WO2023039064A2 - Broadly neutralizing antibodies against sars-like viruses - Google Patents

Abstract

The invention provides novel broadly neutralizing antibodies and related antibody agents against SARS like viruses, e.g., human coronaviruses. Also provided in the invention are polynucleotides and vectors encoding such antibodies, as well as pharmaceutical compositions containing the antibodies or polynucleotides. Therapeutic uses of the antibodies or pharmaceutical compositions in preventing or treating viral infections (e.g., SARS-CoV-2 infection) are also encompassed by the invention.

Description

BROADLY NEUTRALIZING ANTIBODIES AGAINST SARS-LIKE VIRUSES

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application Numbers 63/241,590 (filed September 8, 2021; now pending). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under grant numbers AI144462 and AI161818 awarded by the National Institutes of Health. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Relatively early in the COVID-19 pandemic, it appeared that SARS-CoV-2 was a virus that might be particularly amenable to control by vaccination. Many different vaccine modalities, most notably mRNA vaccination, showed spectacular success in phase 3 protection studies ^19,2°. The success was attributed at least in part to the ability of the different modalities to induce robust neutralizing antibody (nAb) responses ^21-23. However, as the virus has now infected hundreds of millions worldwide, variants have arisen (variants of concern, VOCs), some of which show notable resistance to neutralization by immunodominant nAb responses induced through infection and vaccination ^1,3-6. Current vaccines are still apparently largely effective in preventing hospitalization and death caused by VOCs ^24,25. However, as vaccine-induced nAb responses naturally decline, breakthrough infections are on the increase and there are concerns that these may become more prevalent and perhaps more clinically serious, and that more pathogenic and resistant VOCs may appear. There are also concerns that emerging SARS-like viruses may seed new pandemics from spillover events ^2,7.

[0004] There are an urgent and ongoing needs for nAbs and vaccines that are effective against a greater diversity of sarbecoviruses. The present invention is directed to addressing such unmet needs in the art. SUMMARY OF THE INVENTION

[0005] In one aspect, the invention provides novel broadly neutralizing antibodies or antigen-binding fragments thereof that specifically bind to the spike (S) protein of a SARS like virus (a sarbecovirus). In some embodiments, the antibodies or antigen-binding fragments can neutralize a SARS-like virus by specifically binding to a conserved epitope on the spike protein of the virus described herein. In some embodiments, the antibodies or antigen-binding fragments are capable of binding to and neutralizing one or more human coronaviruses. In some embodiments, the epitope recognized by the antibodies are located in the receptor binding domain (RBD). In some specific embodiments, the antibodies specifically target a conserved RBD epitope of SARS-CoV-2.

[0006] In some embodiments, the antibodies or antigen-binding fragments of the invention contain heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences that are respectively identical to an antibody containing a heavy chain sequence and a light chain sequence shown in Table 1. Some of the antibodies of the invention are not full length antibodies. In some embodiments, the antibodies or antigenbinding fragments of the invention contain one or more amino acid substitutions relative to the heavy chain sequence and/or the light chain sequence shown in Table 1.

[0007] In a related aspect, the invention provides fusion or modified molecules which contain one of the broadly neutralizing sarbecovirus antibodies or antigen-binding fragments described herein and a second moiety that is fused or conjugated to the antibody or antigenbinding fragment thereof. In various embodiments, the second moiety can be a polypeptide or a small organic molecule.

[0008] In a related aspect, the invention provides polynucleotide sequences that encode the broadly neutralizing sarbecovirus antibodies or antigen-binding fragments described herein. Related vectors and cells that harbor such a polynucleotide or vector are also provided in the invention. Further encompassed by the invention are pharmaceutical compositions that contain a broadly neutralizing sarbecovirus antibody or antigen-binding fragment described herein or a polynucleotide encoding the antibody, as well therapeutic applications of the pharmaceutical compositions in preventing or treating infection of a SARS-like virus. [0009] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

DESCRIPTION OF THE DRAWINGS

[0010] Fig. 1. Plasma neutralization and memory B cell responses in infected, vaccinated, and infected/vaccinated donors.

[0011] a. SARS-CoV-2 pseudovirus neutralization of plasma samples from COVID-19 convalescent donors as well as vaccinated individuals with or without a prior history of SARS-CoV-2 infection. Statistical comparisons between the two groups were performed using a Mann-Whitney two-tailed test, (, (*p < 0.05, ****p < 0.0001).

[0012] b. Plasma neutralization for all three groups against distantly related sarbecoviruses. Pangl7, SARS-CoV-1, and WIV1 are shown. RBDs are indicated for all spikes. In contrast to infected only and vaccinated only donors, approximately half of the infected-vaccinated donors have neutralizing titers against SARS-CoV-1 above background (Fig. 5).

[0013] c. Plasma neutralizing activity against SARS-CoV-2 (Wuhan) and SARS-CoV-2 variants of concern (B. l.1.7 (alpha), B.1.351 (beta), P.l (gamma) and B.1.617.2 (delta)).

[0014] d. Flow cytometric analysis of IgG⁺ B cells from PBMCs of human donors CC25 and CC84 isolated at the indicated timepoints (see Fig. 6 for gating strategy). Numbers indicate percentage of cells binding to SARS-CoV-1 and SARS-CoV-2 spike proteins, respectively.

[0015] e. Quantification of SARS-CoV-1 -specific, SARS-CoV-2-specific, and broadly neutralizing IgG⁺ B cells from donor CC25 (top) and CC84 (bottom) donors.

[0016] Fig. 2. Binding, neutralization and immunogenetic properties of sarbecovirus mAbs. A total of 107 mAbs were isolated, 56 mAbs from donor CC25 and 51 mAbs from donor CC84. MAbs were isolated by single B cell sorting using SARS-CoV-1 and SARS- CoV-2 S-proteins as baits.

[0017] a. Heatmap showing IGVH germline gene usage (VH1-46, VH1-69 and VH3-30 and other V-genes), lineage information (unique (sky) and expanded (tangerine) lineages) and V-gene nucleotide somatic mutations (SHMs). ELISA binding activity of mAbs with SARS-CoV-2, SARS-CoV-1 and other b- and a-sarbecovirus derived S-proteins and domains of SARS-CoV-2 S-protein (NTD, RBD-SD1, RBD-SD1-2) (LOD <0.5 OD₄₀₅). Binding of mAbs with clade la (SARS-CoV-2 related: SARS-CoV-2, RatG13, Pangl7), clade lb (SARS-CoV-1 related: SARS-CoV-1, WIV1, SHC014), clade 2 (RmYN02, Rfl, Rs4081, Yunl l) and clade 3 (BM4831, BtKY72) sarbecovirus S-protein derived monomeric RBDs. Percent neutralization of ACE2 -utilizing sarbecoviruses (SARS-CoV-2, Pangl7, SARS-CoV-1 and WIV1) by mAb supernatants (cut-off <60%). Breadth (%) of binding to 12 sarbecovirus RBDs and breadth (%) of neutralization with 4 ACE2 sarbecoviruses is indicated for each mAb. MAb expression levels in the supernatants were quantified by total IgG ELISA and the concentrations were approximately comparable overall. For reproducibility, the binding and neutralization assays were all performed twice with mAb supernatants expressed independently twice.

[0018] b. Number of mAbs (unique clones) neutralizing SARS-CoV-2 and other sarbecoviruses. 40 mAbs neutralized all 4 ACE2 sarbecoviruses tested.

[0019] c. Pie plots showing IGHV gene usage distribution of isolated mAbs with enriched gene families shown, VH1-46, VH1-69 and VH3-30. Dot plots showing % nucleotide mutations (SHMs) in the heavy chain (VH) of isolated mAbs. The mAbs are grouped by neutralization with sarbecoviruses.

[0020] d. Pie and dot plots depicting IGHV gene distribution and VH nucleotide SHMs respectively. The mAbs are grouped by binding to sarbecovirus-derived RBDs.

[0021] e. CDRH3 length distributions of isolated mAbs across broadly neutralizing and broadly neutralizing mAb groups compared to human baseline germline reference. MAbs with 20- and 21- amino acid-CDRH3s are highly enriched.

[0022] f. Sequence conservation logos of 20 (n = 17) and 21 (n = 11) amino acid long CDRH3-bearing mAbs show enrichment of D-gene derived residues, including IGHD2-15 germline D-gene encoded two cysteines in 20 amino acid long CDRH3-bearing mAbs that may potentially form a disulfide bond.

[0023] g. Enrichment of IGHD2-15 and IGHD3-22 germline D-genes in sarbecovirus broadly neutralizing or broadly neutralizing mAbs compared to corresponding human baseline germlines.

[0024] Fig. 3. Binding and neutralization of mAbs in terms of potency and breadth.

[0025] A total of 19 mAbs from donor CC25 and 11 mAbs from donor CC84 were selected to determine specificity, neutralization potency and breadth, kinetic properties, and neutralization activities against sarbecoviruses and SARS-CoV-2 VOCs. [0026] a. Heatmap of binding responses determined by BLI using SARS-CoV-1 and SARS-CoV-2 S and S-protein domains and subdomains with IGHV gene usage for each mAb indicated.

[0027] b. Heatmap of dissociation constants (KD (M) values) for mAb binding to spike- derived monomeric RBDs from four sarbecovirus clades: clade lb (n = 3); clade la (n = 3); clade 2 (n = 4); clade 3 (n = 2).

[0028] c. IC50 neutralization of mAbs against SARS-CoV-2, SARS-CoV-1, Pangl7,

WIV1, and SHC014 determined using pseudotyped viruses.

[0029] d, Neutralization of 18 bnAbs against SARS-CoV-2 (WT) and four major SARS- CoV-2 variants of concern. CC12.1, DH1047, K398.22 and DEN mAbs were used as controls.

[0030] Fig. 4. Epitope specificities of sarbecovirus bnAbs.

[0031] a. Heatmap summary of epitope binning of sarbecovirus bnAbs based on BLI competition of bnAbs with human (CC12.1, CC12.19, CR3022, DH1047 and S309) and macaque (K398.22) RBD-specific nAbs. IGHV gene usage for each mAb is indicated. Geomean neutralization potency and breadth (calculated from Fig. 3B) and RBD binding breadth with clade 2 or all clade sarb ecoviruses (calculated from 3C) for each mAb are indicated. The BLI competition was performed with monomeric SARS-CoV-2 RBD, and the competition levels are indicated. Based on competition with human and one macaque nAb of known specificities, the sarbecovirus bnAbs were divided into group- 1 and group-2.

[0032] b. Binding of human nAbs to SARS-CoV-2 RBD. The RBD is shown as a black chain trace, whereas antibodies are represented by solid surfaces: CC12.1 (PDB 6XC2), CR3022 (PDB 6W41), S309 (PDB 7R6W), DH1047 (7LD1), and K398.22 (PDB unreleased).

[0033] c. Electron microscopy 3D reconstructions of sarbecovirus bnAb Fabs with SARS-CoV-2 S-protein. Fabs of group-1 (n = 9) and group-2 (n = 2) were complexed with SARS-CoV-2 S-protein and 3D reconstructions were generated from 2D class averages. Fabs are shown and the spike SI and S2 subunits and the RBD sites are labelled. Group-1 Fabs are based on competition levels with ACE2 binding site nAb, CC12.1.

[0034] d. The epitope of each antibody is outlined corresponding to panel b. Epitope residues are defined by buried surface area (BSA) > 0 A² as calculated by PDBePISA (https://www.ebi.ac.uk/msd-srv/prot_int/pistart.html). Putative epitope regions of group-1 bnAbs based on the competitive binding assay are indicated by circles.

[0035] e. Conservation of 12 sarbecovirus RBDs. The conservation was calculated by ConSurf. The putative epitope region targeted by group- 1 bnAbs is circled as in panel d.

[0036] Fig. 5. Sera neutralization. Neutralization by sera from COVID-19, 2 x mRNA- spike-vaccinated and SARS-CoV-2 infected/mRNA vaccinated donors with pseudotyped SARS-CoV-2, SARS-CoV-2 variants of concern [B.l.1.7 (alpha), B.1.351 (beta), P. l (gamma) and B.1.617.2 (delta)], as well as other sarbecoviruses (Pangl7, SARS-CoV-1, and WIV1). ID50 neutralization titers are shown. Prior to vaccination, the sera from infected- vaccinated donors were tested for neutralization and the ID50 neutralization titers are shown for comparison.

[0037] Fig. 6. Flow cytometry B cell profiling and sorting strategies.

[0038] a. Gating strategy for analysis of IgG⁺ B cell populations that bind SARS-CoV-1 S-protein only (CD 19⁺CD20⁺CD3 CD4 CD8 CD 14TgMTgG⁺CoV2B V421 'CoV2 AF488' CoVl⁺), SARS-CoV-2 S-protein only (CD19⁺CD20⁺CD3-CD4'CD8-CD14TgMTgG⁺ CoV2BV421⁺CoV2AF488⁺CoVl"), or both SARS-CoV-1 and SARS-CoV-2 S-proteins (CD 19⁺CD20⁺CD3 CD4 CD8 CD 14TgMTgG⁺Co V2B V421 ⁺CoV2 AF488⁺CoVl ⁺).

[0039] b. Gating strategy used to isolate single broadly neutralizing IgG⁺ B cells (indicated in red).

[0040] Fig. 7. Binding, neutralization and immunogenetics information of isolated mAbs. A total of 107 mAbs from two SARS-CoV-2 infected-vaccinated donors CC25 (n = 56 mAbs) and CC84 (n = 51 mAbs) were isolated by single B cell sorting using SARS-CoV- 1 and SARS-CoV-2 S-proteins as baits. MAbs were expressed and tested for antigen binding, pseudovirus neutralization, and analyzed for immunogenetic properties. Germline, lineage, somatic hypermutation (SHM), ELISA binding with S-proteins and RBDs, neutralization with ACE2 -utilizing sarbecoviruses and breadth are indicated according to the key. Paired gene information, including heavy chain CDRH3 and light chain CDRL3 sequences are represented for each mAb.

