation.

Embodiment 85. The antibody or antigen-binding fragment of any one of Embodiments 1-70 and 84 wherein the antibody or antigen-binding fragment binds to two or more sarbecovirus S proteins, as measured using biolayer interferometry.

Embodiment 86. The antibody or antigen-binding fragment of Embodiments 1-70 or 84- 85, wherein the antibody or antigen-binding fragment is capable of neutralizing a sarbecovirus infection and/or of neutralizing an infection of a target cell with an IC50 of about 0.01 to about 11 pg/ml.

Embodiment 87. The antibody or antigen-binding fragment of any one of Embodiments 1-70 or 84-86, 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 a sarbecovirus.

Embodiment 88. A method for in vitro diagnosis of a sarbecovirus infection, the method comprising: (i) contacting a sample from a subject with an antibody or antigen-binding fragment of any one of Embodiments 1-70 or 84-87; and (ii) detecting a complex comprising an antigen and the antibody, or comprising an antigen and the antigen binding fragment.

Embodiment 89. The method of Embodiment 88, wherein the sample comprises blood isolated from the subject.

Embodiment 90. An antibody, or an antigen-binding fragment thereof, that competes for binding to a sarbecovirus surface glycoprotein with the antibody or antigen-binding fragment of any one of Embodiments 1-70 or 84-87.

Embodiment 91. The method of Embodiment 78 or the antibody, antigen-binding fragment, polynucleotide, recombinant vector, host cell, human B cell, and/or composition for use of Embodiment 82 or 83, wherein the sarbecovirus comprises: (i) SARS-CoV; (ii) SARS- CoV-2; (iii) WIV1; (iv) PANG/GD; (v) PANG/GX; (vi) RatG13; (v) ZXC21; (vi) ZC45; (vii) RmYN02; (viii) Anlongll2; (ix) YN2013; (x) SC2018; (xi) SC2011; (xii) BGR2008; (xiii) BtkY72; (xiv) SARS-CoV-2 variant P.l; (xv) SARS-CoV-2 variant B .1.1.7; (xvi) SARS-CoV-2 vari ant B.1.429; (xvii) SARS-CoV-2 variant B.1.351; (xviii) SARS-CoV-2 variant B.1.1.222; (xix) SARS-CoV-2 variant C.37; (xx) SARS-CoV-2 variant AY.1; (xxi) SARS-CoV-2 variant AY.2; (xxii) SARS-CoV-2 S protein mutant N501Y; (xxiii) SARS-CoV-2 S protein mutant Y453F; (xxiv) SARS-CoV-2 S protein mutant N439K; (xxv) SARS-CoV-2 S protein mutant K417V; (xxvi) SARS-CoV-2 S protein mutant E484K; (xxvii) SARS-CoV-2 variant B.1.1.529.; or (xxviii) any combination of (i)-(xvii).

Embodiment 92. A method for producing an antibody or antigen-binding fragment of any one of Embodiments 1-70 and 84-87, wherein the method comprises culturing a host cell expressing the antibody or antigen-binding fragment under conditions and for a time sufficient to produce the antibody, or the antigen-binding fragment. Embodiment 93. The method for producing an antibody or antigen-binding fragment of

Embodiment 92, wherein the host cell comprises a recombinant vector comprising a polynucleotide of any one of Embodiments 71-74.

Embodiment 94. The method for producing an antibody or antigen-binding fragment of Embodiment 92 or 93, wherein the host cell is a mammalian cell. Embodiment 95. The method for producing an antibody or antigen-binding fragment of any one of Embodiments 92-94, wherein the method comprises culturing the host cell in batch cell culture.

Embodiment 96. The method for producing an antibody or antigen-binding fragment of any one of Embodiments 92-95, wherein the method further comprises purifying the antibody or antigen-binding fragment.

Table 2. Sequences

EXAMPLES

EXAMPLE 1

ANTIBODIES AGAINST MULTIPLE SARBECOVIRUSES

The following materials and methods were used in Example 1 and, unless otherwise noted, in Example 2. Cell lines

Cell lines used in Example 1 were obtained from ATCC (HEK293T, Vero and Vero-E6), ThermoFisher Scientific (Expi CHO cells, FreeStyle™ 293-F cells and Expi293F™ cells) or were generated via lentiviral transduction (Expi CHO-S, HEK293T-ACE2).

Recombinant protein production

Wild-type SARS-CoV-2 RBD (with N-terminal signal peptide and ‘ETGT’, and C- terminal 8xHis-tag) was expressed in Expi293F cells at 37°C and 8% C02 in the presence of 10 mM kifunensine. Transfection was performed using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatant was collected four days after transfection and supplemented with lOx PBS to a final concentration of 2.5x PBS (342.5 mM NaCl, 6.75 mM KC1 and 29.75 mM phosphates). SARS-CoV-2 S hexapro protein, used for cryo-EM single particle studies, was expressed and purified using known methods.

Antibody isolation and recombinant production

Antigen specific IgG⁺ memory B cells were isolated and cloned from PBMC of SARS- CoV-2 convalescent individuals. Briefly, CD19⁺ B cells were enriched by staining with CD 19 PE-Cy7 and anti-PE microbeads (Milteniy), followed by positive selection using LS columns. Enriched B cells were stained with anti-IgD, anti-IgM, anti-IgA, anti-CD14, all PE labelled and prefusion SARS-CoV-2 S-Avi tag conjugated with streptavidin Alexa-Fluor 647 (Life Technologies). SARS-CoV-2-specific IgG⁺ memory B cells were sorted and seeded on MSC (mesenchymal stromal cells) at 0.5 cell/well in the presence of CpG2006, IL-2, IL6, IL-10 and IL-21 using known methods. After 7 days, B cell supernatants were screened by ELISA for binding to a panel of RBDs representative of different sarbecovirus clades as well as by neutralization using high-troughtput VSV SARS-CoV-2 S-abesed microneutralization. Abs VH and VL sequences were obtained by reverse transcription PCR (RT-PCR) and mAbs were expressed as recombinant human IgGl, carrying the half-life extending M428L/N434S (LS) mutation in the Fc region fragment. ExpiCHO cells were transiently transfected with heavy and light chain expression vectors using known methods. Using the Database IMGT, the VH and VL gene family and the number of somatic mutations were determined by analyzing the homology of the VH and VL sequences to known human V, D and J genes. UCA sequences of heavy and light variable regions were constructed using IMGT/V-QUEST.

MAbs affinity purification was performed on AKTA Xpress FPLC (Cytiva) operated by UNICORN software version 5.11 (Build 407) using HiTrap Protein A columns (Cytiva) for full length human and hamster mAbs and CaptureSelect CHI -XL MiniChrom columns (ThermoFisher Scientific) for Fab fragments, using PBS as mobile phase. Buffer exchange to the appropriate formulation buffer was performed with a HiTrap Fast desalting column (Cytiva).

The final products were sterilized by filtration through 0.22 pm filters and stored at 4°C. Various comparator antibodies are used in these Examples. These include the S2M11 mAh, which locks SARS-CoV-2 S in the closed state (M. A. Tortorici et al. 2020) and mAbs that target RBD antigenic site la (e.g. S2E12) and Ila (e.g. S2X259) which can mimic receptor attachment and prematurely trigger fusogenic S conformational changes (A. C. Walls et al. 2019; Lempp et al. 2021; Piccoli et al. 2020). The antigen binding site of certain antibodies used in this Example 1 is provided in Table

3.

