RU2832110C1 - Polyvalent vector vaccine hvt - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: invention relates to biotechnology and virology. Disclosed is a recombinant herpes virus of turkeys (rHVT) for producing a vaccine for preventing or reducing infection with MDV, IBDV, NDV and/or AIV or associated disease signs in poultry, expressing the infectious bursitis virus (IBDV) viral protein 2 (VP2) gene and the Newcastle disease virus (NDV) fusion protein (F) gene from the first and second expression cassettes. First and second expression cassettes are inserted into a unique small (Us) region of the genome of said rHVT, both being inserted into the Us2 gene. Said rHVT also expresses the haemagglutinin (HA) gene of the avian influenza virus (AIV) from the third expression cassette, which is inserted into the unique long (UL) region of the genome of said rHVT either between the UL40 and UL41 genes, or between the UL44 and UL45 genes. Disclosed is a host bird cage for producing a vaccine; vaccine for preventing or reducing infection with MDV, IBDV, NDV and/or AIV or associated signs of disease in poultry. Disclosed is a method for producing a vaccine for poultry; use of rHVT, host cells or any combination thereof to produce a vaccine for poultry; use of the vaccine; method of preventing or reducing MDV, IBDV NDV and/or AIV infection or associated disease signs; method for vaccination of poultry.

EFFECT: vector construct provides good replication of the vector and its insertions both in vitro and in vivo, as well as effective expression of all heterologous genes at a sufficiently high level and for a considerable period of time to induce and maintain a protective immune response in the vaccinated target against all putative pathogens.

15 cl, 13 tbl, 3 ex

Description

Sequence Listing Information

The sequence listing associated with this application is provided in text format in lieu of a paper copy and is incorporated herein by reference. The name of the text file containing the sequence listing is 930585_402WO_SEQUENCE_LISTING.txt. The text file is 328 KB, was generated on February 24, 2021, and is submitted electronically via EFS-Web.

Prior art

In late 2019, a new betacoronavirus emerged in Wuhan, China. As of February 19, 2021, approximately 110 million cases of infection with this virus (also known as SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) and originally identified as Wuhan coronavirus) have been confirmed worldwide, resulting in approximately 2.45 million deaths. Methods to prevent or treat SARS-CoV-2 infection and diagnostic tools to diagnose SARS-CoV-2 infection are needed.

Brief description of graphic materials

Fig. 1A and 1B show binding of antibodies (1A) S303 (VH SEQ ID NO:63; VL SEQ ID NO:67) and (1B) S309 (VH SEQ ID NO:105; VL SEQ ID NO:168) of the present invention to the recombinant RBD (receptor binding domain) of SARS-CoV-2, as described in Example 2.

Fig. 2A and 2B show neutralization of SARS-CoV-2 infection by certain antibodies of the present invention, as described in Example 4.

Fig. 3A-3I show neutralization of SARS-CoV-2 infection as described in Example 4. Fig. 3A shows neutralization using donor plasma from surviving SARS-CoV-1 victims. Fig. 3B-3D and 3I show neutralization using supernatant from B cells expressing certain antibodies of the present invention. Fig. 3E-3H show neutralization using certain recombinant IgG1 antibodies.

Figures 4A and 4B show the binding of B cell supernatant containing antibody to SARS-CoV-2 protein expressed on ExpiCHO cells (CHO - Chinese hamster ovary cells) as described in Example 1. Graphs showing the binding profiles of S300-S310 antibodies are indicated by boxes.

Fig. 5A and 5B show binding of S311 and S312 antibodies in the supernatant of cultured B cells to SARS-CoV-1 and SARS-CoV-2 as described in Example 6. Antibody concentrations are approximate. SARS S1 Sino: protein from Sino Biological. RBD2: SARS-CoV-2 RBD produced in-house.

Fig. 6A-6E show curves (top) of binding of some antibodies of the present invention to the RBD of SARS-CoV-1 (SARS1) and the RBD of SARS-CoV-2 (SARS2) measured using the Octet instrument and curves (bottom) of KD values. KD values for antibodies (e.g., less than 1.0×10 ^-12 M) with very strong binding and slow dissociation are approximate. These data and experiments are described further in Example 3.

Fig. 7 shows neutralization of infection by antibodies S304 (VH SEQ ID NO:79; VL SEQ ID NO:83) and S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), alone or in combination, against pseudotyped SARS-CoV-2 virus, as described in Example 5.

Figures 8A-8K show binding curves of selected antibodies to the SARS-CoV-1 RBD, the SARS-CoV-2 RBD, and the ectodomains of various coronaviruses measured by ELISA (enzyme-linked immunosorbent assay). See Example 8.

Fig. 9 shows the neutralization of S309 rIgG1 (VH SEQ ID NO:105; VL SEQ ID NO:168) and S315 rIgG1 (VH SEQ ID NO:178; VL SEQ ID NO:182) infection against pseudotyped SARS-CoV-2 virus as described in Example 7.

Fig. 10 shows neutralization of infection by full-length S309 rIgG1 and S309 rFab (both of which contain VH with SEQ ID NO:105 and VL with SEQ ID NO:168) against pseudotyped SARS-CoV-2 virus as described in Example 7.

Fig. 11 shows the binding of antibody S309 (VH SEQ ID NO:105; VL SEQ ID NO:168) to the spike protein of SARS-CoV-1 and SARS-CoV-2 expressed on ExpiCHO cells as described in Example 9. The stacked histograms of the flow cytometry plots demonstrate dose-dependent binding of antibody S309 to SARS-CoV and SARS-CoV-2.

Figures 12A and 12B show concentration-dependent binding measured by flow cytometry for certain antibodies as described in Example 9. Figure 12A shows binding to SARS-CoV-2. Figure 12B shows binding to SARS-CoV-1.

Fig. 13 shows the neutralization of infection by antibodies S303 (VH SEQ ID NO:63; VL SEQ ID NO:67), S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), S306 (VH SEQ ID NO:87; VL SEQ ID NO:91), S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), S310 (VH SEQ ID NO:155; VL SEQ ID NO:159) and S315 (VH SEQ ID NO:178; VL SEQ ID NO:182) against the pseudotyped SARS-CoV-2 virus, as described in Example 4.