[0041] Fig. 8. Immunoglobulin heavy chain V-gene, D-gene and J-gene usage and enrichment in isolated mAbs compared to a reference human germline database. Baseline germline frequencies of heavy chain genes (VH-, DH- and JH- genes) are shown, and mAb, bnAbs and broadly neutralizing mAbs in a-c panels are indicated according to the key in (a). Arrows indicate gene enrichments compared to human baseline germline frequencies, (a) VH genes, (b) DH genes, and (b) JH genes are shown for all unique clone mAbs isolated from CC25 and CC84 donors.

[0042] Fig. 9. mAb supernatant binding to SARS-CoV-2 RBD and SARS-CoV-2 S and association with SHM, binding and neutralization breadth.

[0043] a. Supernatants from Expi293F cell-expressed mAbs were screened for BLI binding with SARS-CoV-2 RBD and SARS-CoV-2 S-protein. Binding kinetics were obtained using the 1 : 1 binding kinetics fitting model on ForteBio Data Analysis software. [0044] b. Correlations of mAb binding ( D (M) values) to SARS-CoV-2 RBD and S- proteins with heavy chain SHM, neutralization breadth, and sarbecovirus RBD breadth are determined by nonparametric Spearman correlation two-tailed test with 95% confidence interval. The Spearman correlation coefficient (r) and p-value are indicated.

[0045] Fig. 10. Binding of select mAbs to RBDs from sarbecovirus clades. BLI binding kinetics of select CC25 and CC84 mAbs to monomeric RBDs derived from sarbecovirus clades: clade lb (SARS-CoV-2, RatG13, Pangl7), clade la (SARS-CoV-2, WIV1, SHC014), clade 3 (BM-4831, BtKY72) and clade 2 ((RmYN02, Rfl, Rs4081, Yunl l). Binding kinetics were obtained using the 1 : 1 binding kinetics fitting model on ForteBio Data Analysis software and maximum binding responses, dissociations constants (AD) and on-rate (k_on) and off-rate constants (k_off) for each antibody protein interaction are shown. KD, k_on and k_Off values were calculated only for antibody-antigen interactions where a maximum binding response of 0.05nm was obtained.

[0046] Fig. 11. Epitope binning of mAbs using a competition assay. 30 select mAbs (19 mAbs from donor CC25 and 11 mAbs from donor CC84) were assayed in BLI competition binning to evaluate epitope properties shared with previously isolated human (CC12.1, CC12.19, CR3022, DH1047 and S309) and macaque (K398.22) mAbs with known epitope specificities. His-tagged SARS-CoV-2 RBD protein (200nM) was captured on anti-His biosensors and incubated with the indicated mAbs at a saturating concentration of lOOpg/mL for 10 mins and followed by nAb incubation for 5 min at a concentration of 25pg/mL. All BLI measurements were performed on an Octet RED384 system. BLI traces are shown for each binding. The binding inhibition % is calculated with the formula: percent (%) of inhibition in the BLI binding response = 1- (response in presence of the competitor antibody / response of the corresponding control antibody without the competitor antibody).

[0047] Fig. 12. Epitope mapping of bnAbs using negative stain Electron Microscopy (ns- EM). Electron microscopy (EM) images of sarbecovirus cross-neutralizing antibody Fabs with SARS-CoV-2 S-protein. 2D class averages of S-protein bound Fabs for each mAbs are shown.

DETAILED DESCRIPTION

[0048] The emergence of current SARS-CoV-2 variants of concern (VOCs) and potential future spillovers of SARS-like coronaviruses into humans pose a major threat to human health and the global economy ^1-7. Development of broadly effective coronavirus vaccines that can mitigate these threats is needed ⁸’⁹. Notably, several recent studies have revealed that vaccination of recovered CO VID-19 donors results in enhanced nAb responses compared to SARS-CoV-2 infection or vaccination alone ^10-13.

[0049] The present invention relate to broadly neutralizing antibodies against SARS-like viruses (or “SARS-related coronaviruses” or “sarb ecoviruses” as used herein), related polynucleotides, vectors, cells, fusion molecules, and pharmaceutical compositions. The invention is derived from studies undertaken by the inventors to isolate and characterize a number of bnAbs from SARS-CoV-2 vaccinated subjects, which demonstrated excellent potency and breadth to sarbecoviruses. Specifically, the inventors utilized a targeted donor selection strategy to isolate a large panel of broadly neutralizing antibodies (bnAbs) to sarbecoviruses from vaccinated human donors. Many of the bnAbs are remarkably effective in neutralization against sarbecoviruses that use ACE2 for viral entry and also show strong binding to non-ACE2 -using sarbecoviruses. The bnAbs are equally effective against SARS- CoV-2 VOCs compared to the original virus. Neutralization breadth is achieved by bnAb binding to epitopes on a relatively conserved face of the receptor binding domain (RBD) as opposed to strain-specific nAbs to the receptor binding site that are commonly elicited in SARS-CoV-2 infection and vaccination ^14-18.

[0050] The generation of a large panel of potent bnAbs provides new opportunities and choices for next-generation antibody prophylactic and therapeutic applications and, importantly, provides a basis for effective design of pan-sarbecovirus vaccines. The invention accordingly provides novel bnAbs, fusion molecules, antibody-drug conjugates and related vaccines that are based on or derived from these isolated antibodies. Engineered vectors containing polynucleotides expressing these bnAbs and host cells harboring such vectors. Further included in the invention are methods of producing the novel antibodies, as well as therapeutic applications of the antibodies and derivative compositions (e.g., vectors, vaccines, and pharmaceutical compositions) are also provided in the invention.

[0051] The invention can employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. (See, for example, Sambrook et al, ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al, ed. (1992) Molecular Cloning: A Laboratory Manual, (Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds. (1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al, eds., Methods In Enzymology, Vols. 154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-FV;

Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986); and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0052] General principles of antibody engineering are set forth in Borrebaeck, ed. (1995) Antibody Engineering (2nd ed.; Oxford Univ. Press). General principles of protein engineering are set forth in Rickwood et al, eds. (1995) Protein Engineering, A Practical Approach (IRL Press at Oxford Univ. Press, Oxford, Eng.). General principles of antibodies and antibody- hapten binding are set forth in: Nisonoff (1984) Molecular Immunology (2nd ed.; Sinauer Associates, Sunderland, Mass.); and Steward (1984) Antibodies, Their Structure and Function (Chapman and Hall, New York, N.Y.). Additionally, standard methods in immunology known in the art and not specifically described can be followed as in Current Protocols in Immunology, John Wiley & Sons, New York; Stites et al, eds. (1994) Basic and Clinical Immunology (8th ed; Appleton & Lange, Norwalk, Conn.) and Mishell and Shiigi (eds) (1980) Selected Methods in Cellular Immunology (W.H. Freeman and Co., NY). [0053] Standard reference works setting forth general principles of immunology include Current Protocols in Immunology, John Wiley & Sons, New York; Klein (1982) J., Immunology: The Science of Self-Nonself Discrimination (John Wiley & Sons, NY); Kennett et al, eds. (1980) Monoclonal Antibodies, Hybridoma: A New Dimension in Biological Analyses (Plenum Press, NY); Campbell (1984) "Monoclonal Antibody Technology" in Laboratory Techniques in Biochemistry and Molecular Biology, ed. Burden et al, (Elsevier, Amsterdam); Goldsby et al, eds. (2000) Kuby Immunology (4th ed.; W.H. Freeman & Co.); Roitt et al. (2001) Immunology (6th ed.; London: Mosby); Abbas et al. (2005) Cellular and Molecular Immunology (5th ed.; Elsevier Health Sciences Division); Kontermann and Dubel (2001) Antibody Engineering (Springer Verlag); Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press); Lewin (2003) Genes VIII (Prentice Hall, 2003); Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Press); Dieffenbach and Dveksler (2003) PCR Primer (Cold Spring Harbor Press).

[0054] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and Technology, Morris (Ed.), Academic Press (1^st ed., 1992); Oxford Dictionary of Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge (1^st ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos (1994);

Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference) , Martin and Hine (Eds.), Oxford University Press (4^th ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0055] The term "antibody" also synonymously called "immunoglobulins" (Ig), or "antigen -binding fragment" refers to polypeptide chain(s) which exhibit a strong monovalent, bivalent or polyvalent binding to a given antigen, epitope or epitopes. Unless otherwise noted, antibodies or antigen-binding fragments used in the invention can have sequences derived from any vertebrate species. They can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi -synthetic or fully synthetic libraries or combinations thereof. Unless otherwise noted, the term “antibody” as used in the present invention includes intact antibodies, antigen-binding polypeptide fragments and other designer antibodies that are described below or well known in the art (see, e.g., Serafini, J Nucl. Med. 34:533-6, 1993). [0056] An “intact antibody” (or “full length antibody”) typically comprises at least two heavy (H) chains (about 50-70 kD) and two light (L) chains (about 25 kD) inter-connected by disulfide bonds. The recognized immunoglobulin genes encoding antibody chains include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. [0057] Each heavy chain of an antibody is comprised of a heavy chain variable region (VH) and a heavy chain constant region. The heavy chain constant region of most IgG isotypes (subclasses) is comprised of three domains, CHI, C H2 and C H3, some IgG isotypes, like IgM or IgE comprise a fourth constant region domain, CH4 Each light chain is comprised of a light chain variable region (VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system and the first component (Clq) of the classical complement system.

[0058] The VH and VL regions of an antibody can be further subdivided into regions of hypervariability, also termed complementarity determining regions (CDRs), which are interspersed with the more conserved framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The locations of CDR and FR regions and a numbering system have been defined by, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services, U.S. Government Printing Office (1987 and 1991). [0059] An antibody-based binding protein, as used herein, may represent any protein that contains at least one antibody-derived VH, VL, or CH immunoglobulin domain in the context of other non-immunoglobulin, or non-antibody derived components. The antibody -based binding proteins of the invention include, but are not limited to (i) F_c-fusion proteins of binding proteins, including receptors or receptor components with all or parts of the immunoglobulin CH domains, (ii) binding proteins, in which VH and or VL domains are coupled to alternative molecular scaffolds, or (iii) molecules, in which immunoglobulin VH, and/or VL, and/or CH domains are combined and/or assembled in a fashion not normally found in naturally occurring antibodies or antibody fragments.

[0060] “Binding affinity” is generally expressed in terms of equilibrium association or dissociation constants (KA or KD, respectively), which are in turn reciprocal ratios of dissociation and association rate constants (k_Off and k_on, respectively). Thus, equivalent affinities may correspond to different rate constants, so long as the ratio of the rate constants remains the same. The binding affinity of an antibody is usually be expressed as the KD of a monovalent fragment (e.g. a F_ab fragment) of the antibody, with KD values in the single-digit nanomolar range or below (subnanomolar or picomolar) being considered as very high and of therapeutic and diagnostic relevance.

[0061] As used herein, the term "binding specificity" refers to the selective affinity of one molecule for another such as the binding of antibodies to antigens (or an epitope or antigenic determinant thereof), receptors to ligands, and enzymes to substrates. Thus, all monoclonal antibodies that bind to a particular antigenic determinant of an entity (e.g., a specific epitope of SARS-CoV-2 spike) are deemed to have the same binding specificity for that entity.

[0062] A "conservative substitution" with respect to proteins or polypeptides refers to replacement of one amino acid with another amino acid having a similar side chain.

Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Methods of identifying nucleotide and amino acid conservative substitutions which do not eliminate protein activity are well-known in the art (see, e.g., Brummell et ah, Biochem. 32: 1180-1 187 (1993); Kobayashi et ah, Protein Eng. 12(10):879-884 (1999); and Burks et al, Proc. Natl. Acad. Sci. USA 94:.412-417 (1997)).

[0063] The term "conservatively modified variant" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0064] For polypeptide sequences, “conservatively modified variants” refer to a variant which has conservative amino acid substitutions, amino acid residues replaced with other amino acid residue having a side chain with a similar charge. Families of amino acid residues having side chains with similar charges have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), betabranched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0065] The term “contacting” has its normal meaning and refers to combining two or more agents (e.g., polypeptides or chemical compounds), combining agents and cells or biological samples, or combining two populations of different cells. Contacting can occur in vitro, e.g., mixing an antibody and a biological sample, or mixing a population of antibodies with a population of cells in a test tube or growth medium. Contacting can also occur in a cell or in situ, e.g., contacting two polypeptides in a cell by co-expression in the cell of recombinant polynucleotides encoding the two polypeptides, or in a cell lysate. Contacting can also occur in vivo inside a subject, e.g., by administering an agent to a subject for delivery the agent to a target cell.

[0066] A “humanized antibody” is an antibody or antibody fragment, antigen-binding fragment, or antibody-based binding protein comprising antibody VH or VL domains with a homology to human VH or VL antibody framework sequences having a T20 score of greater than 80, as defined by defined by Gao et al. (2013) BMC Biotechnol. 13, pp. 55.

[0067] Human coronaviruses (sarbecoviruses) as used herein refer to viruses in the Orthocoronavirinae subfamily of the Coronaviridae virus family. Seven sarbecoviruses have been so far identified, namely sarbecovirus-229E, sarbecovirus-OC43, sarbecovirus- NL63, sarbecovirus-HKUl, severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV) and the novel coronavirus SARS-CoV-2 (aka “2019-nCoV”). Unlike the highly pathogenic SARS-CoV, MERS-CoV, and SARS-CoV-2, the four so-called endemic (or “common”) sarbecoviruses generally cause mild upper-respiratory tract illness and contribute to 15%— 30% of cases of common colds in human adults, although severe and life-threatening lower respiratory tract infections can sometimes occur in infants, elderly people, or immunocompromised patients.