Table 3: Antibody Characteristics

To assess the role of somatic mutations for S2K146 binding and neutralization, its inferred unmutated common ancestor (S2K146 UCA) was also generated. Alignment with the UCA amino acid sequence reveals that S2K146 harbors seven and two somatic hypermutations in the heavy- and light-chain complementarity determining regions (CDR): VH identity 94.4% and VL identity 98.9%, respectively.

Spike Protein Binding ELIS As Binding of various monoclonal antibodies to the spike protein RBD of different sarbecoviruses was measured by enzyme-linked immunoabsorbant assay (ELISA).

96 half area well-plates (Corning, 3690) were coated over-night at 4°C with 25 pi of sarbecoviruses RBD proteins, prepared at 5 pg/ml in PBS pH 7.2. After a blocking step of 60 min at room temperature with PBS 1% BSA (Sigma-Aldrich, A3059), plates were incubated with mAh serial dilutions for 60 min at room temperature. After 4 washing steps with PBS 0.05% Tween 20 (PBS-T) (Sigma-Aldrich, 93773), goat anti-human IgG secondary antibody (Southern Biotech, 2040-04) was added and incubated for 45 min at room temperature. Plates were then washed 4 times with PBS-T and 4-NitroPhenyl phosphate (pNPP, Sigma-Aldrich, 71768) substrate was added. After 45 min incubation, absorbance at 405 nm was measured by a plate reader (Biotek) and data plotted using Prism GraphPad. IC50 was determined for each antibody/virus combination and is reported in the ELISA tables below. Binding of antibodies to various sarbecoviruses representing Clades la (SARS-CoV), lb

(SARS-CoV-2, RATG13, PANG/GD, and PANG/GX), 2 (Anlongl l2, YN203, SC2018,

SX2011, and XC45) and 3 (BtkY72 and BGR2008) was further evaluated using an ELISA as described above. Results are presented in Table 4 and Table 5.

Table 4: ELISA RBD Binding -1

Table 5: ELISA RBD Binding -2

Spike Protein Binding as Measured by Flow Cytometry

ExpiCHO cells were seeded at 6 ^c cells/ml into 50 ml bioreactor tubes in 5 ml culture medium. Spike coding plasmids (5 pg) were diluted in OptiPRO SFM, mixed with ExpiFectamine CHO Reagent (Life Technologies) and added to the cells. After transfection, cells were incubated at 37°C with 8% C02 with an orbital shaking speed of 120 rpm (orbital diameter of 25 mm) for 48 hours.

Transiently transfected ExpiCHO cells were harvested and washed in wash buffer (PBS 2% FBS, 2 mM EDTA). Cells were counted, distributed into round bottom 96-well plates (Corning) and incubated with serially diluted antibodies in wash buffer (starting concentration: 10 pg/ml, 8 points of dilution 1 :4). Alexa Fluor647-labeled Goat Anti-

Human IgG secondary Ab (Jackson Immunoresearch) was prepared at 2 pg/mL added onto cells after two washing steps. Cells were then washed twice and resuspended in wash buffer for data acquisition at ZE5 cytometer (Biorad).

Binding of selected antibodies to various sarbecoviruses representing Clades la (SARS- CoV), lb (SARS-CoV-2, RATG13, PANG/GD, and PANG/GX), 2 (ZXC21, ZC45, YN2013, and RMYN02) and 3 (BtkY72 and BGR2008) was also evaluated for the same antibodies using flow cytometry as described above. Results are presented in Table 6.

Table 6: FACS Binding

Biolayer Interferometry (BLI) and KD

The KD [M] for the spike protein of various sarbecoviruses was determined using BLI. Results are presented in Table 7. Table 7: KD GMΊ Spike Protein

Neutralization Assays

Pseudotyped viruses were prepared using Lenti-X 293 cells seeded in 15-cm dishes. Briefly, cells in culture medium (DMEM supplemented with 10% heat-inactivated FBS, 1% PenStrep) were transfected with 25 pg of plasmid encoding for the corresponding S glycoprotein using TransIT-Lenti (Mirus) as transfectant reagent. One day post-transfection, cells were infected with vesicular stomatitis virus (VSV) (G*AG-luciferase) for 1 h, washed 3 times in PBS with Ca²⁺/Mg²⁺ (Thermo Fisher) before adding 25 ml of culture medium/dish. Particles were harvested after 18-24 h, clarified from cellular debris by centrifugation at 2,000 x g for 20 min at 4°C, aliquoted and stored at -80°C until use in neutralization experiments.

For neutralization experiments, Vero E6 cells were seeded at 20,000 cells/well in culture medium into white 96-well plates (PerkinElmer, 6005688) and cultured overnight at 37°C 5% C02. Ten-point 3-fold mAh serial dilutions were prepared in culture medium and mixed 1 : 1 with pseudotyped VSV prepared in culture medium in order to infect cells with the desired MOI. After 60 min incubation at 37 °C, cell culture medium was aspirated and 50 mΐ of PVs/mAb mixture was added onto cells and incubated 60 min at 37°C 5% CO2. After 60 min, 100 pi of culture medium was added to the cells and incubation at 37°C 5% CO2 followed in the next 16- 24 h. At the end of the incubation time, culture medium was removed from the cells and 50 mΐ/well of Steadylite (PerkinElmer) diluted 1 :2 with PBS with Ca²⁺Mg²⁺ was added to the cells and incubated in the dark for 10 min.

Luminescence signals were read using a Synergy HI Hybrid Multi-Mode plate reader (Biotek). Measurements were done in duplicate and at least six wells per plate contained untreated infected cells (defining the 0% of neutralization, “MAX RLU” value) and infected cells in the presence of S2E12 and S2X259 at 25 pg/ml each (defining the 100% of neutralization, “MIN RLU” value). Average of 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: (l-(RLUx - MIN RLUave) / (MAX RLUave - MIN RLUave)) x 100. Data were analyzed and visualized with Prism (Version 9.1.1). 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, and a lower constrain equal to 0.

MLV pseudotype viruse studies were conducted in a similar manner using murine leukemia virus (MLV) in Vero E6 cells.

Neutralization of authentic SARS-CoV-2 by entry-inhibition assay neutralization was determined using SARS-CoV-2-Nluc, an infectious clone of SARSCoV-2 (based on strain 2019- nCoV/USA_WAl/2020) which encodes nanoluciferase in place of the viral ORF7 and has demonstrated comparable growth kinetics to wildtype virus (Xie et ah, 2020). Vero E6 cells were seeded into black- walled, clear-bottom 96-well plates at 2 ^c 10⁴ cells/well and cultured overnight at 37°C. The next day, 9-point 4-fold serial dilutions of mAbs were prepared in infection media (DMEM + 10% FBS). SARS-CoV-2-Nluc was diluted in infection media at a final MOI of 0.1 or 0.01 PFU/cell, added to the mAb dilutions and incubated for 30 min at 37°C. Media was removed from the Vero E6 cells, mAb-virus complexes were added and incubated at 37°C for 6 or 24 h. Media was removed from the cells, Nano-Glo luciferase substrate (Promega) was added according to the manufacturer’s recommendations, incubated for 10 min at room temperature and the luciferase signal was quantified on a VICTOR Nivo plate reader (Perkin Elmer).