Fig. 14A-14D show the binding affinity/avidity of antibodies S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), S303 (VH SEQ ID NO:63; VL SEQ ID NO:67), S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), and S315 (VH SEQ ID NO:178; VL SEQ ID NO:182) to the RBDs of SARS-CoV-1 (right) and SARS-CoV-2 (left), as described in Example 10.

Figures 15A and 15B show the competition of antibody pairs of the present invention for binding to the RBDs of SARS-CoV-1 (Figure 15A) and SARS-CoV-2 (Figure 15B), as described in Example 12. For each graph, the x-axis shows time (0 to 1000 seconds) and the y-axis shows binding to the RBD measured by BLI (biolayer interferometry) (0 to 3 nm). The first antibody is indicated on the left side of the array and the second antibody is indicated on the top of the array. The dashed vertical lines in Figure 15B illustrate the switch from the first antibody to the second antibody. On the right ("I" - "IV" in Figure 15A, "II" and "IV" in Figure 15B) are antigenic sites determined using structural information, escape mutant analysis, and BLI-based epitope binding.

Figure 16 shows the ability of S309 to interfere with the binding of SARS-CoV-1 (left) or SARS-CoV-2 (right) RBD to human ACE2 (angiotensin converting enzyme 2) (hACE2) as described in Example 13. hACE2 was loaded onto BLI sensors followed by incubation of the sensors with RBD alone or RBD in combination with antibody. The vertical dotted line indicates the onset of RBD association with or without antibody. In the graph on the left, S230 antibody was used as a positive control for inhibition of SARS-CoV-1 RBD binding to ACE2 based on previous studies (see Walls et al., Cell 176(5):1023–1039.e15 (2019)).

Fig. 17A and 17B show the antibody-dependent effects of certain antibodies of the present invention against an infected cell model as described in Example 14. Fig. 17A shows antibody-dependent cytotoxicity using primary NK effector cells (NK - natural killer cells) and SARS-CoV-2-expressing ExpiCHO cells as target cells. The histogram on the right shows the ADCC (antibody-dependent cell-mediated cytotoxicity) for the indicated antibody(ies), calculated as the area under the curve (AUC). Fig. 17B shows antibody-dependent cellular phagocytosis using PBMC (peripheral blood mononuclear cells) as phagocytic cells and PKF67-labeled SARS-CoV-2-expressing ExpiCHO cells as target cells. Line graphs show the mean fluorescence intensity (MFI) of PBMCs after incubation with target cells and antibodies, determined for one representative donor with high affinity FcγRIIIa (Fc gamma receptor IIIa) (symbols show mean ±SD of replicates).

Fig. 18A-18J show binding curves of selected antibodies to the RBD of SARS-CoV-1, the RBD of SARS-CoV-2, and the ectodomains of various coronavirus strains, measured by ELISA. See Example 8. Recombinant mAbs (monoclonal antibody) were tested by ELISA in the concentration range from 5 to 0.00028 mg/mL. RBD2: the receptor binding domain of SARS-CoV-2. RBD1: the receptor binding domain of SARS-CoV (also referred to herein as SARS-CoV-1). Spike protein: a stabilized prefusion trimer of the indicated coronavirus. Some antibodies were recombinantly expressed as IgG1 (IgG1 immunoglobulin) (rIgG1), and some antibodies were recombinantly expressed as IgG1 with an MLNS mutation (M428L and N434S (EU numbering)) in Fc (rIgG1-LS).

Figures 19A and 19B show the ability of certain antibodies to interfere with RBD binding to human ACE2 as described in Example 13. Human ACE2 (hACE2) was loaded onto BLI sensors, followed by incubation of the sensors with RBD alone or RBD in combination with recombinant antibody. The vertical dotted line indicates the start of RBD loading with or without antibody. RBD: receptor binding domain. Figure 19A shows SARS-CoV-1 RBD binding to ACE2. Figure 19B shows SARS-CoV-2 RBD binding to ACE2.

Figures 20A and 20B show the binding affinity and avidity of S309 IgG antibody (Figure 20A) compared to S309 Fab (Figure 20B) for SARS-CoV-1 RBD (bottom of each figure) and SARS-CoV-2 RBD (top of each figure), as described in Example 10. For both IgG and Fab: VH SEQ ID NO:105; VL SEQ ID NO:168. RBDs were loaded onto BLI cylinders and the association of varying concentrations of S309-IgG-MLNS or S309 Fab was measured. The vertical dotted lines indicate the onset of the dissociation phase when the BLI cylinders were switched to buffer.

Figures 21A-21C show the reactivity of antibodies S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), S306 (VH SEQ ID NO:87; VL SEQ ID NO:91), S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), and S310 (VH SEQ ID NO:155; VL SEQ ID NO:159) against SARS-CoV-2, as described in Example 15. Figure 21A shows the reactivity of antibodies S304, S306, S309, and S310 against T×100 extracted lysate of SARS-CoV-2-infected Vero E6 cells. Figure 21B shows the reactivity of the same antibodies against SDS extracted lysate of SARS-CoV-2-infected Vero E6 cells. Figure 21C shows SARS-CoV-1 convalescent human sera against T×100-extracted or SDS-extracted SARS-CoV-2-infected Vero E6 cell lysate. Figs. 21A and 21B also show data for the comparative antibody LCA57, which is specific for the MERS-CoV spike protein (Corti et al. PNAS 112(33):10473–10478 (2015).

Figures 22A-22D show neutralization of infection by SARS-CoV-2 antibodies as assessed by inhibition of nucleoprotein (NP) expression at 24 and 45 hours post infection. See Example 16. Figure 22A shows neutralization of SARS-CoV-2 infection by S304 S304 (VH SEQ ID NO:79; VL SEQ ID NO:83). Figure 22B shows neutralization of SARS-CoV-2 infection by S309 (VH SEQ ID NO:105; VL SEQ ID NO:168. Fig. 22C shows neutralization of SARS-CoV-2 infection by a combination of S304 and S309. Fig. 22D shows control neutralization of SARS-CoV-2 infection by the comparative antibody LCA57, which is specific for the MERS-CoV (Middle East respiratory syndrome coronavirus) spike protein (Corti et al. PNAS 112(33):10473–10478 (2015).