[0068] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Two sequences are "substantially identical" if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length. [0069] Methods of alignment of sequences for comparison are well known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c, 1970; by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; by the search for similarity method of Pearson and Lipman, Proc. Nat’l. Acad. Sci. USA 85:2444, 1988; by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI); or by manual alignment and visual inspection (see, e.g., Brent et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003)). Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.

[0070] The term "subject" refers to human and non-human animals (especially nonhuman mammals). The term "subject" is used herein, for example, in connection with therapeutic and diagnostic methods, to refer to human or animal subjects. Animal subjects include, but are not limited to, animal models, such as, mammalian models of conditions or disorders associated with coronavirus infections. Other specific examples of non-human subjects include, e.g., cows, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys.

[0071] The terms "treat," "treating," "treatment," and "therapeutically effective" used herein do not necessarily imply 100% or complete treatment. Rather, there are varying degrees of treatment recognized by one of ordinary skill in the art as having a potential benefit or therapeutic effect. In this respect, the inventive method can provide any amount of any level of treatment. Furthermore, the treatment provided by the inventive method can include the treatment of one or more conditions or symptoms of the disease being treated. [0072] A "vector" is a replicon, such as plasmid, phage or cosmid, to which another polynucleotide segment may be attached so as to bring about the replication of the attached segment. Vectors capable of directing the expression of genes encoding for one or more polypeptides are referred to as "expression vectors".

[0073] The invention provides novel broadly neutralizing antibodies against SARS like viruses. In one aspect, the antibodies or antigen-binding fragments are capable of neutralizing a SARS-like virus by specifically binding to an epitope on the viral spike protein. In some embodiments, the antibodies or antigen-binding fragments thereof of the invention are capable of neutralizing one or more human coronaviruses. In some embodiments, the antibodies or antigen-binding fragments thereof specifically recognizes one of the conserved RBD epitopes of SARS-CoV-2 as described herein in Example 4, Fig.

4 and Fig. 11. In some embodiments, the antibodies or antigen-binding fragments of the invention are derived from one of the exemplified antibodies described in the Examples below (e.g., Table 1). Typically, they have identical or substantially identical heavy chain and light chain CDR sequences as that of one of the exemplified antibodies. Defined alternatively, they have the same binding specificity as that of one of the exemplified antibodies. In some embodiments, the antibodies or antigen-binding fragments have heavy chain and light CDR sequences that are respectively identical to the heavy chain and light chain CDR sequences of one of the antibodies listed in Table 1. In some embodiments, the antibodies or antigen-binding fragments thereof contain one or more amino acid substitutions relative to the heavy chain sequence and the light chain sequence shown in Table 1. In various embodiments, the substitutions can be located either in the framework region or in the CDRs of the exemplified antibody sequences shown in Table 1.

[0074] Antibodies of the invention include intact antibodies (e.g., IgGl antibodies exemplified herein), antibody fragments or antigen-binding fragments, antibody-based binding proteins, which contain the antigen-binding portions of an intact antibody that retain capacity to bind to SARS-CoV-2 spike protein and cross-react with the spike protein of one or more of the other Sarbecoviruses (e.g., SARS-CoV spike). Examples of such antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab’)2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an intact antibody; (v) disulfide stabilized Fvs (dsFvs) which have an interchain disulfide bond engineered between structurally conserved framework regions; (vi) a single domain antibody (dAb) which consists of a VH or VL domain (see, e.g., Ward et al., Nature 341 :544-546, 1989); and (vii) an isolated complementarity determining region (CDR) as a linear or cyclic peptide. Examples of antibody -based binding proteins are polypeptides in which the binding domains of the antibodies are combined with other polypeptides or polypeptide domains, e.g. alternative molecular scaffolds, Fc-regions, other functional or binding domains of other polypeptides or antibodies resulting in molecules with addition binding properties, e.g. bi- or multispecific proteins or antibodies. Such polypeptides can create an arrangement of binding or functional domains normally not found in naturally occurring antibodies or antibody fragments.

[0075] Antibodies of the invention also encompass “antibody fragments” (also termed “antigen-binding fragments” herein) that contain portions of an intact IgG antibody (e.g., the variant regions) responsible for target antigen recognition and binding. One example of such antibody fragments is single chain antibodies. The term "single chain antibody" refers to a polypeptide comprising a VH domain and a VL domain in polypeptide linkage, generally linked via a spacer peptide, and which may comprise additional domains or amino acid sequences at the amino- and/or carboxyl-termini. For example, a single-chain antibody may comprise a tether segment for linking to the encoding polynucleotide. As an example, a single chain variable region fragment (scFv) is a single-chain antibody. Compared to the VL and VH domains of the Fv fragment which are coded for by separate genes, a scFv has the two domains joined (e.g., via recombinant methods) by a synthetic linker. This enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules.

[0076] Antibodies of the present invention also encompass single domain antigenbinding units, which have a camelid scaffold. Animals in the camelid family include camels, llamas, and alpacas. Cam elids produce functional antibodies devoid of light chains. The heavy chain variable (VH) domain folds autonomously and functions independently as an antigen-binding unit. Its binding surface involves only three CDRs as compared to the six CDRs in classical antigen-binding molecules (Fabs) or single chain variable fragments (scFvs). Camelid antibodies are capable of attaining binding affinities comparable to those of conventional antibodies.

[0077] The various antibodies, antibody-based binding proteins, and antibody fragments thereof described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies, or identified using phage display libraries. Methods for generating these antibodies, antibodybased binding proteins, and antibody fragments thereof are all well known in the art. For example, single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990; and U.S. Pat. No. 4,946,778). In particular, scFv antibodies can be obtained using methods described in, e.g., Bird et al., Science 242:423-426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988. Fv antibody fragments can be generated as described in Skerra and Pliickthun, Science 240: 1038-41, 1988. Disulfide-stabilized Fv fragments (dsFvs) can be made using methods described in, e.g., Reiter et al., Int. J. Cancer 67: 113-23, 1996. Similarly, single domain antibodies (dAbs) can be produced by a variety of methods described in, e.g., Ward et al., Nature 341 :544-546, 1989; and Cai and Garen, Proc. Natl. Acad. Sci. USA 93:6280-85, 1996. Camelid single domain antibodies can be produced using methods well known in the art, e.g., Dumoulin et al., Nat. Struct. Biol. 11 :500-515, 2002; Ghahroudi et al., FEBS Letters 414:521-526, 1997; and Bond et al., J. Mol. Biol. 332:643-55, 2003. Other types of antigen-binding fragments (e.g., Fab, F(ab’)2 or Fd fragments) can also be readily produced with routinely practiced immunology methods. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998.

[0078] In some embodiments, an antibody or antigen-binding fragment of the invention can be further conjugated to a second moiety, which includes, e.g., a polypeptide and a small organic molecule. In some embodiments, the second moiety is a synthetic molecule such as a marker or detectable moiety (or label). Recombinant engineering and incorporated selenocysteine (e.g., as described in U.S. Patent 8,916,159) can be used to conjugate a synthetic molecule. Other methods of conjugation can include covalent coupling to native or engineered lysine side-chain amines or cysteine side-chain thiols. See, e.g., Wu et al., Nat. Biotechnol, 23: 1 137-1 146 (2005).

[0079] The antibodies or antigen-binding fragments of the invention can be generated in accordance with routinely practiced immunology methods. Some of such methods are exemplified herein in the Examples. General methods for preparation of monoclonal or polyclonal antibodies are well known in the art. See, e.g., Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1998; Kohler & Milstein, Nature 256:495-497, 1975; Kozbor et al., Immunology Today 4:72, 1983; and Cole et al., pp. 77-96 in Monoclonal Antibodies and Cancer Therapy, 1985. [0080] The invention provides substantially purified polynucleotides (DNA or RNA) that are identical or complementary to sequences encoding polypeptides comprising segments or domains of the antibody, antibody-based binding protein or antibody fragment thereof chains described herein. In some embodiments, the polynucleotides of the invention encode the heavy chain or light chain sequences of broadly neutralizing antibodies that are derived from one of the exemplified antibodies, e.g., an antibody derived from the antibody chains in Table 1. When expressed from appropriate expression vectors, polypeptides encoded by these polynucleotides are capable of exhibiting coronavirus broadly neutralizing capacity. Also provided in the invention are polynucleotides which encode at least one CDR region and usually all three CDR regions from the heavy or light chain of the antibodies described herein. Some other polynucleotides encode all or substantially all of the variable region sequence of the heavy chain and/or the light chain of the exemplified antibodies.

[0081] The polynucleotides of the invention can encode only the variable region sequences of the exemplified antibodies. They can also encode both a variable region and a constant region of the antibody. Some of polynucleotide sequences of the invention nucleic acids encode a mature heavy chain variable region sequence that is substantially identical (e.g., at least 80%, 90%, 95% or 99%) to the mature heavy chain variable region sequence shown in Table 1. Some other polynucleotide sequences encode a mature light chain variable region sequence that is substantially identical (e.g., at least 80%, 90%, 95% or 99%) to the mature light chain variable region sequence shown in Table 1. Some of the polynucleotide sequences encode a polypeptide that comprises variable regions of the heavy chain or the light chain of one of the exemplified antibodies. Some other polynucleotides encode two polypeptide segments that respectively are substantially identical to the variable regions of the heavy chain or the light chain of one of the exemplified antibodies.

[0082] The polynucleotide sequences can be produced by de novo solid-phase DNA synthesis or by PCR mutagenesis of an existing sequence (e.g., sequences as described in the Examples below) encoding an exemplified functional antibody. Direct chemical synthesis of nucleic acids can be accomplished by methods known in the art, such as the phosphotriester method of Narang et al., Meth. Enzymol. 68:90, 1979; the phosphodiester method of Brown et al., Meth. Enzymol. 68: 109, 1979; the diethylphosphoramidite method of Beaucage et al., Tetra. Lett., 22: 1859, 1981; and the solid support method of U.S. Patent No. 4,458,066. Introducing mutations to a polynucleotide sequence by PCR can be performed as described in, e.g., PCR Technology: Principles and Applications for DN A Amplification, H.A. Erlich (Ed.), Freeman Press, NY, NY, 1992; PCR Protocols: A Guide to Methods and Applications, Innis et al. (Ed.), Academic Press, San Diego, CA, 1990; Mattila et al., Nucleic Acids Res. 19:967, 1991; and Eckert et al., PCR Methods and Applications 1 : 17, 1991.

[0083] Also provided in the invention are expression vectors and host cells for producing the functional antibodies described herein. Specific examples of plasmid and transposon based vectors for expressing the antibodies are described in the Examples below. Various other expression vectors can also be employed to express the polynucleotides encoding the functional antibody chains or binding fragments. Both viral -based and nonviral expression vectors can be used to produce the antibodies in a mammalian host cell. Nonviral vectors and systems include plasmids, episomal vectors, typically with an expression cassette for expressing a protein or RNA, and human artificial chromosomes (see, e.g., Harrington et al., Nat. Genet. 15:345, 1997). For example, nonviral vectors useful for expression of the antibody polynucleotides and polypeptides in mammalian (e.g., human) cells include pCEP4, pREP4, pThioHis A, B & C, pcDNA3.1/His, pEBVHis A, B & C (Invitrogen, San Diego, CA), MPSV vectors, and numerous other vectors known in the art for expressing other proteins. Other useful nonviral vectors include vectors that comprise expression cassettes that can be mobilized with Sleeping Beauty, PiggyBack and other transposon systems. Useful viral vectors include vectors based on lentiviruses or other retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, vectors based on SV40, papilloma virus, HBP Epstein Barr virus, vaccinia virus vectors and Semliki Forest virus (SFV). See, Brent et al., supra; Smith, Annu. Rev. Microbiol. 49:807, 1995; and Rosenfeld et al., Cell 68: 143, 1992.

[0084] The choice of expression vector depends on the intended host cells in which the vector is to be expressed. Typically, the expression vectors contain a promoter and other regulatory sequences (e.g., enhancers) that are operably linked to the polynucleotides encoding a functional antibody chain or fragment. In some embodiments, an inducible promoter is employed to prevent expression of inserted sequences except under inducing conditions. Inducible promoters include, e.g., arabinose, lacZ, metallothionein promoter or a heat shock promoter. Cultures of transformed organisms can be expanded under noninducing conditions without biasing the population for coding sequences whose expression products are better tolerated by the host cells. In addition to promoters, other regulatory elements may also be required or desired for efficient expression of a functional antibody chain or fragment. These elements typically include an ATG initiation codon and adjacent ribosome binding site (Kozak consensus sequence) or other sequences. In addition, the efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (see, e.g., Scharf et al., Results Probl. Cell Differ. 20: 125, 1994; and Bittner et al., Meth. Enzymol., 153:516, 1987). For example, the SV40 enhancer or CMV enhancer may be used to increase expression in mammalian host cells.

[0085] The expression vectors may also provide a secretion signal sequence position to form a fusion protein with polypeptides encoded by inserted functional antibody sequences. More often, the inserted functional antibody sequences are linked to a signal sequences before inclusion in the vector. Vectors to be used to receive sequences encoding the functional antibody light and heavy chain variable domains sometimes also encode constant regions or parts thereof. Such vectors allow expression of the variable regions as fusion proteins with the constant regions thereby leading to production of intact antibodies or fragments thereof. Typically, such constant regions are human, and preferably of human IgGl antibodies.