Results for a variety of antibodies are presented in Table 8.

Table 8: Antibody Neutralization

ACE2 Binding and FcR Activation

Selected antibodies were also evaluated to determine the ability of the antibodies to block binding of the ACE2 receptor to the RBD of SARS2 or SARS1. SARS-CoV and SARS-CoV-2 mouse/rabbit Fc-tagged RBDs (final concentration 20 ng/ml) were incubated with serially diluted recombinant mAbs (from 25 pg/ml) and incubated for 1 h 37°C. The complex RBDmAbs was then added to a pre-coated hACE2 (2 pg/ml in PBS) 96-well plate MaxiSorp (Nunc) and incubated 1 hour at room temperature. Subsequently, the plates were washed and a goat anti mouse/rabbit IgG (Southern Biotech) coupled to alkaline phosphatase (Jackson Immunoresearch) added to detect mouse Fc-tagged RBDs binding. After further washing, the substrate (p-NPP, Sigma) was added, and plates read at 405 nm using a microplate reader (Biotek). The percentage of inhibition was calculated as follows: (l-((OD sample-OD neg ctr)/(OD pos. ctr-OD neg. ctr))*100. Most antibodies tested were able to inhibit binding of the RBD of both viruses, but S2N27, S2L37, and S2L17 were identified as primarily specific for only one of the two viruses (Figure 3 and Table 9). FcR activation was tested using a NFAT-driven luciferase signal indued in Jurkat cells stably expressing the FcgRIIa HI 31 or the FcgRIIIa VI 48 variant by binding of the indicated antibody to full-length wild type spike protein on CHO target cells.

The ability to bind FcgRIIA V and FcgRIIA after Fcg activation was also tested to determine the ability of the various antibodies to active FC-mediated effector functions. Results are presented in Table 9.

Table 9: Antibodies and ACE2 and FcR Binding

Viral Shedding

CHO cells stably expressing the prototypic SARS-CoV-2 Spike protein were harvested, washed in wash buffer (PBS 1% BSA 2 mM EDTA) and resuspended in PBS. Cells were then counted and 90,000 cells/well were dispensed into a round-bottom 96 well plate (Corning) to be treated with 10 ug/ml TPCK-Trypsin (Worthington Biochem) for 30 min at 37°C. After a washing step, cells were incubated with 15 ug/ml mAbs solution for 180, 120, 60, 30 or 5 min at 37°C. After the incubation for the allotted time, cells were washed with ice-cold wash buffer and stained with 1.5 ug/ml Alexa Fluor647-labeled Goat Anti -Human IgG secondary Ab (Jackson Immunoresearch) for 30 min on ice in the dark. Cells were then washed twice with cold wash buffer and analyzed using a ZE5 cytometer (Biorad) with acquisition chamber T= 4°C. Binding at each time point (MFI) was determined normalizing to the MFI at 5 minutes time point and data plotted using GraphPad Prism v. 9.1.1.

It, therefore, appears that S2X259 and S2E12 inhibit SARS-CoV-2 primarily by inhibiting binding of the virus to the ACE2 receptor and also by causing premature shedding of the SI components of the S protein, which further inhibits the ability of the virus to bind to its cellular targets (Figure 4).

EXAMPLE 2

TESTING OF S2K146 MONOCLONAL ANTIBODIES

All testing was conducted as indicated in Example 1 unless provided otherwise in this Example 2.

Spike Protein Binding ELIS As

ELISA testing of the ability of S2K146 and comparator antibody S2X259 to bind the RDB of various sarbecoviruses representing Clades la, lb, 2 and 3, as well as several variants of SARS-CoV-2 was conducted (Figure 5).

Binding of S2K146 and comparator monoclonal antibodies S2X259-v2, S2X259-vl5, S2H90, S2X259-v5, S2X259-v6, and S2X259-v7 to the RBD of different sarbecoviruses was further tested by ELISA (Figure 6).

Further ELISAs were used to establish the EC50 for S2K146 and S2E12 for the RBD-site I of various Clade la, lb, 2, and 3 sarbecoviruses and variants of SARS-CoV-2. Collectively this data shows that S2K146 targets site I and competes with S2E12 to bind to this site. Like S2E12, S2K146 bound to all SARS-CoV-2 VOC RBDs as well as all clade lb sarbecovirus RBDs tested by ELISA (Figure 7 and Figure 8). In addition, S2K146 binding does not appear to be affected by mutations in the variants tested, which include multiple variants of concern (Figure 7 and Figure 8).

Unlike S2E12 and the other site I-targeting antibodies described so far, S2K146 also cross-reacted with SARS-CoV and WIV-1 RBDs (clade la), which share 73% and 76% sequence identity with the SARS-CoV-2 RBD, respectively (Figure 7 and Figure 8). Accordingly, may be effective against viruses in both Clade la and Clade lb.

S2K146 did not bind to clades 2 and 3 sarbecovirus RBDs, similarly to the broadly neutralizing sarbecovirus S309 mAb (sotrovimab) but in contrast to the S2X259 or S2H97 mAbs (Figure 7 and Figure 8).

With the exception of WIV1, no differences were observed in binding to a panel of RBDs representative of clade 1 sarbecoviruses between S2K146 and its UCA, as determined by ELISA

(Figure 9).

Spike Protein Binding as Measured by Flow Cytometry

Flow cytometry analysis of S2K146 and comparative antibody S2E12 cross-reactivity with a panel of twelve spike glycoproteins representative of sarbecovirus clades la and lb transiently expressed on the surface of mammalian cells was conducted (Figure 10). Based on these and ELISA results, it appears that S2K146 recognizes a previously uncharacterized RBM antigenic site which is conserved among sarbecovirus clades la and lb.

Amino acids likely significant for S2K146 interaction with viral antigens are highlighted in boxes in Figure 2.

Biolayer Interferometry and KD

Biolayer Interferometry (BLI) experiments were carried out using an Octet Red96 (ForteBio) and all reagents were prepared in Kinetics buffer (KB) (PBS 0.01% BSA).

To assess antibody S2K146 competition with S2X259, S309 and S2E12, His-tagged SARS-CoV-2 RBD was prepared at 8 pg/ml in Kinetics buffer (PBS 0.01% BSA) and loaded on pre-hydrated anti-penta-HIS biosensors (Sartorius) for 2.5 min. Biosensors were then moved into a solution containing 20 pg/ml S2K146 mAb and association recorded for 5 min. A second association step was subsequently performed into S2X259, S309 and S2E12 mAbs solutions at 20 pg/ml and recorded for 5 min. Response values were exported and plotted using GraphPad Prism (version 9.1.1). Results are presented in Figure 11 and Table 10. S2K146 (IGHV3-43; IGL1-44), did not compete with either S309 (site IV) or S2X259 (site II) for binding to the SARS-CoV-2 RBD but competed with the RBM-targeted S2E12 mAb (site I).

Table 10: Epitope Binding Results

To asses binding affinities, S2K146 and respective UCA Ab were prepared at 3 ug/ml and immobilized on pre-hydrated protein-A biosensors (Sartorius) for 75 sec. After a 30 sec stabilization step in KB, biosensors were moved in SARS-CoV or SARS-CoV-2 :2 dilution series (starting concentration: 18.5 nM) for the 600 sec association step, and then moved back in KB to record dissociation signals for 540 sec. The data were baseline subtracted, results fitted using the Pall ForteBio/Sartorius analysis software (version 12.0) and plotted using GraphPad Prism (version 9.1.1). Biolayer interferometry revealed that S2K146 binds to SARS-CoV and SARS- CoV-2 prefusion-stabilized S ectodomain trimer with nanomolar affinities whereas a nearly two orders of magnitude reduction was observed with the UCA mAb (Figure 12).