Fig. 23 shows neutralization of infection by antibodies S309 (VH SEQ ID NO:105; VL SEQ ID NO:168) and S315 (VH SEQ ID NO:178; VL SEQ ID NO:182) alone or in combination against pseudotyped SARS-CoV-2 virus, as described in Example 5.

Fig. 24A and 24B show the antibody-dependent effects of certain antibodies of the invention against an infected cell model as described in Example 14. Fig. 24A shows antibody-dependent cell-mediated cytotoxicity (ADCC) using primary NK effector cells and SARS-CoV-2-expressing ExpiCHO cells as target cells. The graph shows the percentage of target cell killing following incubation with the antibody or antibody combination shown in the legend. Fig. 24B shows the ADCC for the indicated antibody(ies) calculated as the area under the curve (AUC). Left: AUC determined using cells with the FcγRIIIa VV genotype; Right: AUC determined for cells with the FcγRIIIa FF or FV genotype.

Figures 25A and 25B show additional antibody-dependent effects of certain antibodies of the invention, as described in Example 14. Figure 25A shows antibody-dependent cellular phagocytosis (ADCP) using PBMCs as phagocytic cells and PKF67-labeled SARS-CoV-2-expressing ExpiCHO cells as target cells. The graphs show the mean fluorescence intensity (MFI) of PBMCs after incubation with target cells and antibodies, determined for one representative high-affinity FcγRIIIa donor (symbols show the mean ±SD of replicates). Figure 24B shows the ADCC for the indicated antibody(ies), calculated as the area under the curve (AUC).

Fig. 26 shows antibody binding measured by flow cytometry. Binding of the S309 antibody to the SARS-CoV-2 spike protein expressed in Expi-CHO cells was detected by fluorescence-activated cell sorting.

Fig. 27 shows the binding of the S309 antibody (labeled "11" in the figure key) and four engineered variants of S309 (labeled "12" through "15", respectively) to the S protein as measured by flow cytometry. See Example 9. The four engineered variant antibodies are as follows: S309 N55Q contains the N55Q mutation in CDRH2, resulting in the VH variant sequence (SEQ ID NO:113) and the wild-type VL sequence (SEQ ID NO:168) of S309; S309 W50F contains the VH sequence of the W50F variant (SEQ ID NO:129) and the wild-type VL sequence (SEQ ID NO:168) of S309; S309 W105F comprises the VH sequence of variant W105F (SEQ ID NO: 119) and the wild-type VL sequence (SEQ ID NO: 168) of S309; and S309 W50F/G56A/W105F comprises the VH sequence of variant W50F/G56A/W105F (SEQ ID NO: 172) and the wild-type VL sequence of S309. In Fig. 27, S309 N55Q is labeled as “12”, S309 W50F is labeled as “13”, S309 W105F is labeled as “14”, and S309 W50F-G56A-W105F is labeled as “15”. Antibody binding to the SARS-CoV-2 spike protein expressed on Expi-CHO cells was detected using a fluorescently labeled secondary antibody. Data from two experiments are shown.

Figure 28 shows neutralization of infection by the S309 antibody (referred to as "Variant-11 (wt)" in the figure) and four variant S309 antibodies against pseudotyped SARS-CoV-2 viruses as described in Example 19. In Figure 28, S309 N55Q is labeled as "Variant-12", S309 W50F is labeled as "Variant-13", S309 W105F is labeled as "Variant-14", and S309 W50F-G56A-W105F is labeled as "Variant-15". The pseudotyped viruses are pseudotyped VSV (vesicular stomatitis virus) with the SARS-CoV-2 spike protein.

Figure 29 shows an overview of the results of pseudovirus binding and neutralization assays using the S309 antibody (“S309-WT”) and four engineered S309 variants (“N55Q”; “W50F”; “W105F”; “W50F/G56A/W105F”). The dotted horizontal line shows the change in function of the engineered variant compared to the S309-WT baseline. The differently shaded bars show binding to the glycosylated RBD measured by SPR (surface plasmon resonance), binding to the deglycosylated RBD measured by SPR, binding to antigen-expressing cells measured by FACS (fluorescence-activated cell sorting), and neutralization measured by SARS-CoV-2 pseudoviruses.

Figures 30A-30F show the binding kinetics of exemplary antibodies to glycosylated or deglycosylated SARS-CoV-2 RBD, measured by SPR. See Example 18. The antibodies S309 (having the VH (SEQ ID NO: 105) and VL (SEQ ID NO: 168) amino acid sequences of wild-type S309), S309 N55Q, S309 W50F, S309 W105F, and S309 W50F/G56A/W105F were tested. Figure 30A shows the binding kinetics of wild-type S309 (2 replicates). Figure 30B shows the binding kinetics of S309 N55Q (bottom) compared to wild-type S309 (top). Figure 30C shows the binding kinetics of S309 W50F (bottom) compared to wild-type S309 antibody (top). Fig. 30D shows the binding kinetics of S309 W105F (bottom) compared to wild-type S309 antibody (top). Fig. 30E shows the binding of S309 W50F/G56A/W105F (bottom) compared to wild-type S309 antibody (top). Fig. 30F shows the binding of S309 W50F/G56A/W105F using a 10-minute injection period (top) or a 3-minute injection period (bottom).

Figure 31 shows the activation of high-affinity FcγRIIIa (158V) (left) or low-affinity FcγRIIIa (158F) (right) by antibodies S303 (VH SEQ ID NO:63; VL SEQ ID NO:67), S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), S306 (VH SEQ ID NO:87; VL SEQ ID NO:91), S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), and a combination of S309 and S315, along with the comparative antibody S230. See Example 20. Activation was measured using SARS-CoV-2 S-expressing ExpiCHO cells as target cells and Jurkat reporter cells stably transfected with a NFAT-driven luciferase reporter gene. Activation of FcγRIIIa results in NFAT-mediated expression of the luciferase reporter gene. Results are based on a single experiment, one measurement, or two measurements per mAb.