[0086] The host cells for harboring and expressing the functional antibody chains can be either prokaryotic or eukaryotic. In some preferred embodiments, mammalian host cells are used to express and to produce the antibody polypeptides of the present invention. For example, they can be either a hybridoma cell line expressing endogenous immunoglobulin genes or a mammalian cell line harboring an exogenous expression vector. These include any normal mortal or normal or abnormal immortal animal or human cell. In addition to the cell lines exemplified herein, a number of other suitable host cell lines capable of secreting intact immunoglobulins are also known in the art. These include, e.g., the CHO cell lines, various HEK 293 cell lines, various Cos cell lines, HeLa cells, myeloma cell lines, transformed B-cells and hybridomas. The use of mammalian tissue cell culture to express polypeptides is discussed generally in, e.g., Winnacker, From Genes to Clones, VCH Publishers, N.Y., N.Y., 1987. Expression vectors for mammalian host cells can include expression control sequences, such as an origin of replication, a promoter, and an enhancer, and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. These expression vectors usually contain promoters derived from mammalian genes or from mammalian viruses. Suitable promoters may be constitutive, cell type-specific, stage-specific, and/or modulatable or regulatable. Useful promoters include, but are not limited to, EFla and human UbC promoters exemplified herein, the metallothionein promoter, the constitutive adenovirus major late promoter, the dexamethasone-inducible MMTV promoter, the SV40 promoter, the MRP pol III promoter, the constitutive MPSV promoter, the tetracycline-inducible CMV promoter (such as the human immediate-early CMV promoter), the constitutive CMV promoter, and promoter-enhancer combinations known in the art.

[0087] Methods for introducing expression vectors containing the polynucleotide sequences of interest vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation may be used for other cellular hosts (see generally Sambrook et al., supra). Other methods include, e.g., electroporation, calcium phosphate treatment, liposome-mediated transformation, injection and microinjection, ballistic methods, virosomes, immunoliposomes, polycationmucleic acid conjugates, naked DNA, artificial virions, fusion to the herpes virus structural protein VP22 (Elliot and O'Hare, Cell 88:223, 1997), agent-enhanced uptake of DNA, and ex vivo transduction. For long-term, high-yield production of recombinant proteins, stable expression will often be desired. For example, cell lines which stably express the antibody chains or binding fragments can be prepared using expression vectors of the invention which contain viral origins of replication or endogenous expression elements and a selectable marker gene. Following introduction of the vector, cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media. The purpose of the selectable marker is to confer resistance to selection, and its presence allows growth of cells which successfully express the introduced sequences in selective media. Resistant, stably transfected cells can be proliferated using tissue culture techniques appropriate for the cell type.

[0088] The invention further provides eukaryotic or non-eukaryotic cells (e.g., T lymphocytes) that have been recombinantly engineered to produce the antibodies, antibodybased binding proteins or antibody fragments thereof of the invention. The eukaryotic or non-eukaryotic cells can be used as an expression system to produce the antibody of the invention. In some embodiments, the invention provides coronavirus spike targeting immune cells that are engineered to recombinantly express a broadly neutralizing antibody of the invention. For example, the invention provides a T cell engineered to express an antibody of the invention (e.g., an scFv, scFv-Fc, or (scFv)2), which is linked to a synthetic molecule containing one or more of the following domains: a spacer or hinge region (e.g., a CD28 sequence or a IgG4 hinge-Fc sequence), a transmembrane region (e.g., a transmembrane canonical domain), and an intracellular T-cell receptor (TCR) signaling domain, thereby forming a chimeric antigen receptor (CAR) or T-body. Intracellular TCR signaling domains that can be included in a CAR (or T-body) include, but are not limited to, CD3(^, FcR-y, and Syk-PT signaling domains as well as the CD28, 4- IBB, and CD 134 co-signaling domains. Methods for constructing T-cells expressing a CAR (or T-body) are known in the art. See, e.g., Marcu-Malina et al., Expert Opinion on Biological Therapy, Vol. 9, No. 5 (posted online on April 16, 2009).

[0089] The broadly neutralizing antibodies or antigen-binding fragments thereof disclosed herein can be used in various therapeutic and diagnostic applications. For example, they can be used alone or in a combination therapy in the prophylactic or therapeutic treatment of coronavirus infections (e.g., SARS-CoV-2 infection). In some embodiments, the invention provides methods of using the broadly neutralizing antibodies or fragments thereof to treat patients having infection by one or more coronaviruses (e.g., SARS-CoV-2 and SARS-CoV) or patients having other diseases or conditions associated with coronavirus infections. In some embodiments, the antibodies or antigen-binding fragments of the invention can be used to prevent infections by one or more coronaviruses, or to reduce or manage coronavirus-induced symptoms in a subject infected with one or more coronaviruses. In some other embodiments, the invention provides diagnostic methods for detecting coronavirus related infections or the presence of coronavirus in biological samples obtained from human subjects.

[0090] Pharmaceutical compositions containing one or more of the broadly neutralizing antibodies or antigen-binding fragments described herein are encompassed by the invention. In some embodiments, the pharmaceutical compositions are employed in therapeutic methods for treating coronavirus infections. Typically, the subject or patient suitable for treatment is one who has been or is suspected of having been exposed to one or more coronaviruses (e.g., SARS-CoV-2 or SARS-CoV), is infected or suspected of being infected with one or more coronavirus, has a coronavirus related disease, has a symptom of a coronavirus related disease, or has a predisposition toward contracting a coronavirus related disease. For example, the subject to be treated can be one who has been diagnosed of SARS- CoV-2 infection and/or possess symptoms associated with infections by one or more Sarbecoviruses. The broadly neutralizing antibody or antigen-binding fragment thereof for use in the methods of the invention can a human or humanized antibody. In some embodiments, the broadly neutralizing antibody or antigen-binding fragment thereof comprises a binding domain that binds to the same epitope as, or competitively inhibits binding of, one or more of the antibodies exemplified herein. In addition to the antibodies, pharmaceutical compositions typically also contain a pharmaceutically acceptable, non-toxic, sterile carrier such as physiological saline, non-toxic buffers, preservatives and the like. [0091] Therapeutic methods of the invention typically involve administering to a subject in need of treatment a pharmaceutical composition that contains a therapeutically amount of a broadly neutralizing antibody or antigen-binding fragment described herein (e.g., an antibody shown in Table 1). A therapeutically effective amount refers to an amount sufficient to achieve a therapeutic benefit, e.g., to ameliorate symptoms associated with Sarbecovirus infections. Suitable amount to be administered can be readily determined by one of ordinary skill in the art without undue experimentation given the invention. Factors influencing the mode of administration and the respective amount of a broadly neutralizing sarbecovirus neutralizing antibody or antigen-binding fragment thereof include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Similarly, the amount of a broadly neutralizing sarbecovirus immunotherapeutic to be administered will be dependent upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent. In some embodiments, the therapeutic methods of the invention can be employed in combination with other regimen for treating or controlling sarbecovirus infections. These include, e.g., remdesivir, Bamlanivimab, Casirivimab and Imdevimab cocktail, hydroxychloroquine and chloroquine, interferon P-la, Azithromycin, Tocilizumab and other IL-6 inhibitors, Interferon-y, or intravenous fluids and balancing electrolytes. [0092] Methods of preparing and administering a broadly neutralizing antibody or antigen-binding fragment thereof provided herein, to a subject in need thereof are well known to or can be readily determined by those skilled in the art. The route of administration of a broadly neutralizing sarbecovirus neutralizing antibody or antigen-binding fragment thereof can be, for example, oral, parenteral, by inhalation or topical. The term parenteral as used herein includes, e.g., intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. While all these forms of administration are clearly contemplated as suitable forms, another example of a form for administration would be a solution for injection, in particular for intravenous or intraarterial injection or drip. In some cases a suitable pharmaceutical composition can comprise a buffer (e.g. acetate, phosphate or citrate buffer), a surfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. human albumin), etc. In other methods compatible with the teachings herein, a broadly neutralizing antibody or antigen-binding fragment thereof as provided herein can be delivered directly to a site where the binding molecule can be effective in virus neutralization, e.g., the endosomal region of a coronavirus-infected cell. Preparation of pharmaceutical compositions of the invention and their various routes of administration can be carried out in accordance with methods well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

[0093] The invention also provided methods for using the broadly neutralizing antibodies or related antigen-binding fragments described herein in diagnostic methods for detecting sarbecovirus infections or the presence of sarbecoviruses. Various assays routinely practiced in the art can be employed for performing the diagnostic methods. These include, e.g., competitive and non-competitive assay systems using techniques such as Western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few. Such assays are routine and well known in the art (see, e.g., Ausubel et al., eds, (1994) Current Protocols in Molecular Biology (John Wiley & Sons, Inc., NY) Vol. 1, which is incorporated by reference herein in its entirety). Methods and reagents suitable for determination of binding characteristics of a broadly neutralizing sarbecovirus antibody or antigen- binding fragment thereof are known in the art and/or are commercially available. Equipment and software designed for such kinetic analyses are commercially available (e.g., BIAcore®, BIAevaluation® software, GE Healthcare; KINEXA® Software, Sapidyne Instruments). [0094] The diagnostic methods of the invention typically involve obtaining a biological sample from a subject that has or is suspected of having been infected with a coronavirus spike. Preferably, the subject is a human. In various embodiments, the biological sample suitable for the assays can be blood or any fraction thereof (e.g., serum, plasma, or whole blood), urine, feces, saliva, vomitus, or any combination thereof. Utilizing the novel antibodies disclosed herein, presence of a coronavirus spike or spike derived antigen in the biological sample can be readily determined with any of the various immunoassays described herein, e.g., ELISA.

[0095] The invention further provides kits that contain a broadly neutralizing sarbecovirus immunotherapeutic of the invention for performing the therapeutic or diagnostic applications described herein. Typically, the kits contain two or more components required for performing the therapeutic or diagnostic methods of the invention. Kit components include, but are not limited to, one or more the disclosed antibodies or antibody fragments thereof, appropriate reagents, and/or equipment. In some embodiments, the kits can contain an antibody or antibody fragment thereof of the invention and an immunoassay buffer suitable for detecting sarbecovirus spike proteins (e.g. by ELISA, flow cytometry, magnetic sorting, or FACS). The kit may also contain one or more microtiter plates, standards, assay diluents, wash buffers, adhesive plate covers, magnetic beads, magnets, and/or instructions for carrying out a method of the invention using the kit. The kit scan include an antibody or antigen-binding fragment thereof of the invention bound to a substrate (e.g., a multi-well plate or a chip), which is suitably packaged and useful to detect sarbecovirus spike antigens. In some embodiments, the kits include an antibody or antibody fragment thereof of the invention that is conjugated to a label, such as, a fluorescent label, a biologically active enzyme label, a luminescent label, or a chromophore label. The kits can further include reagents for visualizing the conjugated antibody or antibody fragment thereof, e.g., a substrate for the enzyme. In some embodiments, the kits include an antibody or antibody fragment thereof of the invention that is conjugated to a contrast agent and, optionally, one or more reagents or pieces of equipment useful for imaging the antibody in a subject.

[0096] Generally, the broadly neutralizing antibodies or antibody fragments thereof of the invention in a kit are suitably packaged, e.g., in a vial, pouch, ampoule, and/or any container appropriate for a therapeutic or detection method. Kit components can be provided as concentrates (including lyophilized compositions), which may be further diluted prior to use, or they can be provided at the concentration of use. For use of the antibody of the invention in vivo, single dosages may be provided in sterilized containers having the desired amount and concentration of components.

[0097] In various applications, the broadly neutralizing antibodies of the invention can be employed to produce antibody derivatives such as immunoconjugates. In some embodiments, the antibodies of the invention can be linked to a therapeutic moiety, such as a cytotoxin, a drug or a radioisotope. When conjugated to a cytotoxin, these antibody conjugates are referred to as "immunotoxins." A cytotoxin or cytotoxic agent includes any agent that is detrimental to (e.g., kills) cells. Techniques for conjugating such therapeutic moiety to antibodies are well known in the art. In some embodiments, antibodies of the invention can be conjugated to an appropriate detectable agent to form immunoconjugates for use in diagnostic applications and in vivo imaging. The detectable agents can be any chemical moieties that contain a detectable label, e.g., radioisotopes, enzymes, fluorescent labels and various other antibody tags. In some other embodiments, the broadly neutralizing antibodies of the invention can be further modified to contain additional non-proteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers, e.g., polyethylene glycol (PEG).

EXAMPLES

[0098] The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims.

Example 1. Donors for bnAb isolation

[0099] To identify donors for bnAb isolation, we first screened sera from 3 different groups for SARS-CoV-2 neutralization. The groups were: i) COVID-19 convalescent donors (n = 21); ii) spike-mRNA vaccinated (2X) donors (n = 10) and iii) COVID-19 convalescent donors (n = 15) who had subsequently been mRNA vaccinated (IX) (Fig. 5). Consistent with earlier studies, we observed significantly higher levels of plasma nAbs in donors who were previously infected then vaccinated (“infected-vaccinated”) compared to donors who were only infected or only vaccinated (Fig. la and Fig. 5). To examine the breadth of nAb responses across these 3 groups, we tested sera for neutralization against ACE2 receptorutilizing sarbecoviruses (Pangl7, SARS-CoV-1 and WIV1) and against SARS-CoV-2 VOCs (B.l.1.7 (alpha), B.1.351 (beta), P. l (gamma) and B.1.617. V2 (delta)) and (Fig. Ib-c and Fig. 5). Sera from infected-vaccinated donors showed greater breadth of neutralization and more effective neutralization of VOCs than sera from donors who were only previously infected or only vaccinated. Consistent with previous studies, neutralization efficacy of infected-vaccinated sera against VOCs was similar to that against the ancestral strain of SARS-CoV-2 ¹²>⁴³>⁴⁸>⁴⁹ (Fig i_c) Neutralization of SARS-CoV-1, which is relatively phylogenetically distinct from SARS-CoV-2 compared to other sarbecoviruses (Fig. 5) ^2,5°, was relatively low but was clearly above background for about half of the infected- vaccinated donors (Fig. 5). None of the convalescent-only or vaccinated-only donors in our cohort had sera that could neutralize SARS-CoV-1, as also noted by us earlier ⁵¹. Of note, many of the existing SARS-CoV-2 broadly neutralizing or cross-neutralizing antibodies were isolated from SARS-CoV-1 convalescent donors ²⁷>^{28 52}-⁵⁵ and only more recently from SARS-CoV-2 infected donors ²⁶>²⁹'^{31 56} These bnAbs appear to target more conserved spike epitopes, and as also illustrated by SARS-CoV-1 cross-neutralizing bnAbs elicited in S- protein vaccinated macaques ⁵¹>⁵⁷>⁵⁸. Hence, SARS-CoV-1/2 cross-neutralization appears to be a good indicator of the presence of pan-sarbecovirus activity and possibly greater CoV neutralization breadth ⁵⁹. Accordingly, for isolation of bnAbs in this study, we focused on two SARS-CoV-2 infected-vaccinated donors (CC25 and CC84) with most potent SARS- CoV-1 cross-neutralizing antibody titers.