Neutralization Assays

To evaluate neutralization potency of the S2K146, dose-response inhibition assays were carried out using a vesicular stomatitis virus (VSV) pseudotyping platform as described in Example 1. S2K146 and S2K146 UCA neutralization against SARS-CoV and SARS-CoV-2 was tested using a VSV-based pseudovirus system. (Figure 13) S2K146-mediated neutralization of VSV pseudotypes harboring Wuhan-Hu-1 SARS-CoV-2 S, and variants B.1.351 (beta), B.l.1.7 (alpha), P.l (gamma), B.1.429 (epsilon), C.37 (lambda), AY.2 (delta+) SARS-CoV-2 S or SARS-CoV S was aslo evalutated (Figure 14). S2K146 efficiently blocked wildtype SARS-CoV and SARS-CoV-2 S-mediated entry into cells with half maximum inhibitory concentration (IC50) of 108 and 16 ng/mL, respectively (Figure 13 and Figure 14). However, S2K146 UCA showed a marked loss in neutralizing activity against both SARS-CoV and SARS-CoV-2 S VSV pseudotypes, although S2K146 displayed a slightly lower affinity (Figure 13). Thus, somatic hypermutations associated with S2K146 affinity maturation are important to enhance antibody potency but not necessarily breadth.

Comparative results with antibody S2E12 for neutralization of SARS-CoV-2 MLV-based pseudotypes are presented in Figure 15.

Neutralizing ability of Antibody S2K146 and comparator antibodies S2H90 and S2E12 against VSV pseudovirus and MLV pseudovirus in different cell lines was also evaluated

(Figure 16, Figure 17, and Figure 18).

Neutralization of Antibody S2K146 against SARS-CoV-2 variants and WT virus was also analyzed. S2K146 retains effectiveness against current variants of concern (Figure 19). Specific neutralization data of antibody S2K146 and comparator antibody S2E12 in a VSV-based pseudotype assay is provided for multiple variants in Figure 20.

S2K146- and SE12-mediated neutralization of replication-competent nanoluciferase SARS-CoV-2 Wuhan-Hu-1 and SARS-CoV-2 variant of concern viruses was also tested.

S2K146 showed neutralizing activity against authentic Wuhan-1 SARS-CoV-2 and SARS-CoV- 2 VOC (Wuhan-1, IC50= 10 ng/mL; Alpha, IC50= 9 ng/mL; Beta, IC50= 9 ng/mL; Delta, IC50= 8 ng/mL; Kappa, IC50= 30 ng/mL) comparable to that observed with the ultrapotent S2E12 mAb (Wuhan-1, IC50= 3.5 ng/mL; Alpha, IC50= 2.5 ng/mL; Beta, IC50= 2 ng/mL; Delta, IC50= 1.5 ng/mL; Kappa, IC50= 4.5 ng/mL) (Figure 21).

ACE2 Binding and FcR Activation

The ability of S2K146 to block ACE2 receptor binding to the SARS-CoV-2 RBD and the SARS-CoC RBD, as compared to S2E12, was also evaluated by competition ELISA. S2K146 inhibited binding of the SARS-CoV-2 and SARS-CoV RBDs to human ACE2 in a concentration- dependent manner (Figure 22). Furthermore, S2K146 binds both viruses, while S2E12 binds only SARS-CoV-2.

FcgR activation was tested using a NFAT-driven luciferase signal induced in Jurkat cells stably expressing the FcgRIIa H131 (Figure 23) or the FcgRIIIa V158 variant (Figure 24) by S2K146 or comprarator antibody binding to full-length wild type SARS-CoV-2 spike protein on CHO target cells. Without wishing to be bound by theory, efficient S2K146-induced SI shedding might explain the lack of FcyRIIa and FcyRIIIa activation, which was used as a proxy for antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity.

However, FcgR activation was further tested using a NFAT-driven luciferase signal induced in Jurkat cells stably expressing the FcgRIIa H131 variant (Figure 25) or the FcgRIIIa VI 58 variant (Figure 26) by S2K146 mAb binding to uncleavable full-length wild-type SARS- CoV-2 spike protein on CHO target cells. Although this spike protein could not release the SI subunit, preventing SI shedding, S2K146 was still not able to activate FcyRIIa and triggered only a marginal activation of FcyRIIIa. Accordingly, factors other than SI shedding may also influence antibody-dependent cellular phagocytosis and antibody-dependent cellular cytotoxicity.

Viral Shedding

Figure 27 shows the effects of S2K146 and comparator antibodies S2M11 and S2E12 on SI shedding by SARS-CoV-2 over time. From these data, S2K146 blocks shedding comparably to S2E12. Without wishing to be bound by theory, it therefore appears that S2K146 inhibits SARS-CoV-2 primarily by inhibiting binding of the virus to the ACE2 receptor and also by causing premature shedding of the SI components of the S protein, which further inhibits the ability of the virus to bind to its cellular targets. Due to its ability to trigger SI shedding,

S2K146 does not promote activation of effector functions in vitro.

Hamster Studies

Therapeutic activity of S2K146 against challenge with the B.1.351 (beta) variant of SARS-CoV-2 was tested in a Syrian hamster model of infection. S2K146 was administered at 1, 5 and 10 mg/kg via intraperitoneal injection 24h after intranasal challenge with SARS-CoV-2 and the lungs of the animals were collected 3 days later for the quantification of viral RNA and replicating virus. S2K146 10 mg/kg n = 6; S2K146 5 mg/kg n = 5; S2K146 5 mg/kg n = 5. In parallel, 6 animals were administered 1 mg/kg of the ultrapotent comparator antibody S2E12 mAh. Viral RNA loads in the lungs were reduced more than 1.5, 3 and 4 orders of magnitude after receiving 1, 5 and 10 mg/kg of S2K146, respectively (Figure 28). Viral replication in the lungs was completely abrogated for the 5 and 10 mg/kg groups and reduced by greater than 2.5 orders of magnitude for the 1 mg/kg group (Figure 29).

Overall serum mAh concentration measured at day 4 post-infection inversely correlated with viral RNA loads and infectious virus in the lungs (Figure 30 and Figure 31). S2K146 therefore effectively protects against SARS-CoV-2 challenge in vivo in a stringent therapeutic setting.

EXAMPLE 3

TESTING OF ANTIBODIES AGAINST SARS-CoV-2 OMICRON VARIANT

The following materials and methods wereused in Example 3. Cell lines

Cell lines were obtained from ATCC (HEK293T and Vero E6) or ThermoFisher Scientific (Expi CHO cells, FreeStyle™ 293-F cells and Expi293F™ cells).