Fig. 32 shows the activation of FcγRIIa by exemplary antibodies S303 (VH SEQ ID NO:63; VL SEQ ID NO:67), S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), S306 (VH SEQ ID NO:87; VL SEQ ID NO:91), S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), and a combination of S309 and S315, along with the comparative monoclonal antibody S230. See Example 20. Activation was measured using SARS-CoV-2 S-expressing ExpiCHO cells as target cells and Jurkat reporter cells stably transfected with a NFAT-driven luciferase reporter gene. Activation of FcγRIIa results in NFAT-mediated expression of the luciferase reporter gene.

Fig. 33A and 33B show the binding of antibodies S303 (VH SEQ ID NO:63; VL SEQ ID NO:67), S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), S306 (VH SEQ ID NO:87; VL SEQ ID NO:91), S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), S310 (VH SEQ ID NO:155; VL SEQ ID NO:159), and S315 (VH SEQ ID NO:178; VL SEQ ID NO:182), along with comparative antibodies S110, S230, and S109, to the S protein expressed on the cell surface. See Example 9. Fig. Figure 33A shows binding to ExpiCHO cells transfected with SARS-CoV-2 S protein. Figure 33B shows binding to ExpiCHO cells transfected with SARS-CoV-1 S protein. The mean fluorescence intensity was measured by flow cytometry for each antibody. Antibody concentrations tested are indicated on the x-axis.

Fig. 34 shows neutralization of infection by exemplary antibodies S303 (VH SEQ ID NO:63; VL SEQ ID NO:67), S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), S306 (VH SEQ ID NO:87; VL SEQ ID NO:91), S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), S310 (VH SEQ ID NO:155; VL SEQ ID NO:159), and S315 (VH SEQ ID NO:178; VL SEQ ID NO:182) against a pseudotyped SARS-CoV-2 virus, as described in Example 4. See Example 4.

Figures 35A and 35B show the conservation of S protein residues as described in Example 21. Figure 35A shows spike protein variants occurring with a frequency n greater than 1 as spheres displayed in the closed (left) and open (right) forms of the complete trimeric spike ectodomain. The RBD and other domains of the spike protein are shown as indicated. Forty mutations (out of 2229 total) are shown. Only residue 367 (represents 8) is isolated in the RBD, but not residues 476 (n is 7) and 483 (n is 17). Figure 35B shows the prevalence of spike glycoprotein variants by amino acid. Each dot is an individual variant. The locations of the A domain and RBD are shown. Variants meeting the 0.1% frequency cutoff are as indicated.

Fig. 36 shows the neutralization of SARS-CoV-2-MLV (MLV - murine leukemia virus) by the S309 antibody (VH SEQ ID NO:105; VL SEQ ID NO:168) in combination with an equivalent amount of the S304 antibody (VH SEQ ID NO:79; VL SEQ ID NO:83). For antibody cocktails, the concentration shown on the x-axis is equal to the concentration of the individual antibodies. See Example 4.

Fig. 37 shows the neutralization of SARS-CoV2-MLV by the S309 antibody (VH SEQ ID NO:105; VL SEQ ID NO:168) in combination with an equimolar amount of the S315 antibody (VH SEQ ID NO:178; VL SEQ ID NO:182). For antibody cocktails, the concentration shown on the x-axis is equal to the concentration of the individual antibodies. See Example 4.

Figures 38A-38D show binding of specific antibodies to the RBDs of SARS-CoV-2 and SARS-CoV-1 as described in Example 6. Antibodies were recombinantly expressed and binding was analyzed using ELISA. 96-well ELISA plates were coated with SARS-CoV-2 RBD (produced in-house; residues 331-550 of spike protein from BetaCoV/Wuhan-Hu-1/2019, Cat. No. MN908947) at 10 μg/mL and SARS-CoV-1 RBD (Sino Biological, 40150-V08B1) at 1 μg/mL. After blocking with 1% BSA (bovine serum albumin) in PBS (phosphate-buffered saline), antibodies were added to the plates and incubated at room temperature for one hour. The plates were washed and goat anti-human IgG-AP secondary antibody (Southern Biotechnology, 2040-04) was added. The substrate p-nitrophenyl phosphate (pNPP, Sigma-Aldrich, 337 71768) was used to create color. OD405 was analyzed on an ELx808IU plate reader (Biotek). The left side of each figure shows binding to the SARS-CoV-2 RBD, and the right side shows binding to the SARS-CoV-1 RBD.

Figures 39A and 39B show binding of specific antibodies to the SARS-CoV-2 and SARS-CoV-1 spike protein as described in Example 9. Expi-CHO cells were transiently transfected with phCMV1-SARS-CoV-2-S, SARS-spike-pcDNA (DNA (deoxyribonucleic acid) elonating plasmid).3 (SARS strain), or empty phCMV1 using Expifectamine CHO Enhancer. Two days after transfection, cells were harvested for antibody immunostaining. Alexa647-labeled anti-human IgG Fc secondary antibody was used for detection. Antibody binding to transfected cells was analyzed by flow cytometry using a ZE5 Cell Analyzer (BioRad). Figure 39A and 39B show binding of specific antibodies to the SARS-CoV-2 and SARS-CoV-1 spike proteins as described in Example 9. 39A shows the binding of recombinant antibody S300 (VH: SEQ ID NO:1; VL: SEQ ID NO:5). Fig. 39B shows the binding of recombinant antibody S307. Symbols represent the values of one measurement. The left panel of each figure shows the data presented as % of positive cells, and the right panel shows the data presented as mean fluorescence intensity (MFI).

Figures 40A and 40B show binding of type antibodies to SARS-CoV-2 (Figure 40A) or SARS-CoV-1 (Figure 40B) S-glycoproteins expressed on the surface of ExpiCHO cells as described in Example 9. Symbols represent replicates of a single experiment.