Example 2. Isolation and characterization of a large panel of sarbecovirus bnAbs [00100] Using SARS-CoV-1 and SARS-CoV-2 S-proteins as baits, we sorted antigenspecific single B cells to isolate 107 mAbs from two COVID-19 recovered donors who had been recently vaccinated with the Modema spike-mRNA vaccine (CC25 (n = 56) and CC84 (n = 51)) (Fig. Id and Fig. 6a) ⁶⁰. Briefly, from the peripheral blood mononuclear cells (PBMCs) of the donors, we sorted CD19⁺CD20⁺ IgG⁺ IgM" B cells that bound to both SARS-CoV-2 and SARS-CoV-1 S-proteins. Flow cytometry profiling of pre-vaccination (post-infection) PMBCs of CC25 and CC84 donors revealed that SARS-CoV-1/2 broadly neutralizing IgG⁺ memory B cells were likely seeded after infection and got recalled upon vaccination (Fig. Id), as also observed in other studies ^43,61. Heavy (HC) and light (LC) chain sequences from 107 S-protein sorted single B cells were recovered and expressed as IgGs (Fig. 7).

[00101] All 107 mAbs exhibited broadly neutralizing binding to SARS-CoV-2 and SARS-CoV-1 S-proteins (Fig. 2a, Fig. 7). Very few of the mAbs showed weak but detectable binding to P-sarbecovirus (MERS-CoV sarbecovirus-HKU-1 and sarbecovirus- OC43) and a-sarbecovirus (sarbecovirus-NL63 and sarbecovirus-229E)-derived S-proteins (Fig. 2a, Fig. 7). To determine the epitope specificities of the mAbs, we tested binding with SARS-CoV-2 SI subunit domains and observed that the vast majority of the mAbs (>80%) displayed RBD-specific binding (Fig. 2a). To determine the cross-reactivity of RBD binding, we investigated 12 diverse RBDs representing all the 4 major sarbecovirus clades; clades la, lb, 2 and 3 ²>²⁸>⁵⁰ (Fig 2b, Fig. 7). The mAbs showed the greatest degree of cross-reactivity with clades la and lb and the least with clade 2. Approximately a third of the mAbs (31%) showed cross-reactivity with all 12 RBDs derived from all 4 sarbecovirus clades (Fig. 2b, Fig. 7).

[00102] Next, we evaluated cross-clade neutralization with mAb supernatants on a panel of clade la (SARS-CoV-1 and WIV-1) and clade lb (SARS-CoV-2 and Pangl7) ACE2- utilizing sarbecoviruses. Two-thirds of mAbs neutralized both SARS-CoV-1 and SARS- CoV-2 and 43% (40 out of 93 mAbs) neutralized all 4 sarbecoviruses in the panel and are categorized as bnAbs in this study (Fig. 2b, Fig. 7). More comprehensive and quantitative cross-neutralization is described with a smaller panel of mAbs below.

[00103] In terms of antibody sequences, of the 107 isolated mAbs, 93 were encoded by unique immunoglobulin germline gene combinations and 11 were expanded lineages (CC25 [n = 6] and CC84 [n = 5]) that had 2 or more clonal members (Fig. 2a, Fig. 7). There was a notable enrichment of IGHV3-30, particularly, and also IGHV1-46 and IGHV1-69 germline gene families for both donors as compared to human baseline germline frequencies (Fig. 2a, c-d, Fig. 8) ^62,63. Interestingly, the mAbs showed modest levels of V-gene nucleotide somatic hypermutation (SHM): for VH, median = 5.0% and for VL, median = 4.0% (Fig. 7). We sought to determine whether the IGHV germline gene usage and/or VH SHM levels were correlated with the extent of neutralization breadth (Fig. 2a, c-d). We observed enrichment of IGHV3-30 in mAbs that bind to clade 2 RBDs and all 12 sarbecovirus RBDs, but otherwise no notable trends (Fig. 2c-d). VH-gene SHM levels did not distinguish broad versus less broad or non-neutralizing mAbs. Overall, we observed that some IGHV genes were enriched in bnAbs, but diverse human immunoglobulin gene combinations were capable of encoding for sarbecovirus bnAbs.

[00104] To further investigate the potential contribution of SHM to broad reactivity to sarbecoviruses, we tested the binding of mAbs to SARS-CoV-2 S-protein and to monomeric RBD by BLI (Table 1). We found no association of SHM with S-protein binding and a weak correlation with binding to RBD. Consistent with this lack of correlation, we did not observe any correlation of SARS-CoV-2 RBD mAb binding with sarbecovirus neutralization breadth or binding breadth (Table 1), although some modest correlation was observed for S-protein binding. These results suggest that critical antibody paratope features for sarbecovirus breadth may be germline-encoded and limited affinity maturation is needed. While recent findings demonstrate that the accumulation of SHM may increase potency and breadth ^13,64, this may not be a requisite feature of sarbecovirus bnAbs. The predominant use of certain germline gene segments in bnAbs suggests that a germline-targeting approach ^38-40 to pan- sarbecovirus vaccines may be rewarding and the relatively low levels of SHM are promising for successful vaccine deployment provided appropriate immunogens can be designed.

[00105] Next, we examined the CDRH3 loop lengths of the isolated Abs and observed a strong enrichment for 20- and 21 -residue long CDRH3s compared to the human baseline reference database (Fig. 2e) ^62,63. These long CDRH3s were found to contain high proportions of two D genes, IGH D2-15 and D3-22, that were notably enriched in bnAbs (Fig. 2g). Notably, 71% (12/17) of mAbs with 20 amino acid CDRH3s utilized germline IGHD2-15 D-gene, and the majority of mAbs bearing 21 amino acid length-CDRH3s utilized either IGHD2-15 or IGHD3-22 germline D-genes (Fig. 2f, Figs. 7 and 8). We noted that D3-22 D-gene was also selected in bnAbs isolated in other studies ²⁶>^{56 65 66}. Therefore, vaccine design strategies will likely need to take these germline features into consideration 38-40

[00106] Altogether, we have isolated the largest set of human sarbecovirus bnAbs to date. The isolated bnAbs, although encoded by diverse immunoglobulin germline gene families, are strongly enriched for certain germline gene features that will inform pan-sarbecovirus vaccine strategies.

Example 3. Further characterization of exemplary bnAbs [00107] We further performed a detailed binding and neutralization characterization of a smaller panel of bnAbs. We selected 30 SARS-CoV-l/SARS-CoV-2 RBD broadly neutralizing mAbs for more detailed characterization (Fig. 3a). Amino acid sequences of the heavy chain (HC) and light chain (LC or KC) of these antibodies are shown in Table 1. Selection was made based on a high degree of broadly neutralizing binding with sarbecovirus clades; the large panel included nAbs that likely had more potent neutralization of SARS-CoV-1 and/or SARS-CoV-2 individually but lacked cross-reactivity (Fig. 2a). To determine sarbecovirus cross-reactivity more extensively, we evaluated 12 soluble monomeric RBDs representing the major sarbecovirus clades, as above in Figure 2. Almost all mAbs bind SARS-CoV-2 and other clade Ib-derived RBDs, with most binding in a nanomolar (nM) to picomolar (pM) KD affinity range (Fig. 3b, Fig. 10). The mAbs that bound most effectively to clade lb RBDs tended to also bind well to clade la and clade 3 RBDs, albeit with somewhat lower affinities, yet still in the nM-pM KD affinity range. Cross-reactivity was least to the clade 2 RBDs, although there was generally some level of reactivity and some mAbs did show high affinity binding. Remarkably, several mAbs showed consistently high affinity binding to RBDs from all 4 sarbecovirus clades.

[00108] Neutralization was investigated only for clade la and lb ACE2 -utilizing viruses, since neutralization assays were not available for clade 2 and 3 viruses, and we found that 22 of 30 mAbs neutralized SARS-CoV-2 with a range of IC50 neutralization titers (IC50 range = 0.05-4.9 pg/mL) (Fig. 3c) and that 28 of 30 mAbs showed neutralization against SARS- CoV-1, including all mAbs that neutralized SARS-CoV-2. Neutralization potency was typically stronger against SARS-CoV-1 than SARS-CoV-2. All mAbs showed neutralization against WIV1, while a majority exhibited cross-neutralization with Pang 17, and to a lesser degree with SHC014. 13 out of 30 mAbs neutralized all 5 ACE2 -utilizing sarbecoviruses tested with a geomean IC50 potency of 0.12 pg/ml. The three most potent SARS-CoV-2 bnAbs, CC25.52, CC25.54 and CC25.3, neutralized all 5 ACE2-utilizing sarbecoviruses with a geomean potencies of 0.03, 0.04 and 0.04 pg/ml, respectively. Although neutralization assays differ, this suggests they are amongst the most potent and broad individual nAbs described to date (compare also control nAbs in Fig. 3c).

[00109] We tested neutralization of SARS-CoV-2 VOCs by select SARS-CoV-2 bnAbs. Consistent with the donor CC25 and CC84 sera neutralization above, the bnAbs were consistently effective against SARS-CoV-2 VOCs tested (Fig. 3d). In comparison, SARS- CoV-2 strain-specific nAb, CC12.1 showed substantial loss of neutralization with VOCs. The results suggest that these bnAbs target more conserved RBD epitopes that are likely more resistant to SARS-CoV-2 escape mutations. Overall, we have identified multiple potent sarbecoviruses bnAbs that exhibit broad reactivity to diverse sarbecovirus lineages.

Table 1. Sequences of 30 characterized SARS-CoV-1 and SARS-CoV-2 bnAbs

CC25.1

HC

QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGWINAY DSNTDYAQKFQGRVTMTTDTSTSTAFMDLRGLRSDDTAVYYCARDLGRYYDNSG YYYRYYYFGMDIWGQGTTVTVSS (SEQ ID NO:1)

LC

QLVLTQPPSASGSPGQSVTISCTGTSSDVGDYDYVSWYQQHPGKAPELIIYEVTKRPS GVPDRFSGSKSGNTASLTVSGLQAEDEADYYCSSYAGRDNLVFGGGTKVTVL (SEQ ID N0:31)

CC25.3

HC

EVQLVQSGAEVKKPGASVKVSCKASGYIFIDYYIHWVRQAPGQGLEWMGWINPHS GGANFAPKFQDRVTMTRDTSISTAYMELSRLRSDDTAVYYCAKDRFRYYYDRSGN YQREPNSWFDPWGQGTLVTVSS (SEQ ID NO:2)

_KC

DIQMTQSPSSLSASVGDRVTITCRASQGIRDDLGWYQQKSGKAPKLLIYAASSLQSG VPSRFSGSGSGTDFTLTINSLQPEDFATYYCLQDYSYPYTFGQGTKVEIK (SEQ ID NO:32)

CC25.4

HC

QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYFLHWVRQAPGQGLEWMGWINPD SGGTNYAQRFQGRVTMTRDTSISTAYMEVSRLRSDDTAVYYCARDNERYQMQNY YHYYGMDVWGQGTTVTVSS (SEQ ID NO:3)

LC QSVLTQPPSASGTPGQRVTISCSGSSSNIGSNTVNWYQQLPGTAPKLLIYSNNQRPSG

VPDRFSGSKSGTSASLANSGLQSEDEADYYCAAWDDSLNGYVVFGGGTKLTVL (SEQ ID NO:33)

CC25.11

HC

EVQLVQSGAEVKKPGSSVKVSCKASEVTFNSYGISWVRQAPGQGLEWMGRLIPIFD

TTDYAQKFQDRVTISADTSTNTTYMELSSLKSEDTAVYYCAREVVVIGATYLDSWG QGTLVTVSS (SEQ ID NO:4)

_KC

EIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTISSLETEDFAVYYCQQRSSWPPGFTFGPGTKLEIK (SEQ ID NO:34)

CC25.13

HC

QVQLVESGGGLVKPGGSLRLSCAASGFSLRDYFMSWIRQAPGKGLEWVGYISRRSS

DTNYADSLKGRFTISRDNAKNSLYLQMNSLRVEDTAVYYCARQYYDILTGYSTGEY

WFDPWGQGTLVTVSS (SEQ ID NO: 5)

LC

QSVLTQPPSVSGAPGQRVTISCTGTSSNIGAGYDVNWYQQLLGTAPKLLIYGNKNRP SGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGSMVFGGGTKVTVL (SEQ ID NO: 35)

CC25.17

HC

QVQLVESGGGVVQPGRSLKLSCAVSGFTFRTYGMHWVRQAPGKGLEWVAVISYD

GSDTHYADSVKGRFTISRDSSKNTLYLQMNSLRPEDTAVYYCTKMGGGPYCGGGN CYSGYLDYWGQGTLVTISS (SEQ ID NO: 6)

_KC

DIVMTQSPSTLSASVGDRVTITCRATQSIGSWLAWYQQRPGKVPKLLIYEASNLESG VPSRFSGSGSGTEFTLTISSLHPDDFATYYCQQYESYSTFGGGTKLEIK (SEQ ID CC25.21