Omicron prevalence analysis

The viral sequences and the corresponding metadata were obtained from GISAID EpiCoV project (https://www.gisaid.org/). Analysis was performed on sequences submitted to GISAID up to Dec 09, 2021. S protein sequences were either obtained directly from the protein dump provided by GISAID or, for the latest submitted sequences that were not incorporated yet in the protein dump at the day of data retrieval, from the genomic sequences with the exonerate⁴⁸ 2 2.4.0— haf93efl_3 (https://quay.io/repository /biocontainers/exonerate?tab=tags ) using protein to DNA alignment with parameters -m protein2dna —refine full — minintron 999999 —percent 20 and using accession YP 009724390.1 as a reference. Multiple sequence alignment of all human spike proteins was performed with mafft⁴⁹ 7.475— h516909a_0

(https://quay.io/repository/biocontainers/mafft7tabMags) with parameters —auto —reorder — keeplength — addfragments using the same reference as above. S sequences that contained >10% ambiguous amino acid or that were < than 80% of the canonical protein length were discarded. Figures were generated with R 4.0.2 (https://cran.r-project.org/) using ggplot2 3.3.2 and sf 0.9-7 packages. To identify each mutation prevalence, missingness (or ambiguous amino acids) was taken into account in both nominator and denominator.

Monoclonal Antibodies

VIR-7831 (sotrovimab) and VIR-7832 (a variant mAb similar to sotrovimab) were produced at WuXi Biologies (China). Antibody VH and VL sequences for mAbs cilgavimab (PDB ID 7L7E), tixagevimab (PDB ID 7L7E, 7L7D), casirivimab (PDB ID 6XDG), imdevimab (PDB ID 6XDG) and ADI-58125 (PCT application WO2021207597, seq. IDs 22301 and 22311) were subcloned into heavy chain (human IgGl) and the corresponding light chain (human IgKappa, IgLambda) expression vectors respectively and produced in transiently expressed in Expi-CHO-S cells (Thermo Fisher, #A29133) at 37°C and 8% C02. Cells were transfected using ExpiFectamine. Transfected cells were supplemented 1 day after transfection with ExpiCHO Feed and ExpiFectamine CHO Enhancer. Cell culture supernatant was collected eight days after transfection and filtered through a 0.2 pm filter. Recombinant antibodies were affinity purified on an Af TA xpress FPLC device using 5 mL HiTrap™ MabSelect™ PrismA columns followed by buffer exchange to Histidine buffer (20 mM Histidine, 8% sucrose, pH 6) using HiPrep 26/10 desalting columns. Antibody VH and VL sequences for bamlanivimab (LY-CoV555), etesevimab (LY-C0VOI6), regdanvimab (CT-P59) were obtained from PDB IDs 7KMG, 7C01 and 7CM4, respectively and mAbs were produced as recombinant IgGl by ATUM. The mAbs composing the NTD- and RBD-specific were discovered at VIR Biotechnology and have been produced as recombinant IgGl in Expi-CHO-S cells as described above. The identity of the produced mAbs was confirmed by LC-MS analysis.

IgG mass quantification by LC/MS intact protein mass analysis

Fc N-linked glycan from mAbs were removed by PNGase F after overnight non denaturing reaction at room temperature. Deglycosylated protein (4 pg) was injected to the LC- MS system to acquire intact MS signal. Thermo MS (Q Exactive Plus Orbitrap) was used to acquire intact protein mass under denaturing condition with m/z window from 1,000 to 6,000. BioPharma Finder 3.2 software was used to deconvolute the raw m/z data to protein average mass. The theoretical mass for each mAb was calculated with GPMAW 10.10 software. Many of the protein post-translational modifications such as N-terminal pyroglutamate cyclization, and c- terminal lysine cleavage, and formation of 16-18 disulfide bonds were added into the calculation.

Sample donors

Samples were obtained from SARS-CoV-2 recovered and vaccinated individuals under study protocols approved by the local Institutional Review Boards (Canton Ticino Ethics Committee, Switzerland, Comitato Etico Milano Area 1). All donors provided written informed consent for the use of blood and blood derivatives (such as PBMCs, sera or plasma) for research. Samples were collected 14-28 days after symptoms onset and 14-28 days or 7-10 months after vaccination.

Serum/plasma and mAbs pseudovirus neutralization assays

VSV pseudovirus generation

The plasmids encoding the Omicron SARS-CoV-2 spike variant was generated by overlap PCR mutagenesis of the wild-type plasmid, pcDNA3.1(+)-spike-D19⁵⁰. Replication defective VSV pseudovirus expressing SARS-CoV-2 spike proteins corresponding to the ancestral Wuhan- 1 virus and the Omicron VOC were generated as previously described⁸ with some modifications. Lenti-X 293T cells (Takara) were seeded in 15-cm² dishes at a density of 10e6 cells per dish and the following day transfected with 25 pg of spike expression plasmid with TransIT-Lenti (Mims, 6600) according to the manufacturer’s instructions. One day post transfection, cells were infected with VSV-luc (VSV-G) with an MOI 3 for 1 h, rinsed three times with PBS containing Ca2+/Mg2+, then incubated for additional 24 h in complete media at 37°C. The cell supernatant was clarified by centrifugation, aliquoted, and frozen at -80°C.

VSV pseudovirus neutralization

Vero-E6 were grown in DMEM supplemented with 10% FBS and seeded into clear bottom white 96 well plates (PerkinElmer, 6005688) at a density of 20Ό00 cells per well. The next day, mAbs or plasma were serially diluted in pre-warmed complete media, mixed with pseudoviruses and incubated for 1 h at 37°C in round bottom polypropylene plates. Media from cells was aspirated and 50 mΐ of virus-mAb/plasma complexes were added to cells and then incubated for 1 h at 37°C. An additional 100 pL of prewarmed complete media was then added on top of complexes and cells incubated for an additional 16-24 h. Conditions were tested in duplicate wells on each plate and eight wells per plate contained untreated infected cells (defining the 0% of neutralization, “MAX RLU” value) and infected cells in the presence of S309 and S2X259 at 20 pg/ml each (defining the 100% of neutralization, “MIN RLU” value). Virus-mAb/plasma-containing media was then aspirated from cells and 100 pL of a 1:2 dilution of SteadyLite Plus (Perkin Elmer, 6066759) in PBS with Ca⁺⁺ and Mg⁺⁺ was added to cells. Plates were incubated for 15 min at room temperature and then were analyzed on the Synergy -HI (Biotek). Average of 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: (l-(RLUx - MIN RLUave) / (MAX RLUave - MIN RLUave)) x 100. Data were analyzed and visualized with Prism (Version 9.1.0). IC50 (mAbs) and ID50 (plasma) 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, and a lower constrain equal to 0. Each neutralization experiment was conducted on two independent experiments, i.e., biological replicates, where each biological replicate contains a technical duplicate. IC50 values across biological replicates are presented as arithmetic mean ± standard deviation. The loss or gain of neutralization potency across spike variants was calculated by dividing the variant IC50/ID50 by the parental IC50/ID50 within each biological replicate, and then visualized as arithmetic mean ± standard deviation. Recombinant protein production

SARS-CoV-2 RBD proteins for SPR binding assays (with N-terminal signal peptide and C-terminal thrombin cleavage site-TwinStrep-8xHis-tag) were expressed in Expi293F (Thermo Fisher Scientific) cells at 37°C and 8% C02. Transfections were performed using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific). Cell culture supernatants were collected two to four days after transfection and supplemented with 1 Ox PBS to a final concentration of 2.5x PBS (342.5 mM NaCl, 6.75 mM KC1 and 29.75 mM phosphates). SARS- CoV-2 RBDs were purified using cobalt-based immobilized metal affinity chromatography and buffer exchanged into PBS using a HiPrep 26/10 (Cytiva) desalting column. Recombinant human ACE2 (residues 19-615 from Uniprot Q9BYF1 with a C-terminal AviTag-lOxHis-GGG-tag, and N-terminal signal peptide) was produced by ATUM. Protein was purified viaNi Sepharose resin followed by isolation of the monomeric hACE2 by size exclusion chromatography using a Superdex 200 Increase 10/300 GL column (Cytiva) pre-equilibrated with PBS.