Figures 41A and 41B show the binding affinity and avidity of S309 IgG (Figure 41A) and S309 Fab (Figure 41B) for SARS-CoV-2 RBD (top) or SARS-CoV-2 spike protein (bottom). For both IgG and Fab: VH SEQ ID NO:105; VL SEQ ID NO:168. See Example 11. Biotinylated SARS-CoV-2 RBD or biotinylated SARS-CoV-2 S-ectodomain prefusion trimer were loaded onto streptavidin biosensors and the association of different concentrations of S309-IgG-MLNS (containing the M428L and N434S Fc mutations (EU numbering)) or S309 Fab was measured. The vertical dotted lines indicate the start of the dissociation phase when the biosensors were switched to the buffer.

Figures 42A and 42B show the binding of antibodies S303, S304, S306, S309, S310, and S315, along with comparative antibodies S110, S124, S230, and S109, to the S protein expressed on the cell surface. See Example 9. Figure 42A shows binding to ExpiCHO cells transfected with the SARS-CoV-2 S protein. Figure 42B shows binding to ExpiCHO cells transfected with the SARS-CoV-1 S protein. The mean fluorescence intensity was measured by flow cytometry for each antibody. The antibody concentrations tested are indicated on the x-axis.

Fig. 43 shows the conservation of the spike protein residues as described in Example 21. Spike protein variants supported by at least two sequences as indicated, spheres displayed in closed (left) and open (right) forms of the complete trimeric spike ectodomain. The RBD and other domains of the spike protein are shown as indicated. 171 variants (out of 11,839 spike protein sequences analyzed) are shown.

Fig. 44A-44C show the selection for resistance to SARS-CoV-2 as described in Example 23. Fig. 44A shows a flow chart illustrating the method for selection for resistance. Fig. 44B shows a timeline illustrating the procedure used for each passage of the resistance selection process. Fig. 44C shows the results of selection for resistance of SARS-CoV-2 to the S309 N55Q MLNS GAALIE antibody containing the VH of SEQ ID NO: 113 and the VL of SEQ ID NO: 168, with the G236A, A330L, I332E, M428L, and N434S mutations in Fc (EU numbering).

Fig. 45 shows the antibody-dependent cytotoxicity of certain antibodies of the invention using primary NK cells as effector cells and SARS-CoV-2-expressing ExpiCHO cells as target cells. See Example 14. The graph shows the percentage of target cell killing following incubation with the S309 LS (also referred to herein as S309 MLNS), S309 GRLR (G236R/L328R; non-FcR binding variant), or S309 LS GAALIE (also referred to herein as S309 MLNS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L, and N434S (EU numbering) antibodies).

Fig. 46 shows neutralization of SARS-CoV-2 infection in human lung Calu-3 cells (a human lung cancer cell line) and VeroE6 cells using the S309 N55Q MLNS antibody as described in Example 24.

Figure 47 shows the neutralization of SARS-CoV-2 infection by the S309 antibody (VH SEQ ID NO:105; VL SEQ ID NO:168) as detected by the nanoluciferase assay. See Example 25. The x-axis shows the antibody concentration. The three curves represent assays using three different virus concentrations in MOI (multiplicity of infection) units, as shown in the figure on the right. Data were collected six hours after SARS-CoV-2 virus infection. The calculated IC50 (half-maximal inhibitory concentration) values for each MOI are shown in the boxes below the graph.

Figures 48A and 48B show neutralization of SARS-CoV-2 infection by the S309 antibody (VH SEQ ID NO:105; VL SEQ ID NO:168) as analyzed by IFA (immunofluorescence assay). See Example 25. Data were collected six hours after infection with the SARS-CoV-2 virus. Figure 48A shows photographs of representative wells in which cell nuclei were stained blue and SARS-CoV-2 nucleocapsids were stained red, which is more clearly visible in the photographs. Each red spot in each image represents an individual infected cell. Neutralization of infection by the S309 antibody can be observed as a decrease in the number of bright spots indicating infected cells as the concentration of S309 was increased. Antibody concentrations are shown on the top and virus concentrations in MOI (multiplicity of infection) units are shown on the left. Fig. 48B shows quantitative data from the IFA assay. The calculated IC50 values for each MOI are shown in the boxes below the graph.

Fig. 49 shows the neutralization of SARS-CoV-2 infection by the antibodies S309 N55Q LS (also referred to herein as S309 N55Q MLNS, comprising the Fc mutations M428L/N434S (EU numbering)) and S309 N55Q LS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE, comprising the Fc mutations G236A, A330L, I332E, M428L, and N434S (EU numbering)). See Example 26. S309 N55Q LS and S309 N55Q LS GAALIE each comprise a VH having the amino acid sequence set forth in SEQ ID NO: 113 and a VL having the amino acid sequence set forth in SEQ ID NO: 168. Data are the mean of four replicates, +/- standard deviation. The graph shown is representative of three independent experiments.

Figures 50A and 50B show neutralization of SARS-CoV-2 pseudotyped viruses using S309 N55Q LS (also referred to herein as S309 N55Q MLNS, containing the Fc mutations M428L/N434S (EU numbering)) (Figure 50A) and S309 N55Q LS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L, and N434S (EU numbering)) antibodies (Figure 50B). See Example 27. Pseudotyped viruses are pseudotyped VSVs with the SARS-CoV-2 spike protein. Data are the mean of four replicates, +/- standard deviation. Each graph shown is representative of four independent experiments.

Figures 51A and 51B show the binding of S309 N55Q MLNS (Figure 51A) and S309 N55Q MLNS GAALIE (Figure 51B) antibodies to the SARS-CoV-2 RBD measured by surface plasmon resonance (SPR). See Example 28. Values are from two independent experiments.

Figures 52A and 52B show the binding of S309 N55Q LS (also referred to herein as S309 N55Q MLNS, containing the Fc mutations M428L/N434S (EU numbering)) (Figure 52A) and S309 N55Q LS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L, and N434S (EU numbering)) (Figure 52B) antibodies to the cell surface expressed SARS-CoV-2 spike protein as measured by flow cytometry. See Example 29. Data are expressed as the percentage of cells identified as positive for antibody binding. The results presented are obtained from one experiment and are representative of three independent separate experiments.