HC

EVQLVESGGGVVQPGRSLRLSCAASGFTFRHYGMHWVRQAPGKGLEWVAVISYDG

GHKYYGNSVKGRLTISRDDSKNTLYLQINSLRAEDTAVYFCVKQGGPYCSGGNCYS GYFDYWGQGTLVTVSS (SEQ ID NO:7)

_KC

DVVMTQSPSSLSASVGDRVTITCQASQDISNHLNWYQQKPGKAPKLLIYDASNLET GVPPRFSGSGSVTDFTFIISSLQPEDFATYYCHQYHNVPLTFGGGTKLEIK (SEQ ID NO:37)

CC25.22

HC

QVQLVESGGGVVRPGRSLRLSCAASGFTFSHYGMHWVRQAPGKGLEWVAVILYDG

SNKLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKADSPYCSAGDCY SSDFDYWGQGTLVTVSS (SEQ ID NO: 8)

_KC

DVVMTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKAGQAPRLLIYGASSRA TGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGPGTKVEIK (SEQ ID NO:38)

CC25.36

HC

EVQLVESGGGL VQPGGSLRLSC AASGFTFS SRNMNWVRQAPGKGLEWVS YIS S SGSI YYADSVKGRFSISRDNVKNSLYLQMNSLRDEDTAVYYCARGVVGYYDMLTGPPDN WLDAWGQGTLVTVSS (SEQ ID NO: 9)

LC

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRP SGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDSSLSGWVFGGGTKLTVL (SEQ ID NO: 39) CC25.38

HC

QVQL VESGGGL VQPGGSLRL SC AASGFIF S S YNMNWVRQ APGKGLEW VS YIS SD S S TKYYADSMKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARVVGQRSYYYYGM DVWGQGTTVTVSS (SEQ ID NO: 10)

_KC

DIVMTQSPSSLSASVGDRVTITCRASQSIHNYLNWYQQKPGKAPKLLIYAASSLQSE VPSRFSGSESGTDFTLTISSLQPEDFATYYCQQSYSTPPTFGQGTKLEIK (SEQ ID NO:40)

CC25.42

HC

QVQL VESGGGL VQPGGSLRLSCAASGFIFSNYWMSWVRQAPGKGLEWVANIKPVG NEKYYVDSVKGRFSISRDNARNSLYLQMNSLRVEDTAVYYCAREGLGYTGDDNFD YWGQGTLVTVSS (SEQ ID NO: 11)

_KC

AIRMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSN RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGSHWQGQITFGQGTRLEIK (SEQ ID NO:41)

CC25.43

HC

EVQLVESGGGLVQPGGSLKLSCSASGFTLSDSAMHWVRQASGKGLEWVGRISSNV

NNDATVYAASLKGRFTISRDESKNMAYLQMNSLKNEDTAVYYCTVVPVLEYYQYG

MDVWGQGTTVTISS (SEQ ID NO: 12)

_KC

DIQLTQSPSTLSASVGDRVIITCRASQSISTWLAWYQQRPGQAPKLLIYKASILQSGVP

PRFSGSGSGTDFTLTISRLQPDDFATYYCQQYDSDSQPLTFGGGTKLEIK (SEQ ID NO:42)

CC25.44

HC EVQLVESGGGLVQPGRSLRLSCAASGFTFENYAMHWVRQAPGKGLEWVAGITWNS

GNREYADSVKGRFTISRDNAKKSLYLQMSSLRAEDTALYFCAKDPNSGILIYGLDV

WGQGTTVTVSS (SEQ ID NO: 13)

_KC

DIVMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYQVSN

RDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMEGTHWPPTFGQGTKLEIK (SEQ ID NO:43)

CC25.48

HC

QVQLQESGPGLVKPSETLSLTCTVSGASIGRSNNYWGWIRQPPGKGLEWIGTIFYSG

NTYYNPSLRSRLSISVDTSKNQFSLRLSSMTAADTAVYYCARQDFYYDSSGYYFREY

HWFDPWGQGTLVTVSS (SEQ ID NO: 14)

_KC

DIVMTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATG IPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPTFGQGTRLEIK (SEQ ID NO:44)

CC25.52

HC

QVQLQESGPGLVKPSETVSLTCSVSGGSLSSYHWSWIRQPPGKGLEWIGHIYISASTN

YNPSNYNPSLRGRVVISVDRSKNQFSLKLTSVTAADTAVYYCASLSYCGADCYTEF

DYWGQGTLLSVSS (SEQ ID NO: 15)

LC

QSALTQPPSASGTPGQRVTISCSGSSSNIGNNFVFWYQQFPGTAPKLLIYRSNQRPSG VPDRFSGSKSGTSASLAISGLRSEDETDYYCAAWDDSLSAMVFGGGTKLTVL (SEQ ID NO:45)

CC25.53

HC

EVQLQESGPGLVKPSETLSLTCTVSGGSVNSRNFYWSWIRQPPGKGLVWIGYISNSG

STKYNPSLKSRVTMTVDTSKNQFSLRLNSVTAADTAIYYCAREVFYYDRSGYFSSD AFDIWGQGTMVTVSS (SEQ ID NO: 16)

_KC

DIVMTQFPDSLAVSLGERATINCKSSQSVLHSSNNKNYLAWYQQRPGQCPKLLIYW ASTRESGVPDRFSGSGSGTDFTLTVSSLQAEDVALYYCQQYYRTPYTFGQGTKVEIK (SEQ ID NO:46)

CC25.54

HC

EVQLQESGPGLVKPSETLSLTCTVSGGSVSSHNFHWSWIRQPPGKGLELIGEIYYSGS

TIHSPSLKSRTTNYNPSLKSRVTMSVDTSKNQVSLKLGSVTAADTAVYYCARELYY YDRSGYYVHDGFDIWGPGTTVTVSS (SEQ ID NO: 17)

_KC

DIQMTQSPSTLSASVGDSVSITCRASQSISSWLAWYQQKPGTAPKLLIYKASSLESGV PSRFSGRGSGTEFTLTISSLQPDDFATYYCQQYNTYPWTFGQGTKVEIK (SEQ ID NO:47)

CC25.55

HC

EVQLVQSGAEVKRPGDSLRISCKGSGYIFTNYWINWVRQMPGKGLEWMGRIDPSDS

YTNYSPSFEGLVIISVDKSINTAYLQWSSLKASDTAMYYCTRQSPVSGYYPIPTYYLD

YWGQGTLVTVSS (SEQ ID NO: 18)

LC

QPVLTQPPSLSGAPGQRVSIACTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNTNRP SGVPDRFSVSKSGTSASLAITGLQAEDEADYYCQSYDTSLSAWVFGGGTKLTVL (SEQ ID NO:48)

CC25.56

HC

EVQLVQSGAEVKKPGESLKISCKGSGYTFTRHWIGWVRQMPGKGLEWMGVIYPGD

SDTRYSPSFQGQVTVSADKSISTAYLQWSSLKASDTAMYFCARGGIAVASGAFDIW GQGTMVTVSS (SEQ ID NO: 19)

LC QSALTQPPSVSVAPGKTARITCGGNNIGSKSVHWYQQKAGQAPVVVIYYPSDRPSGI

PERFSGSNSENTATLTISGVEAGDEADYYCQLWDTNSDHWVFGGGTKLTVL (SEQ ID NO:49)

CC84.2

HC

QVQLVQSGAEVKKPGASVKVSCKASGYTFTRYAMHWVRQAPGQSLECMGWINAG

NGNTKYSKNFQGRVTITRDTSANTVYMELSSLRSEDTAVYYCARDLYYYDSSGYQ

HINYQLDYWGQGTLVTVSS (SEQ ID NO:20)

LC

QSVLTQPPSVSGAPGQRVAISCTGSSPNIGAGYDVHWYQQLPGTAPKLVIYGNTNRP SGVPDRFSASKSGTSASLAITGLQTEDEADYYCQSYDINLSGSPVIFGGGTKLTVL (SEQ ID NO:50)

CC84.4

HC

EVQLVQSGSEVKKPGASVKISCKASGYTFSSHYMHWVRQAPGQGLEWMGIINPDA

GSTTYAPNFQGRLTMTSDTSTTTVYLEMSSLRSEDTAMYYCTRDQGFVPNRDGIDF

WGQGTLVTVSS (SEQ ID NO:21)

_KC

DIQMTQSPATLSVSPGERATLSCRASQSISSNLAWYQQKPGQAPRLLIFGASTRATGI PARFSGSGSGTEFTLTISGLQSEDFAVYYCLQYNKWPPYSFGQGTKVEIK (SEQ ID NO:51)

CC84.5

HC

EVQLVQSGAEVKRPGASVKISCKTSGYTFTTFYIHWVRQVPGQGLQWMGIINPSGG

QTTYEQPFQGRITMTRGTSTSTVYMELSSLRSEDTAIYYCARDSTAFDFRSGYSLQEP

TWFAPWGQGTLVTVSS (SEQ ID NO:22)

_KC

AIRMTQSPATLSVSPGERATLSCRASQSLSSNLAWYQQKPGQAPRLLIYGASTRATGI

PARFSGSGSGTEFTLTISSLQSEDFAVYYCQQYNYWPGTFGQGTKVDIK (SEQ ID NO: 52)

CC84.9

HC

QVQLVQSGAEVKKPGSSVKVSCKSSGDFLSNYAISWLRQAPGQGLEWVGGIIPIFGS

ENYAQKFQGRVTITADESTSTAYVELTSLRSEDTAVYYCARFEGATYYFDSRGYPDS SGNSPKMDVWGQGTTVTISS (SEQ ID NO:23)

_KC

DIQMTQSPLSLPVTLGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQSPRRLIYKVSN

RDSGVPDRFSGSGSETDFTLKISRVEAEDVGVYYCMQGTNWPPTFGPGTKLEIK (SEQ ID NO:53)

CC84.10

HC

EVQLVQSGAEMKKPGSSVKVSCKSSGGTFNNYPISWVRQAPGQGLEWMGGIIPLFS

TTNYAQNFQGRVTITADESTGTAYMELTGLRSEDTAVYFCARDGGPFFYDRNGHPR

KENYFDPWGQGTLVTVSS (SEQ ID NO:24)

LC

QSVLTQPPSVSGAPGQRLTISCTGVSSNIGADSDVHWYQQVPGAAPKLLIYGNSNRP SGVPDRFSGSKSGTSASLTITGLQAEDEADYYCQSYDSSLSSVIFGGGTKLTVL (SEQ ID NO:58)

CC84.12

HC

EVQLVQSGAEVKKPGS S VKVSCKASGGTF S SD AISWVRQAPGQGLEWMGGLIPISG

KADYAQKFQGRVTIDADESTKTAYMELSSLRPEDTAVYYCARERSDSHQLYYYYG

MDVWGQGTTVTVSS (SEQ ID NO:25)

_KC

EIVLTQSPATLSLSPGERATLSCRASQSVSTYLAWYQQKPGQAPRLLIYDASNRATGI

PARFSGSGFGTDFTLTISSLEPEDFAIYYCQQRSSPAFGQGTKVDIK (SEQ ID NO:55)

CC84.13 HC

QVQLVQSGAEVKKPGSSVKVSCKGSGDTFTSYAISWVRQAPGQGLEWMGVIIPLFG

TANYAQRFQGRVTITADEFTSTAYMELRSLTSEDTAVYYCTRDGSSGRYPHNWFDP

WGQGTLVTVSS (SEQ ID NO:26)

LC

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQQLPGTVPKLLIYANTNRP SGVPDRFSGSKSGTSASLAITGLQAEDEADYYCQSYDTSLSGVFGTGTKVTVL (SEQ ID NO:56)

CC84.21

HC

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRLAPGQGLEWMGGIILILG

TANYAQKFQGRVTITADKSPSTAYLELSSLRSEDTAVYYCARSTFYYDRSGNPRPDD

VFDIWGQGTMVTISS (SEQ ID NO:27)

LC

QSVLTQPPSVSGAPGQRVTISCTGSSSNIGAGYDVHWYQHLPGTAPKLLIFGNSNRPS GVPERFSGSKSGTSASLAITGLQAEDEAQYYCQSYDSSLSGWVFGGGTKLTVL (SEQ ID NO:57)

CC84.24

HC

EVQLVESGGGLVKPGGSLRLSCAASGFTFSPYSMNWVRQAPGKGLEWVSSIRSSGN

YISYADSVKGRFTISRDNAKNSLYLQMNSLRAEDMAVYYCARAGRDYYDRSGYQR

FPGFDYWGQGTLVTVSS (SEQ ID NO:28)

LC

QSALTQPPSVSVAPGQTARITCGGNNIGSKGVQWYQQKPGQAPVLVVYDDSDRPSG IPERFSGSNSGNTATLTISRVEAGDEADYYCQVWDSSSDHWVFGGGTKLTVL (SEQ ID NO:58)

CC84.28

HC

EVQLVESGGGVVQPGRSLRLSCAVSGLTFKNYGFHWVRQAPGKGLEWVAVISYDG SDKYYVDSVKGRFTVSRDDSKNTLYLQMNSLRREDTAVYYCAKSGGGGFCSGGSC YRNYLDYWGQGTLVTVSS (SEQ ID NO:29)

_KC

DIQMTQSPSSLSASVGDRVTINCHASQDITHYLNWYQQKPGKAPKLLIYDASNLETG VPSRFRGSGSGTNFTFTIISLQPEDFTTFYCQQYDNLPLTFGGGTKVEIK (SEQ ID NO:59)

CC84.51

HC

QLQLVQSGAEVKKPGESLKISCKGSGYSFTTYWVGWVRQMPGKGLEWMGIIYPGD SETTYSPSFEGQVTISADKSISTAYLQWSRLRASDTAMYFCARQWNYPDGAFDVWG HGTMVTVSS (SEQ ID NO: 30)

_KC

DIVMTQSPSTLSASVGDRVTITCRASQNIRNWVAWYQQKPGKAPKFLIYRASSLETG VPSRFSGSGSGTEFTLTISSLQPDDFATYYCQQYNSYFPTFGQGTKLEIK (SEQ ID NO: 60)

Example 4. Epitope specificity of sarbecovirus bnAbs

[00110] To help map the epitopes recognized by the sarbecovirus bnAbs, we first epitope binned them using BLI competition with RBD nAbs of known specificities (Fig. 4a, Fig.