Transient expression of animal ACE2

The mouse (GenBank: Q8R0I0), american mink (GenBank: QPL12211.1), and pangolin (XP 017505752.1) ACE2 ectodomains constructs were synthesized by GenScript and placed into a pCMV plasmid. The domain boundaries for the ectodomain are residues 19-615. The native signal tag was identified using SignalP-5.0 (residues 1-18) and replaced with a N-terminal mu- phosphatase signal peptide. These constructs were then fused to a sequence encoding thrombin cleavage site and a human Fc fragment or a 8x His tag at the C-terminus. All ACE2-Fc, and ACE2 His constructs were produced in Expi293 cells (Thermo Fisher A14527) in Gibco Expi293 Expression Medium at 37°C in a humidified 8% C02 incubator rotating at 130 rpm. The cultures were transfected using PEI-25K (Polyscience) with cells grown to a density of 3 million cells per mL and cultivated for 4-5 days. Proteins were purified from clarified supernatants for using a 1 mL HiTrap Protein A HP affinity column (Cytiva) or a 1 mL HisTrap HP affinity column (Cytiva), concentrated and flash frozen in lx PBS, pH 7.4 (10 mM Na2HP04, 1.8 mM KH2P04, 2.7 mM KC1, 137 mM NaCl).

ACE 2 binding measurements using surface plasmon resonance

Measurements were performed using a Biacore T200 instrument. A CM5 chip covalently immobilized with StrepTactin XT was used for surface capture of StrepTag-containing RBDs. Running buffer was HBS-EP+ pH 7.4 (Cytiva) and measurements were performed at 25 °C. Experiments were performed with a 3 -fold dilution series of monomeric human ACE2 (300, 100, 33, 11 nM) or animal ACE2 (900, 300, 100, 33 nM) and were run as single-cycle kinetics. Data were double reference-subtracted and fit to a 1:1 binding model using Biacore Evaluation software.

Statistical analysis Neutralization measurements were done in duplicate and relative luciferase units were converted to percent neutralization and plotted with a non-linear regression model to determine IC50/ID50 values using GraphPad PRISM software (version 9.0.0). Comparisons between two groups of paired data were made with Wilcoxon rank test. Comparisons between multiple groups of unpaired data were made with Kruskal -Wallis rank test and corrected with Dunn’s test. The recently emerged SARS-CoV-2 Omicron variant (B.1.1.529.1) harbors 37 amino acid substitutions in the spike (S) protein of which 15 are in the receptor-binding domain (RBD), thereby raising concerns about the efficacy of available vaccines and antibody therapeutics.

Analysis of the substitutions within the Omicron variant showed substantial changes from any previously documented SARS-CoV-2 strain, including 37 S protein mutations in the current most frequent haplotype (Figure 32, Figure 33 and Table 11).

Table 11: Mutations in SARS-CoV-2 Variants of Concern or Interest

In particular, 15 of these mutations are localized in the RBD, which is the major target of neutralizing antibodies, suggesting that Omicron may efficiently escape from vaccine-induced and therapeutic monoclonal antibodies (mAbs). Five of these mutations are in the receptor binding motif (RBM) of the RBD which is the domain that directly interacts with the receptor ACE2.

To study the effect of this large number of mutations which occur in Omicron S protein, a pseudovirus-based assay was employed to study the neutralization by mAbs and polyclonal antibodies as well as surface-plasmon resonance to measure binding of RBD to human and animal ACE2 receptors.

Omicron RBI) binds with increased affinity to human ACE 2 and gains binding to mouse ACE2

The rapid emergence of Omicron and the unprecedented number of substitutions raise questions about its origin. A search was performed for any intermediate SARS-CoV-2 sequence that would be indicative of a progressive accrual of mutations or recombination and by the identification of any variant that could be indicative of inter-species ping-pong. Twenty -three of 37 mutations in Omicron have been observed previously in another sarbecovirus or in a VOC/VOI, while the remaining 14 are novel or have not been previously reported in association with VOC/VOIs (Figure 43). Analysis of the GISAID database indicated that there were rarely more than 10-15 Omicron S mutations present in a given non-Omicron haplotype or Pango lineage (Figure 44, Figure 45, and Figure 46A-46D). While the possibility of recombination events was assessed, persistent replication in immunocompromised individuals or inter-species ping-pong are possible scenarios for the rapid accumulation of multiple mutations that may be then selected for fitness and immune escape.

To assess the latter scenario, experiments were performed to investigate whether RBD mutations found in Omicron may have resulted from adaptation of SARS-CoV-2 to animal receptors. To this end, RBD binding to mouse, mink, and pangolin ACE2 receptors was tested by SPR (Figure 34 and Figure 47). Binding to animal ACE2 by tested RBD variants was not detected, with the only exception of murine ACE2 that was found to bind with high affinity to Omicron RBD. Several of the mutations found in the Omicron RBD are at positions that are key contact sites of the Wuhan-Hu-1 RBD with human ACE2, such as K417N, Q493K and G496S. Individually, with the single exception of N501 Y that increases the binding affinity by 6-fold, all other substitutions have been shown by deep mutational scanning (DMS) to reduce binding to human ACE2, resulting in a predicted decrease of binding by more than 200-fold (Table 12). Table 12: Characteristics of single point mutations present in Omicron RBD relative to

Wuhan-Hu-1 RBD

At variance with this prediction, Omicron RBD was found to display a 2.6-fold increased binding affinity to human ACE2 (Figure 35), indicating a strong synergistic effect of mutated RBM residues. This cooperative effect has not been observed before for RBD from other VOC/VOI, most likely due to a low number of concurrent mutations. Additive effect of individual mutations was observed for the Beta VOC where the 3-fold reduction by K417N and the 6.4-fold increase by N501 Y resulted in a cumulative 2.4-fold increase (Table 12). Collectively, these findings suggest that mutations in the RBD of Omicron may have enabled adaptation to rodents as well as contributed to increased transmission in humans. Differential reduction of Omicron neutralizing titers in infected or vaccinated individuals To investigate the impact of the 37 mutations present in the Omicron S on the escape from antibody neutralization, the plasma neutralizing titers of antibodies against Wuhan-Hu-1 and Omicron VSV pseudoviruses in samples collected from convalescent patients or from individuals immunized with BNT162b2 mRNA vaccine were determined (Figure 36, Figure 37, Figure 48, Figure 49, Figure 50, Figure 51, and Table 13).

Table 13: Demographics of donors used to obtain data in Figures 36, 37, and 48-51

The decrease in neutralizing titers was marked (57-fold) for samples collected 2 to 4 weeks after symptomatic infections but was less pronounced for vaccinated individuals (11.6- fold) and especially for infected and vaccinated individuals (only 4.6-fold). Furthermore, samples collected 7 to 10 months after vaccination, although showing overall low neutralizing activity, displayed a modest decrease against Omicron (3.6-fold). Collectively, these findings demonstrate a substantial decrease in plasma neutralizing activity against Omicron, that may fall below the suggested protective titers. The results are also consistent with the notion that affinity maturation driven by multiple antigenic stimulations overtime can increase the breadth of neutralization.