Figure 53 shows the binding of S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to human FcγRIIa (both low affinity R131 and high affinity H131 alleles), FcγRIIIa (both low affinity F158 and high affinity V158 alleles), and FCγRIIb using SPR. See Example 30. Biotinylated purified FcγRs were captured on the sensor chip surface prior to antibody injection. The association and dissociation profiles (separated by the vertical dashed line in each graph) were measured in real time as the change in SPR signal.

Fig. 54 shows the binding of antibodies S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) LS (also referred to herein as S309 MLNS, containing the Fc mutations M428L/N434S), S309 N55Q (VH: SEQ ID NO:113; VL: SEQ ID NO:168) LS (also referred to herein as S309 N55Q MLNS, containing the Fc mutations M428L/N434S (EU numbering)) and S309 N55Q LS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L and N434S (EU numbering)) to complement component C1q, as measured by BLI at Octet instrument. See Example 31. The association and dissociation profiles (separated by the vertical dotted line in the graph) were measured in real time as the change in the interference pattern.

Fig. 55A-55D show in vitro activation of human FcγR by the antibodies S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) LS (also referred to herein as S309 MLNS, containing the Fc mutations M428L/N434S), S309 N55Q (VH: SEQ ID NO:113; VL: SEQ ID NO:168) LS (also referred to herein as S309 N55Q MLNS, containing the Fc mutations M428L/N434S) (EU numbering), and S309 N55Q LS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L, and N434S (EU numbering) EU), along with the negative control antibody S309 GRLR. See Example 32. CHO cells stably transfected with the SARS-CoV-2 spike protein served as antibody targets. Serial dilutions of the antibody were incubated with target cells at room temperature for 15 min. Jurkat effector cells expressing the indicated FcγR and engineered with the NFAT-mediated luciferase reporter were resuspended in assay buffer and then added to the assay plates. After incubation at 37°C for 18 h, Bio-Glo™ Luciferase Assay Reagent (Promega) was added and luminescence was quantified using a Liminometer (Bio-Tek). Graphs show activation of human FcγRIIa (top left), FcγRIIb (top right), low-affinity FcγRIIIa allele F158 (bottom left), and high-affinity FCγRIIb allele V158 (bottom right). Means +/- standard deviation of replicates are shown.

Fig. 56A and 56B show NK cell-mediated killing (ADCC) of SARS-CoV-2 spike protein-expressing cells in vitro in the presence of antibodies S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) LS (also referred to herein as S309 MLNS, containing the Fc mutations M428L/N434S), S309 N55Q (VH: SEQ ID NO:105; VL: SEQ ID NO:168) LS (also referred to herein as S309 N55Q MLNS, containing the Fc mutations M428L/N434S (EU numbering)) or S309 N55Q LS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L, and N434S (EU numbering) or the S309 GRLR control antibody. See Example 33. Serial dilutions of the antibody (serially diluted 10-fold in AIM-V medium from 40,000 ng/mL to 0.075 ng/mL) were incubated with CHO-CoV-2 spike protein cells for 10 min before mixing with NK cells for 4 h. NK cells were freshly isolated from two donors previously genotyped for homozygous expression of low affinity (F/F158; Fig. 56A) or high affinity (V/V158; Fig. 56B). ADCC was measured using an LDH (lactate dehydrogenase) release assay. Shown are mean values +/- standard deviation of four replicates.

Fig. 57 shows monocyte-mediated phagocytosis (ADCC) of cells expressing the SARS-CoV-2 spike protein in vitro in the presence of antibodies S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) LS (also referred to herein as S309 MLNS, containing the Fc mutations M428L/N434S), S309 N55Q (VH: SEQ ID NO:113; VL: SEQ ID NO:168) LS (also referred to herein as S309 N55Q MLNS, containing the Fc mutations M428L and N434S (EU numbering)) or S309 N55Q LS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L and N434S (EU numbering) or the S309 GRLR control antibody. See Example 33. Antibodies were incubated with PKH67-labeled CHO-CoV-2 spike protein-labeled cells for 10 min before mixing with freshly isolated Cell Trace Violet-labeled PBMCs. ADCP activity was measured after overnight incubation by flow cytometry as the percentage of CD14+ monocytes that were double positive for PKH67 and cell trace violet. Mean values +/- standard deviation of replicates are shown.

Fig. 58A and 58B show that antibody S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) inhibits SARS-CoV-2 spike protein-mediated cell fusion. See Example 34. Fig. 58A shows photomicrographs of cells engineered to overexpress SARS-CoV-2 spike protein in the presence (bottom) or absence (top) of S309. Fig. 58B shows quantitative data from the fusion inhibition assay at varying antibody concentrations.

Figure 59 shows plaque-forming unit (FFU) assay data demonstrating that the S309 N55Q antibody variant does not cause antibody-mediated enhancement of SARS-CoV-2 replication in donor-derived PBMCs or human dendritic cells. See Example 35.

Fig. 60 shows the expression (immunofluorescence) of DC-SIGN (dendritic cell intercellular adhesion molecule 3-grasping non-integrin)/L-SIGN (liver/lymph node intercellular adhesion molecule 3-grasping non-integrin), DC-SIGN, and ACE2 transgenes in HEK293T (human embryonic kidney 293 expressing SV40 large T antigen) cells engineered to overexpress the indicated protein. See Example 37.

Figure 61 shows the levels of VSV pseudovirus infection in wild-type HEK293T cells and in HEK293T cells engineered to overexpress DC-SIGN, L-SIGN, or ACE2. The pseudovirus expressed recombinant SARS-CoV-2 spike protein with a luciferase reporter. See Example 37.

Fig. 62 shows neutralization of VSV pseudovirus infection by monoclonal antibody S309 (VH SEQ ID NO:105, VL SEQ ID NO:168) in HEK293T cells engineered to overexpress DC-SIGN, L-SIGN, or ACE2. In this example, antibody S309 contains Fc mutations M428L and N434S (EU numbering). See Example 37.

Fig. 63 shows levels of infection with live SARS-CoV-2 in wild-type HEK293T cells and in HEK293T cells engineered to overexpress DC-SIGN, L-SIGN, or ACE2. Infection was determined using a recombinant S protein with a luciferase reporter. See Example 37.