11), including 5 human nAbs: (1) CC12.1, an RBS-A or class 1 nAb targeting the ACE2 binding site ⁶’^14,17; (2) CC12.19, which recognizes a complex RBD epitope and competes with some non-RBD Abs ¹⁵; (3) CR3022, which recognizes the class 4 epitope site ^{6 14}; (4) S309, which recognizes the class 3 epitope site ^{6 14}; and (5) DH1047, which recognizes a conserved site and is class 4 ²⁷. In addition, we included K398.22, a macaque bnAb ⁵¹, which targets an RBD bnAb epitope distinct from that recognized by human bnAbs characterized to date but has features characteristic of class 4 bnAbs (Fig. 4a-b). The bnAbs we describe here can be clustered for convenience into two major groups. Group-1 bnAbs strongly competed with SARS-CoV-2 class 4 human bnAbs, CR3022 and DH1047, and macaque bnAb K398.22, showed more sporadic competition with CC12.1 and did not compete with CC12.19 or S309. Group-2 mAbs competed strongly with CC12.19, weakly with macaque K398.22, and only infrequently and/or weakly with any of the other bnAbs. Group-1 bnAbs were potent and broad against ACE2 -utilizing sarbecoviruses, but many lineage members displayed limited reactivity with clade 2 sarbecovirus RBDs. The group-2 mAbs showed broader reactivity with sarbecoviruses but were relatively less potent compared to group- 1 bnAbs (Fig. 4a). Notably, one group-2 bnAb, CC25.11 showed strong competition with human class 3 RBD bnAb, S309 ⁵², and the macaque bnAb, K398.2 ⁵¹. The findings suggest that both group-1 and 2 bnAbs target more conserved RBD epitopes but group-1 bnAbs are overall more potent but less broad against clade 2 sarbecoviruses with some exceptions. [00111] To further investigate the epitopes recognized by the bnAbs, we utilized singleparticle, negative-stain electron microscopy (nsEM) and confirmed that the 9 Group-1 and 2 Group-2 bnAb Fabs bound to the RBD of SARS-CoV-2 S-protein (Fig. 4c, Fig. 12). The binding modes of bnAbs to SARS-CoV-2 S-protein were largely similar with some differences in the angles of approaches, but not distinct enough to clearly segregate group-1 epitope bnAbs. The group-2 bnAb reconstructions are consistent with an epitope that spans the RBD, and other parts of the S-protein as described for the competitive Ab CC12.19 ¹⁵. These bnAb Fabs showed binding to S-protein with all three stoichiometries (Fab: trimer; 1 : 1, 1 :2 and 1 :3) with some of the Fabs exhibiting destabilizing effects on the S-trimer (Fig. 4c, Fig. 12). This destabilization is seen as dimers and flexible densities in the 2D class averages (Fig. 12).

Example 5. Strategies for generating pan-sarbecovirus passive antibodies and vaccines [00112] Here we characterized the largest set of sarbecovirus bnAbs described to date. The bnAbs recognize a relatively conserved face of the RBD and many are highly potent with fine differences in recognition properties that may provide useful in the face of viral variation. In particular, as variants emerge during this and future CoV pandemics, the availability of a selection of potent bnAbs provides choice of optimal reagents for prophylaxis and therapy to respond to the viral threats.

[00113] In terms of vaccine design, the generation of HIV immunogens typically draws heavily on the availability of multiple bnAbs to a given site to provide the best input for design strategies ^36,67. The same consideration is likely to apply to pan-sarbecovirus vaccine design. Further, although the bnAbs that we isolated were encoded by diverse gene families, certain V and D gene families were highly enriched, and rational vaccine design strategies may seek to target these genetic features ³⁶’³⁸'⁴⁰. Some of the potent bnAbs compete with the immunodominant human SARS-CoV-2 RBS-A/class 1 nAb CC12.1 that shows relatively low cross-reactivity. Elicitation of nAbs like CC12.1 may then reduce the elicitation of bnAbs and rational vaccine design modalities may need to mask RBS-A/class 1 immunodominant sites ^68-70 whilst leaving the bnAb sites intact. Resurfaced RBD-based immunogens in various flavors ⁵⁰>⁵⁷>⁵⁸ may achieve a similar goal.

[00114] Given the strong bnAb responses induced through infection-vaccination as indicated from serum studies and by our mAbs, are there lessons here for vaccine design? The higher frequency of bnAbs in infection-vaccination may have a number of causes. First, the spike S-protein may have subtle conformational differences, particularly in the sites targeted by bnAbs, between the native structure on virions and the stabilized form presented by mRNA immunization. This may favor the activation of bnAbs in the infection step followed by recall during mRNA boosting. Second, the long time-lag between infection and vaccination may have favored the accumulation of key mutations associated with bnAbs. There is evidence that intact virions can be maintained on follicular dendritic cells in germinal centers over long time-periods in a mouse model ⁷¹. Third, T cell help provided by the infection may be superior to that provided by mRNA vaccination alone. Overall, there is an intriguing possibility that pan-sarbecovirus nAb activity may be best achieved by a hybrid approach ⁴² to immunization that seeks to mimic infection-vaccination, once the key contributing factors to breadth development in that approach can be determined.

[00115] In summary, we isolated multiple potent sarbecovirus cross-neutralizing human antibodies and provide a molecular basis for broad neutralization. The bnAbs identified may themselves have prophylactic or therapeutic utility and the bnAb panel delineates the boundaries and requirements for broad neutralization and will be an important contributor to rational vaccine design.

Example 5. Materials and methods

[00116] Convalescent COVID-19 and human vaccinee sera: Sera from convalescent COVID-19 donors ³⁴, spike-mRNA-vaccinated humans, and from COVID-19-recovered vaccinated donors, were provided through the “Collection of Biospecimens from Persons Under Investigation for 2019-Novel Coronavirus Infection to Understand Viral Shedding and Immune Response Study” UCSD IRB# 200236. The protocol was approved by the UCSD Human Research Protection Program. Convalescent serum samples were collected based on COVID-19 diagnosis regardless of gender, race, ethnicity, disease severity, or other medical conditions. All human donors were assessed for medical decision-making capacity using a standardized, approved assessment, and voluntarily gave informed consent prior to being enrolled in the study.

[00117] Plasmid construction: To generate soluble S ectodomain proteins from SARS- CoV-1 (residues 1-1190; GenBank: AAP13567) and SARS-CoV-2 (residues 1-1208;

GenBank: MN908947), we constructed the expression plasmids by synthesizing the DNA fragments from GeneArt (Life Technologies) and cloned them into the phCMV3 vector (Genlantis, USA). To keep the soluble S proteins in a stable trimeric prefusion state, the following changes in the constructs were made: double proline substitutions (2P) were introduced in the S2 subunit; the furin cleavage sites (in SARS-CoV-2 residues 682-685, and in SARS-CoV-1 residues 664-667) were replaced by “GSAS” linker; the trimerization motif T4 fibritin was incorporated at the C-terminus of the S proteins. To purify and biotinylate the spike proteins, the HRV-3C protease cleavage site, 6x HisTag, and AviTag spaced by GS-linkers were added to the C-terminus after the trimerization motif. To produce truncated proteins of SARS-CoV-1 and SARS-CoV-2 spike, the PCR amplifications of the gene fragments encoding SARS-CoV-1 RBD (residue 307-513), SARS-CoV-2 NTD (residue 1- 290), RBD (residue 320-527), RBD-SD1 (residue 320-591), and RBD-SD1-2 (residue 320-681) subdomains were carried out using the SARS-CoV-1 and SARS-CoV-2 plasmids as templates. To generate pseudoviruses of non-human sarb ecoviruses, the DNA fragments encoding the spikes of the sarb ecoviruses without the ER retrieval signal were codon-optimized and synthesized at GeneArt (Life Technologies). The spike encoding genes of Pangl7 (residues 1-1249, GenBank: QIA48632.1), WIV1 (residues 1-1238, GenBank: KF367457) and SHC014 (residue 1-1238, GenBank: AGZ48806.1) were constructed into the phCMV3 vector (Genlantis, USA) using the Gibson assembly (NEB, E2621L) according to the manufacturer’s instructions. To express the monomeric RBDs of sarbecovirus clades (clades, lb, la, 2 and 3), the conserved region aligning to SARS-CoV-2 RBD (residue 320- 527) were constructed into phCMV3 vector with 6x HisTag, and AviTag spaced by GS- linkers on C-terminus. The sarbecovirus RBD genes encoding RaTG13 (residues 320-527, GenBank: QHR63300.2), Pangl7 (residues 318-525, GenBank: QIA48632.1), WIV1 (residues 308-514, GenBank: KF367457), RsSHC014 (residues 308-514, GenBank: AGZ48806.1), BM-4831 (residues 311-514, NCBI Reference Sequence: NC_014470.1), BtKY72 (residues 310-516, GenBank: KY352407), RmYN02 (residues 299-487, GSAID EPI ISL 412977), Rfl (residues 311-499, GenBank: DQ412042.1), Rs4081 (residues 311- 499, GenBank: KY417143.1) and Yunl l (residues 311-499, GenBank: JX993988) were synthesized at GeneArt (Life Technologies) and constructed using the Gibson assembly (NEB, E2621L).

[00118] Cell lines: HEK293F cells (Life Technologies) and Expi293F cells (Life Technologies) were maintained using 293FreeStyle expression medium (Life Technologies) and Expi293 Expression Medium (Life Technologies), respectively. HEK293F and Expi293F cell suspensions were maintained in a shaker at 150 rpm, 37°C with 8% CO2. Adherent HEK293T cells were grown in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin and maintained in an incubator at 37°C with 8% CO2. A stable hACE2-expressing HeLa cell line was generated using an ACE2 lentivirus protocol previously described. Briefly, the pBOB-hACE2 plasmid and lentiviral packaging plasmids (pMDL, pREV, and pVSV-G (Addgene #12251, #12253, #8454)) were co-transfected into HEK293T cells using the Lipofectamine 2000 reagent (ThermoFisher Scientific, 11668019). [00119] Transfection for protein expression: For expression of mAbs, HC and LC gene segments that were cloned into corresponding expression vectors were transfected into Expi293 cells (Life Technologies) (2-3 million cells/mL) using FectoPRO PolyPlus reagent (Polyplus Cat # 116-040) for a final expression volume of 2, 4 or 50 mL. After approximately 24 hours, sodium valproic acid and glucose were added to the cells at a final concentration of 300 mM each. Cells were allowed to incubate for an additional 4 days to allow for mAb expression. For expression of spike proteins, RBDs, and NTDs, cloned plasmids (350 pg) were transfected into HEK293F cells (Life Technologies) (1 million cells/mL) using Transfectagro reagent (Corning) and 40K PEI (1 mg/mL) in a final expression volume of 1 L as previously described. Briefly, plasmid and transfection reagents were combined and filtered preceding PEI addition. The combined transfection solution was allowed to incubate at room temperature for 30 mins before being gently added to cells. After 5 days, supernatant was centrifuged and filtered.

[00120] Protein purification: For mAb purification, a 1 : 1 solution of Protein A Sepharose (GE Healthcare) and Protein G Sepharose (GE Healthcare) was added to Expi293 supernatant for 2h at room temperature or overnight at 4°C. The solution was then loaded into an Econo-Pac column (BioRad #7321010), washed with 1 column volume of PBS, and mAbs were eluted with 0.2 M citric acid (pH 2.67). The elution was collected into a tube containing 2 M Tris Base. Buffer was exchanged with PBS using 30K Amicon centrifugal filters (Millipore, UFC903008). His-tagged proteins were purified using HisPur Ni-NTA Resin (Thermo Fisher). Resin-bound proteins were washed (25 mM Imidazole, pH 7.4) and slowly eluted (250 mM Imidazole, pH 7.4) with 25 mL elution buffer. Eluted proteins were buffer-exchanged with PBS, and further purified using size-exclusion chromatography using Superdex 200 (GE Healthcare).

[00121] ELISA: ELISAs were performed on 96-well half-area microplates (ThermoFisher Scientific) as described previously ¹⁵. The plate was coated with 2 pg/mL mouse anti-His antibody (Invitrogen cat. #MA1-21315-1MG, ThermoFisher Scientific) overnight at 4°C. The following day, plates were washed three times with PBST (PBS + 0.05% Tween20) and incubated for Ih with blocking buffer (3% bovine serum albumin (BSA)). Following removal of blocking buffer, plates were treated with His-tagged proteins (5 pg/mL in PBST + 1% BSA) for 1.5h at room temperature. Plates were washed and serum was added at threefold dilutions (beginning at 1 :30) and allowed to incubate for 1.5h. Following washes, secondary antibody (AffiniPure Goat anti-human IgG Fc fragment specific, Jackson ImmunoResearch Laboratories cat. #109-055-008) was added for an additional Ih.

Secondary antibody was washed, and staining substrate (alkaline phosphatase substrate pNPP tablets, Sigma) was added. Absorbance at 405 nm was measured after 8, 20, and 30 min using VersaMax microplate reader (Molecular Devices) and analyzed using SoftMax version 5.4 (Molecular Devices).