Broadly neutralizing sarbecovirus antibodies retain activity against SARS-CoV-2 Omicron

Neutralizing mAbs with efficacy in prevention or treatment of SARS-CoV-2 can be grouped into two classes with regard to their capacity to block Spike protein binding to ACE2. Out of the eight currently authorized or approved mAbs, seven (bamlanivimab, etesevimab, casirivimab, imdevimab, cilgavimab, tixagevimab and regdanvimab) block binding of S to ACE2 and are often used in combination. These mAbs bind to epitopes overlapping the RBM (Figure 38) which is structurally plastic and has exhibited a significant mutation rate over the course of the pandemic. Combining two such ACE2 blocking mAbs provided greater resistance to variant viruses that carry RBM mutations. The second class of mAbs, represented by sotrovimab, do not block ACE2 binding but neutralize SARS-CoV-2 by targeting non-RBM epitopes shared across many sarbecoviruses, including SARS-CoV.

In vitro neutralizing activity of the two classes of therapeutic mAbs on the original Wuhan-Hu-1 and Omicron strains was compared using VSV pseudoviruses. While sotrovimab showed only a 2.7-fold decrease in IC50 values, all RBM-specific mAbs showed a complete loss of activity against Omicron, with the exception of the cocktail of cilgavimab and tixagevimab which showed a ~200-fold decrease (Figure 39, Figure 40, and Table 14). These findings are consistent with two recent reports and, together with serological data, support the notion of antigenic shift in Omicron.

Next, a larger panel of 36 neutralizing NTD- or RBD-specific mAbs (for which the epitope has been characterized structurally or assigned to a given antigenic site through competition studies) was tested (Figure 41, Figure 52, Figure 53, Figure 54, and Table 14). The 4 NTD-specific antibodies completely lost activity on Omicron, in line with the presence of several mutations and deletions in the antigenic supersite of the NTD. Out of the 22 mAbs assigned to site I (RBM), three retained potent neutralizing activity against Omicron. One of these mAbs, S2K146 binds as a molecular mimic of ACE2; structural data are still lacking for S2N28 and S2X324 mAbs. Out of the 9 mAbs specific for the conserved site II of the RBD (class 4 mAbs), only S2X259 mAb retained activity against Omicron, while neutralization was decreased by more than 10-fold or abolished for the remaining mAbs. Finally, neutralization of Omicron was also maintained with S2H97 mAb, which recognizes the highly conserved cryptic site V. Collectively, these findings identify three additional classes of broadly neutralizing sarbecovirus mAbs besides sotrovimab that retain neutralization of Omicron (Figure 42).

Table 14: Binding properties and V gene usage of tested mAbs

DSO, days after symptom onset. N/A, not available.

The high number of substitutions in Omicron delineates a dramatic shift in SARS-CoV-2 which has led to change in both tropism and immune escape. While influenza antigenic shift is defined by the specific mechanism of genetic reassortment, the mechanism for the introduction of such a high number of mutations in Omicron remains to be established.

Consistent with the variable decrease in plasma neutralizing antibody titers, only 6 out of a panel of 44 neutralizing anti-SARS-CoV-2 mAbs retained potent neutralizing activity against Omicron. These mAbs define 4 sites in the RBD that appear to be relatively conserved in Omicron and in other sarbecoviruses. Three potent neutralizing mAbs were found that bind to the RBM and are not affected by the multiple mutations of the Omicron, of which one (S2K146) is a molecular mimic of the ACE2 receptor.

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 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 above-detailed 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

1. An antibody, or an antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising a CDRH1, a CDRH2, and a CDRH3, and a light chain variable domain (VL) comprising a CDRL1, a CDRL2, and a CDRL3, wherein:

(i) the CDRH1 comprises or consists of the amino acid sequence according to SEQ ID NO: 144, 24, 34, 44, 54, 64, 74, 84, 94, 104, 114, 124, 134, or 170, or a functional variant thereof comprising one, two, or three acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline-encoded amino acid;

(ii) the CDRH2 comprises or consists of the amino acid sequence according to SEQ ID NO: 145, 25, 35, 45, 55, 65, 75, 85, 95, 105, 115, 125, 135, or 171, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid;

(iii) the CDRH3 comprises or consists of the amino acid sequence according to SEQ ID NO: 146, 26, 36, 46, 56, 66, 76, 86, 96, 106, 116, 126, 136, or 172, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid;

(iv) the CDRL1 comprises or consists of the amino acid sequence according to SEQ ID NO: 148, 28, 38, 48, 58, 68, 78, 88, 98, 108, 118, 128, 138, or 174, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid;

(v) the CDRL2 comprises or consists of the amino acid sequence according to SEQ ID NO: 149, 29, 39, 49, 59, 69, 79, 89, 99, 109, 119, 129, 139, or 175, or a functional variant thereof comprising one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid; and/or

(vi) the CDRL3 comprises or consists of the amino acid sequence according to SEQ ID NO: 150, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 176, or a functional variant thereof comprising having one, two, or three amino acid substitutions, one or more of which substitutions is optionally a conservative substitution and/or is a substitution to a germline- encoded amino acid, wherein the antibody or antigen-binding fragment is capable of binding to the surface glycoprotein of a sarbecovirus.

2. The antibody or antigen-binding fragment of claim 1, wherein the antibody or antigen-binding fragment is capable of binding to the surface glycoprotein when the surface glycoprotein is expressed on a cell surface of a host cell and/or is comprised on a virion.

3. The antibody or antigen-binding fragment of claim 1 or 2, which is capable of binding to a surface glycoprotein from two or more ( e.g ., two, three, four, five, or more) sarbecoviruses.

4. The antibody or antigen-binding fragment of any one of claims 1-3, which is capable of neutralizing an infection by one or more sarbecoviruses in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.

5. The antibody or antigen-binding fragment of any one of claims 1-4, which is capable of neutralizing an infection by two or more sarbecoviruses in an in vitro model of infection and/or in an in vivo animal model of infection and/or in a human.

6. The antibody or antigen-binding fragment of any one of claims 1-5, comprising CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 amino acid sequences according to SEQ ID NOs.:

(i) 144-146 and 148-150, respectively;

(ii) 24-26 and 28-30, respectively;

(iii) 34-36 and 38-40, respectively;

(iv) 44-46 and 48-50, respectively;

(v) 54-56 and 58-60, respectively;

(vi) 64-66 and 68-70, respectively;

(vii) 74-76 and 78-80, respectively;

(viii) 84-86 and 88-90, respectively;

(vix) 94-96 and 98-100, respectively;

(x) 104-106 and 108-110, respectively;

(xi) 114-116 and 118-120, respectively;

(xii) 124-126 and 128-130, respectively; (xiii) 134-136 and 138-140, respectively; or (xiv) 170-172 and 174-176, respectively.

7. The antibody or antigen-binding fragment of any one of claims 1-6, wherein:

(i) the VH comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO: 143, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, and 169, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid; and/or

(ii) the VL comprises or consists of an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence according to SEQ ID NO: 147, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, and 173, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more substitution to a germline-encoded amino acid.