Fig. 64 shows neutralization of live SARS-CoV-2 infection in HEK293T cells engineered to overexpress DC-SIGN, L-SIGN, or ACE2 by an exemplary monoclonal antibody S309 (VH SEQ ID NO:105, VL SEQ ID NO:168). In this example, antibody S309 contains Fc mutations M428L and N434S (EU numbering). See Example 37.

Fig. 65 shows the expression (immunofluorescence) of L-SIGN, DC-SIGN, SIGLEC1, and ACE2 transgenes in HEK293T cells engineered to overexpress the indicated protein(s). See Example 37.

Fig. 66 shows levels of infection with live SARS-CoV-2 in wild-type HEK293T cells and in HEK293T cells engineered to overexpress DC-SIGN, L-SIGN, SIGLEC-1, or ACE2. Infection was determined using a recombinant S protein with a luciferase reporter. See Example 37.

Fig. 67 shows neutralization of live SARS-CoV-2 virus infection in HEK293T cells engineered to overexpress DC-SIGN, L-SIGN, SIGLEC-1, or ACE2 by an exemplary monoclonal antibody S309 (VH SEQ ID NO:105, VL SEQ ID NO:168). In this example, antibody S309 contains Fc mutations M428L and N434S (EU numbering). See Example 37.

Fig. 68A and 68B show an analysis of the expression of receptor proteins, including CD209 (DC-SIGN) and SIGLEC proteins, in several cell types. The size of the dot correlates with the percentage of cells of the indicated cell type that express the protein, and the intensity of the shading of the dot correlates with the level of protein expression. See Example 37.

Figure 69 shows infection with live SARS-CoV-2 expressing N-luciferase in HEK293T cells (“parent”) or HEK293T cells stably expressing DC-SIGN, L-SIGN, SIGLEC-1, or ACE2. The data represent experiments testing SARS-CoV-2 at three multiplicities of infection (MOI). See Example 37.

Fig. 70 shows VSV infection with pseudotyped SARS-CoV-2 in HEK293T cells, HeLa cells, and MRC5 cells transiently transduced with lentivirus to express DC-SIGN, L-SIGN, SIGLEC-1, or ACE2. Uninfected cells are shown as a negative control. See Example 37.

Fig. 71A and 71B show binding (measured by biolayer interferometry) of wild-type S309, S309 N55Q MLNS, S309 N55Q MLNS GAALIE (Fig. 71A), and comparative antibodies REGN10933 and REGN10987 (Fig. 71B) to mutated RBD variants. See Example 39.

Fig. 72 shows the neutralization of SARS-CoV-2 ("WT" is Wuhan-Hu-1; "UK" is SARS-CoV-2 variant B.1.1.7; and "SA" is variant B.1.351) pseudovirus MLV in Vero-E6 cells by S309 antibody as described in Example 39. Comparative antibodies REGN10987, REGN10933, and the combination of REGN10987 + REGN10933 were also evaluated.

Figures 73A-73D show that S309 (VH SEQ ID NO:105; VL SEQ ID NO:168) provides robust protection against in vivo SARS-CoV-2 infection. Syrian hamsters were administered the indicated amount of mAb 48 hours prior to intranasal SARS-CoV-2 infection. (A) Quantification of viral RNA (ribonucleic acid) in the lungs 4 days post-infection. (B) Quantification of replicating virus in lung homogenates collected 4 days post-infection using a TCID50 assay. (C) Histological score of lung tissue was assessed 4 days post-infection. (D) mAb concentration measured in serum before infection (day 0) is inversely correlated with viral RNA load in the lung 4 days after infection. See Example 38.

Detailed description of the invention

Provided herein are antibodies and antigen-binding fragments that are capable of binding to SARS-CoV-2 (e.g., to the SARS-CoV-2 surface glycoprotein and/or RBD as described herein, in the SARS-CoV-2 virion and/or expressed on the surface of a host cell, such as a cell infected with SARS-CoV-2). The host cell can be, for example, a lung cell, a CHO cell (such as, for example, an ExpiCHO cell transfected to express the surface glycoprotein), or the like. In some embodiments, the antibodies and antigen-binding fragments disclosed herein can neutralize SARS-CoV-2 infection in an in vitro infection model and/or in a human subject. Also provided are polynucleotides encoding antibodies and antigen-binding fragments, vectors, host cells and related compositions, as well as methods of using antibodies, nucleic acids, vectors, host cells and related compositions for treating (e.g., reducing, delaying, eliminating or preventing) a SARS-CoV-2 infection in a subject and/or for producing a medicament for treating a SARS-CoV-2 infection in a subject.

Before describing this invention in more detail, it may be helpful to understand it by defining some terms that will be used herein. Additional definitions are provided throughout this description of the invention.

In the context of this document, "SARS-CoV-2", also referred to herein as "Wuhan coronavirus" or "Wuhan seafood market pneumonia virus" or "Wuhan CoV" or "novel CoV" or "nCoV" or "2019 nCoV" or "Wuhan nCoV", is a betacoronavirus presumed to belong to the B genus (sarbecovirus). SARS-CoV-2 was first identified in Wuhan, Hubei Province, China, in late 2019 and had spread to China and other parts of the world by early 2020. Symptoms of SARS-CoV-2 include fever, dry cough, and shortness of breath.

The genomic sequence of SARS-CoV-2 isolate Wuhan-Hu-1 is presented as SEQ ID NO:163 (see also GenBank MN908947.3, 2020-01-23), and the amino acid translation of the genome is presented as SEQ ID NO:164 (see also GenBank QHD43416.1, 2020-01-23). Like other coronaviruses (e.g., SARS CoV), SARS-CoV-2 contains a type I transmembrane "spike" or surface ("S") glycoprotein containing a receptor-binding domain (RBD). The RBD is thought to mediate entry of lineage B SARS coronavirus into respiratory epithelial cells by binding to angiotensin-converting enzyme 2 (ACE2). Specifically, the receptor-binding motif (RBM) in the viral RBD is thought to interact with ACE2.