[00122] Biotinylation of proteins: To randomly biotinylate the proteins described in this paper, we used an EZ-Link NHS-PEG Solid-Phase Biotinylation Kit (Thermo Scientific #21440). To dissolve the reagents supplied in the kit for stock solutions, 10 pL DMSO was added into each tube. To make a working solution, 1 pL stock solution was diluted by 170 pL water freshly before use. To concentrate the proteins before biotinylation, the proper sized filter Amicon tubes were used. The proteins were adjusted to 7-9 mg/mL in PBS. For each 30 pL aliquoted protein, 3 pL of working solution was added and mixed thoroughly following by a 3h incubation on ice. To stop the reaction and remove the free NHS-PEG4- Biotin, the protein solution was buffer exchanged into PBS using Amicon tubes. All proteins were evaluated by BioLayer Interferometry after biotinylation.

[00123] BirA biotinylation of proteins for B cell sorting: For B cell sorting, the spike probes with the His and Avi-tag at the C-terminus were biotinylated by the intracellular biotinylating reaction during transfection step. To biotinylate the recombinant Avi-tagged spike probes, the BirA biotin-protein ligase encoding plasmid was co-transfected with the spike probe-Avi-tag encoding plasmids in the FreeStyle™ 293-F cell. 150ug BirA plasmid and 300ug spike probe plasmids were transfected with PEI reagent as described in the Transient transfection section. The spike probes were purified with HisPur Ni-NTA Resin (Thermo Fisher) as described in the Protein purification section. After the purification, the biotinylated proteins were evaluated by BioLayer Interferometry.

[00124] BioLayer Interferometry (BLI): Binding assays were performed on an Octet RED384 instrument using Anti -Human IgG Fc Capture (AHC) biosensors (ForteBio). All samples were diluted in Octet buffer (PBS with 0.1% Tween 20) for a final concertation of 10 pg/mL for mAbs and 200 nM for viral proteins. For supernatant mAb binding screening, 125 pL of expression supernatant was used. For binding assays, antibodies were captured for 60 s and transferred to buffer for an additional 60 s. Captured antibodies were dipped into viral proteins for 120 s in order to obtain an association signal. For dissociation, biosensors were moved to Octet buffer only for an additional 240 s. Randomly biotinylated SARS- CoV-2 S and SARS-CoV-2 RBD were diluted to 200 nM and captured for 5 min. Antigen- captured biosensors were placed in a saturating concentration of mAbs (100 pg/mL) for 10 min. Biosensors were subsequently moved to competing antibodies (25 pg/mL) for an additional 5 min. All BLI results were analyzed using the 1 : 1 binding kinetics fitting model on ForteBio Data Analysis software.

[00125] Isolation of monoclonal antibodies (mAbs): To isolate antigen-specific memory B cells, we used SARS-CoV-1 and SARS-CoV-2 spike proteins as probes to perform single cell sorting in a 96-well format. PBMCs from post-infection vaccinated human donors were stained with fluorophore labeled antibodies and spike proteins. To generate spike probes, streptavidin-AF647 (Thermo Fisher S32357) was coupled to BirA biotinylated SARS-CoV-1 spike. Streptavidin-AF488 (Thermo Fisher S32354) and streptavidin-BV421 (BD Biosciences 563259) were coupled to BirA biotinylated SARS-CoV-2 spike separately. The conjugation reaction was carried freshly before use with spike protein versus streptavidin- fluorophores at 2: 1 or 4: 1 molecular ratio. After 30 min incubation at room temperature, the conjugated spike proteins were stored on ice or at 4 °C for up to 1 week. To prepare PBMCs, the frozen PBMCs were thawed in lOmL recover medium (RPMI 1640 medium containing 50% FBS) immediately before staining. The cells were washed with lOmL FACS buffer (PBS, 2% FBS, 2 mM EDTA) and each 10 million cells were resuspended in lOOpL of FACS buffer. To isolate SARS-CoV-1 and SARS-CoV2 broadly neutralizing IgG+ B cells, PBMCs were stained for CD3 (APC Cy7, BD Pharmingen #557757), CD4 (APC-Cy7, Biolegend, #317418), CD8 (APC-Cy7, BD Pharmingen #557760), CD14 (APC-H7, BD Pharmingen #561384, clone M5E2), CD19 (PerCP-Cy5.5, Biolegend, #302230, clone FHB19), CD20 (PerCP-Cy5.5, Biolegend, #302326, clone 2H7), IgG (BV786, BD Horizon, #564230, Clone G18-145) and IgM (PE, Biolegend, #314508, clone MHM-88). Antibodies were incubated with PBMCs on ice for 15 min. After the 15 min staining, SARS-CoV-1- S- AF647, SARS-CoV-2-S-AF488, and SARS-CoV-2-S-BV421 were added to the PBMC solution incubating on ice. After another 30 min incubation, FVS510 Live/Dead stain (Thermo Fisher Scientific, #L34966) 1 : 1000 diluted with FACS buffer was added to the PBMC solution for 15 min. Subsequently, cells were washed with 10 mL ice cold FACS buffer. Each 10 million cells were resuspended with 500pL FACS buffer and then filtered through 70um nylon mesh FACS tube caps (Fisher Scientific, #08-771-23). A BD FACSMelody sorter (BRV 9 Color Plate 4way) was used for the single cell sorting process. To isolate broadly neutralizing B cells, the gating strategy was set as follows: lymphocytes (SSC-A vs. FSC-A) and singlets (FSC-H vs. FSC-A) were gated first, and then live cells were selected by FVS510 Live/Dead negative gating. B cells were identified as CD19+CD20+CD3-CD4-CD8-CD14-IgM-IgG+ live singlets. Cross-reactive S-protein specific B cells were sequentially selected for SARS-CoV-2-S-BV421/SARS-CoV-2-S- AF488 double positivity and SARS-CoV-l-S-AF647/SARS-CoV-2-S-AF488 double positivity. Single cells were sorted into 96-well plates on a cooling platform. To prevent degradation of mRNA, plates were moved onto dry ice immediately after sorting. Reverse transcription was done right after. Superscript IV Reverse Transcriptase (Thermo Fisher), dNTPs (Thermo Fisher), random hexamers (Gene Link), Ig gene-specific primers, DTT, and RNAseOUT (Thermo Fisher), and Igepal (Sigma) were used in the reverse transcription PCR reaction as described previously ^72,73. To amplify IgG heavy and light chain variable regions, two rounds of nested PCR reactions were carried out using the cDNAs as template and Hot Start DNA Polymerases (Qiagen, Thermo Fisher) and specific primer sets described previously ^72,73. The PCR products of the heavy and light chain variable regions were purified with SPRI beads according to the manufacturer’s instructions (Beckman Coulter). Then, the purified DNA fragments were constructed into expression vectors encoding human IgGl, and Ig kappa/lambda constant domains, respectively. Gibson assembly (NEB, E2621L) was used according to the manufacturer’s instructions in the construction step. To produce mAbs, the paired heavy and light chain were co-transfected into 293Expi cells. [00126] Pseudovirus production: To generate pseudoviruses, plasmids encoding the SARS-CoV-1, SARS-CoV-2 or other variants spike proteins with the ER retrieval signal removed were co-transfected with MLV-gag/pol and MLV-CMV-Luciferase plasmids into HEK293T cells. Lipofectamine 2000 (Thermo Fisher Scientific, 11668019) was used according to the manufacturer’s instructions. 48 hours post transfection, supernatants containing pseudoviruses were collected and filtered through a 0.22 pm membrane to remove debris. Pseudoviruses could be stored at -80°C prior to use.

[00127] Pseudovirus entry and serum neutralization assays: To generate hACE2- expressing stable cell lines for the pseudovirus infection test, we used lentivirus to transduce the hACE2 into HeLa cells. Stable cell lines with consistent and high hACE2 expression levels were established as HeLa-hACE2 and used in the pseudovirus neutralization assay. To calculate the neutralization efficiency of the sera or mAbs, the samples were 3-fold serially diluted and 25 pL of each dilution was incubated with 25 pL of pseudovirus at 37 °C for 1 h in 96-half area well plates (Coming, 3688). Just before the end of the incubation, HeLa- hACE2 cells were suspended with culture medium at a concentration of 2 x 10⁵/mL. The DEAE-dextran (Sigma, # 93556-1G) was added to the cell solutions at 20 pg/mL. 50 pL of the cell solution was distributed into each well. The plates were incubated at 37 °C for 2 days and the neutralization efficiency was calculated by measuring the luciferase levels in the HeLa-hACE2 cells. After removal of the supernatant, the HeLa-hACE2 cells were lysed by luciferase lysis buffer (25 mM Gly-Gly pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100) at room temperature for 10-20 mins. After adding Bright-Glo (Promega, PRE2620) to each well, luciferase activity was inspected by a luminometer. Each experiment was carried out with duplicate samples and repeated independently at least twice. Percentage of neutralization was calculated according to the equation:

% Neutralization

[00128] The neutralization percentage was calculated and plots against antibody concentrations or sera dilution ratio were made in Graph Pad Prism. The curves were fitted by nonlinear regression and the 50% pseudovirus neutralizing (IC50) or binding (ID50) antibody titer was calculated.

[00129] Competition BLI: To determine the binding epitopes of the isolated mAbs compared with known human SARS-CoV-2 mAbs, we did in-tandem epitope binning experiments using the Octet RED384 system. 200 nM of randomly biotinylated SARS-CoV- 2 S or RBD protein antigen was captured using SA biosensors (18-5019, Sartorius). The biosensor was loaded with antigen for 5 min and then moved into the saturating mAbs at a concentration of 100 pg/mL for 10 min. The biosensors were then moved into bnAb solution for 5 min to measure binding in the presence of saturating antibodies. As control, biosensors loaded with antigen were directly moved into bnAb solution. The percent (%) inhibition in binding is calculated with the formula: [Percent (%) binding inhibition = 1- (bnAb binding response in presence of the competitor antibody / binding response of the corresponding control bnAb without the competitor antibody)].

[00130] Fab production: To generate the Fab from the IgG, a stop codon was inserted in the heavy chain constant region at “KSCDK”. The truncated heavy chains were cotransfected with the corresponding light chains in 293Expi cells to produce the Fabs. The supernatants were harvested 4 days post transfection. Fabs were purified with Capture Select™ CHI -XL MiniChrom Columns (#5943462005). Supernatants were loaded onto columns using an Econo Gradient Pump (Bio-Rad #7319001). Following a wash with lx PBS, Fabs were eluted with 25 mL of 50 mM acetate (pH 4.0) and neutralized with 2 M Tris Base. The eluate was buffer exchanged with lx PBS in 10K Amicon tubes (Millipore, LTFC901008) and filtered with a 0.22 pm spin filter.

[00131] Negative stain electron microscopy: S-protein was complexed with Fab at three times molar excess per trimer and incubated at room temperature for 30 mins. Complexes were diluted to 0.03mg/ml in lx Tris-buffered saline and 3 pl applied to a 400mesh Cu grid, blotted with filter paper, and stained with 2% uranyl formate. Micrographs were collected on a Thermo Fisher Tecnai Spirit microscope operating at 120kV with an FEI Eagle CCD (4k x 4k) camera at 52,000 X magnification using Leginon automated image collection software ⁷⁴. Particles were picked using DogPicker ⁷⁵ and data was processed using Relion 3.0 ⁷⁶. Map segmentation was performed in UCSF Chimera ⁷⁷.

[00132] Statistical Analysis: Statistical analysis was performed using Graph Pad Prism 8 for Mac, Graph Pad Software, San Diego, California, USA. Median area-under-the-curve (AUC) or reciprocal 50% binding (ID50) or neutralization (IC50) titers were compared using the non-parametric unpaired Mann-Whitney -U test. The correlation between two groups was determined by Spearman rank test. Data were considered statistically significant at * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001.

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***

[00211] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00212] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WE CLAIM:

1. An antibody or antigen-binding fragment thereof that neutralizes a SARS- like virus and specifically binds to an epitope on the spike protein of the virus, wherein the epitope is recognized by antibodies comprising a heavy chain variable region sequence selected from SEQ ID NOs: l-30 and a light chain variable region sequence selected from SEQ ID NOs:31-60.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the SARS-like virus is a human coronavirus.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the epitope is in the receptor binding domain (RBD).

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the SARS-like virus is SARS-CoV-2.

5. The antibody or antigen-binding fragment thereof of claim 1, comprising heavy chain CDR1, CDR2 and CDR3 sequences and light chain CDR1, CDR2 and CDR3 sequences that are respectively identical to an antibody containing a heavy chain variable region selected from SEQ ID NOs: l-30 and a light chain variable region selected from SEQ ID NOs:31-60.

6. The antibody or antigen-binding fragment thereof of claim 5, which is not a full length antibody.

7. The antibody or antigen-binding fragment thereof of claim 5, comprising one or more amino acid substitutions relative to an antibody having a heavy chain variable region sequence selected from SEQ ID NOs: l-30 and a light chain variable region sequence selected from SEQ ID NOs:31-60.

8. The antibody or antigen-binding fragment thereof of claim 7, wherein the one or more amino acid substitutions are in CDRs of the antibody.

9. The antibody or antigen-binding fragment thereof of claim 7, wherein the one or more amino acid substitutions are in framework regions of the antibody.

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10. A molecule comprising the antibody or antigen-binding fragment thereof of claim 5 and a second moiety that is fused or conjugated to the antibody or antigen-binding fragment thereof.

11. The molecule of claim 10, wherein the second moiety is a polypeptide or a small organic molecule.

12. The molecule of claim 10, which is an antibody-drug conjugate.

13. A polynucleotide encoding the antibody or antigen-binding fragment thereof of claim 1.

14. A vector comprising the polynucleotide of claim 13.

15. A host or engineered cell harboring the polynucleotide of claim 13 or the vector of claim 14.

16. A pharmaceutical composition, comprising the antibody or antigen-binding fragment thereof of claim 1 or the polynucleotide of claim 13.

17. A method for preventing or treating infection of a SARS-like virus in a subject, comprising administering to the subject the pharmaceutical composition of claim 16.

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