8. The antibody or antigen-binding fragment of any one of claims 1-7, wherein the

VH and the VL comprise or consist of amino acid sequences having 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 to, or comprising or consisting of, the amino acid sequences according to SEQ ID NOs.:

(i) 143 and 147, respectively;

(ii) 23 and 27, respectively;

(iii) 33 and 37, respectively;

(iv) 43 and 47, respectively;

(v) 53 and 57, respectively;

(vi) 63 and 67, respectively;

(vii) 73 and 77, respectively;

(viii) 83 and 87, respectively;

(vix) 93 and 97, respectively;

(x) 103 and 107, respectively; (xi) 113 and 117, respectively;

(xii) 123 and 127, respectively;

(xiii) 133 and 137, respectively; or

(xiv) 169 and 173, respectively.

9. The antibody or antigen-binding fragment of any one of claims 1-8, which:

(i) recognizes an epitope in the Spike protein of two or more, three or more, four or more, or five or more sarbecoviruses;

(ii) is capable of blocking an interaction between the Spike protein of two or more, , three or more, four or more, or five or more sarbecoviruses and their respective cell surface receptor(s), wherein, optionally, a cell surface receptor comprises a human ACE2;

(iii) recognizes an epitope that is conserved in the Spike protein of two or more, , three or more, four or more, or five or more sarbecoviruses;

(iv) is cross-reactive against two or more, , three or more, four or more, or five or more sarbecoviruses; or

(v) any combination of (i)-(iv).

10. The antibody or antigen-binding fragment of any one of claims 1-9, 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.

11. The antibody or antigen-binding fragment of claim 10, comprising:

(i) a first VH and a first VL; and

(ii) a second VH and a second VL, wherein the first VH and the second VH are different and each independently comprise an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO: 143, 23, 33, 43, 53, 63, 73, 83, 93, 103, 113, 123, 133, and 169, and wherein the first VL and the second VL are different and each independently comprise an amino acid sequence having 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 to, or comprising or consisting of, the amino acid sequence set forth in SEQ ID NO: 147, 27, 37, 47, 57, 67, 77, 87, 97, 107, 117, 127, 137, and 173; and wherein the first VH and the first VL together form a first antigen-binding site, and wherein the second VH and the second VL together form a second antigen-binding site.

12. The antibody or antigen-binding fragment of any one of claims 1-11, wherein the antibody or antigen-binding fragment further comprises a Fc polypeptide or a fragment thereof.

13. The antibody or antigen-binding fragment of claim 12, 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;

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

(iii) a mutation that enhances binding to human FcyRIIa and/or decreases binding to a human FcyRIIb as compared to a reference Fc polypeptide that does not comprise the mutation; and/or

(iv) a mutation that enhances binding to a human Clq compared to a reference Fc polypeptide that does not comprise the mutation.

14. An isolated polynucleotide encoding the antibody or antigen-binding fragment of any one of claims 1-13, or encoding a VH, a heavy chain, a VL, and/or a light chain of the antibody or the antigen-binding fragment.

15. The polynucleotide of any claim 14, comprising a 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 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 to, or comprising or consisting of, the polynucleotide sequence according to any one of SEQ ID NOs.: 151, 152, 31, 32, 41, 42, 51, 52, 61, 62, 71, 72, 81, 82, 91, 92, 101, 102, 111, 112, 121, 122, 131, 132, 141, 142, 177, and 178.

16. A recombinant vector comprising the polynucleotide of any one of claims 14 and

15

17. A host cell comprising the polynucleotide of any one of claims 14 and 15 and/or the vector of claim 16, wherein the polynucleotide is heterologous to the host cell.

18. A human B cell comprising the polynucleotide of any one of claims 14 and 15, wherein polynucleotide is heterologous to the human B cell and/or wherein the human B cell is immortalized.

19. A composition comprising:

(i) the antibody or antigen-binding fragment of any one of claims 1-13;

(ii) the polynucleotide of any one of claims 14 and 15;

(iii) the recombinant vector of claim 16;

(iv) the host cell of claim 17; and/or

(v) the human B cell of claim 18, and a pharmaceutically acceptable excipient, carrier, or diluent.

20. A method of treating a sarbecovirus infection in a subject, the method comprising administering to the subject an effective amount of

(i) the antibody or antigen-binding fragment of any one of claims 1-13;

(ii) the polynucleotide of any one of claims 14 and 15;

(iii) the recombinant vector of claim 16;

(iv) the host cell of claim 17; and/or

(v) the human B cell of claim 18, and/or

(vi) the composition of claim 19.

21. The antibody or antigen-binding fragment of any one of claims 1-13, the polynucleotide of any one of claims 14 and 15, the recombinant vector of claim 16, the host cell of claim 17, the human B cell of claim 18, and/or the composition of claim 19 for use in a method of treating a sarbecovirus infection in a subject.

22. The antibody or antigen-binding fragment of any one of claims 1-13, the polynucleotide of any one of claims 14 and 15, the recombinant vector of claim 16, the host cell of claim 17, the human B cell of claim 18, and/or the composition of claim 19 for use in the preparation of a medicament for the treatment of a sarbecovirus infection in a subject.

23. A method for in vitro diagnosis of a sarbecovirus infection, the method comprising:

(i) contacting a sample from a subject with an antibody or antigen-binding fragment of any one of claims 1-13; and

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

24. An antibody, or an antigen-binding fragment thereof, that competes for binding to a sarbecovirus surface glycoprotein with the antibody or antigen-binding fragment of any one of claims 1-13.

25. The method of claim 20 or the antibody, antigen-binding fragment, polynucleotide, recombinant vector, host cell, human B cell, and/or composition for use of claim 21 or 22, wherein the sarbecovirus comprises:

(i) SARS-CoV;

(ii) SARS-CoV-2;

(iii) WIV1;

(iv) PANG/GD;

(v) PANG/GX;

(vi) RatG13;

(v) ZXC21;

(vi) ZC45;

(vii) RmYN02;

(viii) Anlongll2;

(ix) YN2013;

(x) SC2018;

(xi) SC2011;

(xii) BGR2008;

(xiii) BtkY72;

(xiv) SARS-CoV-2 variant P.1 ;

(xv) SARS-CoV-2 variant B.1.1.7;

(xvi) SARS-CoV-2 variant B.1.429;

(xvii) SARS-CoV-2 variant B.1.351;

(xviii) SARS-CoV-2 variant B.1.1.222; (xix) SARS-CoV-2 variant C.37;

(xx) SARS-CoV-2 variant AY.1 ;

(xxi) SARS-CoV-2 variant AY.2;

(xxii) SARS-CoV-2 S protein mutant N501Y;

(xxiii) SARS-CoV-2 S protein mutant Y453F;

(xxiv) SARS-CoV-2 S protein mutant N439K;

(xxv) SARS-CoV-2 S protein mutant K417V;

(xxvi) SARS-CoV-2 S protein mutant E484K;

(xxvii) SARS-CoV-2 variant B.1.1.529.; or (xxviii)any combination of (i)-(xvii).

26. A method for producing an antibody or antigen-binding fragment of any one of claims 1-13, wherein the method comprises culturing a host cell expressing the antibody or antigen-binding fragment under conditions and for a time sufficient to produce the antibody, or the antigen-binding fragment.

27. The method for producing an antibody or antigen-binding fragment of claim 26, wherein the host cell comprises a recombinant vector comprising a polynucleotide of any one of claims 14 and 15.