The amino acid sequence of the SARS-CoV-2 Wuhan-Hu-1 surface glycoprotein (S) is shown in SEQ ID NO:165. The antibodies and antigen-binding fragments of the present invention are capable of binding to a SARS CoV-2 surface glycoprotein (S), such as Wuhan-Hu-1. For example, in some embodiments, the antibody or antigen-binding fragment binds to an epitope in the RBD of the Wuhan-Hu-1 S protein.

The amino acid sequence of the RBD of SARS-CoV-2 Wuhan-Hu-1 is shown as SEQ ID NO:166. The S protein of SARS-CoV-2 Wuhan-Hu-1 shares approximately 73% amino acid sequence identity with the S protein of SARS-CoV. The amino acid sequence of the RBM of SARS-CoV-2 Wuhan-Hu-1 is shown as SEQ ID NO:167. The SARS-CoV-2 RBD shares approximately 75 to 77% amino acid sequence similarity with the RBD of SARS coronavirus, and the SARS-CoV-2 Wuhan Hu-1RBM shares approximately 50% amino acid sequence similarity with the RBM of SARS coronavirus.

Unless otherwise stated herein, SARS-CoV-2 Wuhan Hu-1 refers to a virus comprising the amino acid sequence set forth in any one or more of SEQ ID NOs:164, 165, and 166, optionally with the genomic sequence set forth in SEQ ID NO:163.

There are a number of emerging SARS-CoV-2 variants. Some SARS-CoV-2 variants contain the N439K mutation, which has increased binding affinity for the human ACE2 receptor (Thomson, E.C., et al., The circulating SARS-CoV-2 spike variant N439K maintains fitness while evading antibody-mediated immunity. bioRxiv, 2020). Several SARS-CoV-2 variants contain the N501Y mutation, which is associated with increased transmissibility, including the B.1.1.7 (also known as 20I/501Y.V1 and VOC 202012/01; (del69-70, del144, N501Y, A570D, D614G, P681H, T716I, S982A, and D1118H mutations)) and B.1.351 (also known as 20H/501Y.V2; L18F, D80A, D215G, R246I, K417N, E484K, N501Y, D614G, and A701V mutations) lineages, which were detected in the United Kingdom and South Africa, respectively (Tegally, H., et al. al, Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv, 2020: p. 2020.12.21.2024864; Leung, K., et al., Early empirical assessment of the N501Y mutant strains of SARS-CoV-2 in the United Kingdom, October to November 2020. medRxiv, 2020: p. 2020.12.20.20248581). B.1.351 also contains two other mutations in the RBD domain of the SARS-CoV2 spike protein, K417N and E484K (Tegally, H., et al., Emergence and rapid spread of a new severe acute respiratory syndrome-related coronavirus 2 (SARS-CoV-2) lineage with multiple spike mutations in South Africa. medRxiv, 2020: p. 2020.12.21.202486400). Other SARS-CoV-2 variants include the B.1.1.28 lineage, which was first reported in Brazil; the P.1 variant, B.1.1.28 lineage (also known as 20J/501Y.V3), which was first reported in Japan; variant L452R, which was first reported in California in the United States (Pan American Health Organization, Epidemiological update: Occurrence of variants of SARS-CoV-2 in the Americas, January 20, 2021, available at reliefweb.int/sites/reliefweb.int/files/resources/2021-jan-20-phe-epi-update-SARS-CoV-2.pdf). Other SARS-CoV-2 variants include SARS CoV-2 group 19A; SARS CoV-2 group 19B; SARS CoV-2 group 20A; SARS CoV-2 group 20B; SARS CoV-2 group 20C; SARS CoV-2 group 20D; SARS CoV-2 group 20E (EU1); SARS CoV-2 group 20F; SARS CoV-2 group 20G; and SARS CoV-2 B1.1.207; and other SARS CoV-2 lineages described in Rambaut, A., et al., A dynamic nomenclature proposal for SARS-CoV-2 lineages to assist genomic epidemiology. Nat Microbiol 5, 1403–1407 (2020). The above SARS-CoV-2 variants and their amino acid and nucleotide sequences are incorporated herein by reference.

In the present specification, any concentration range, percentage range, ratio range, or integer range recited herein shall be understood to include the value of any whole number within the stated range and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise stated. In addition, any range of numbers recited herein that relates to any physical feature, such as polymer subunits, size, or thickness, shall be understood to include any whole number within the stated range, unless otherwise stated. As used herein, "about" means ±20% of the stated range, value, or structure, unless otherwise stated. It shall be understood that the terms "a" or "the" as used herein are intended to mean "one or more" of the listed terms. Thus, the use of an alternative (e.g., "or") shall be understood to mean one, both, or any combination of these alternatives. In the context of this document, "include", "have" and "contain" are used synonymously, and these terms and their variations are to be construed as non-limiting.

“Optional” or “optional” means that the element, component, event, or circumstance described below does not necessarily have to occur and that the description includes instances in which the element, component, event, or circumstance occurs and instances in which it does not.

It should also be understood that individual constructs or groups of constructs derived from various combinations of the structures and subunits described herein are disclosed herein to the same extent as if each construct or group of constructs were set forth separately. Thus, the selection of particular structures or particular subunits is within the scope of the present invention.

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

As used herein, "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogues and amino acid mimetics that function similarly to naturally occurring amino acids. Natural amino acids are those amino acids encoded by the genetic code as well as those amino acids that are subsequently modified, such as hydroxyproline, γ-carboxyglutamate and O-phosphoserine. Amino acid analogues refer to compounds that have the same basic chemical structure as a naturally occurring amino acid, that is, an α-carbon that is bonded to a hydrogen, a carboxyl group, an amino group and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogues have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refer to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function similarly to the naturally occurring amino acid.

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

"Conservative substitution" refers to amino acid substitutions that do not significantly affect or change the binding characteristics of a particular protein. Generally, conservative substitutions are substitutions in which the substituted amino acid residue is replaced with an amino acid residue that has a similar side chain. Conservative substitutions include substitutions based on one of the following groups: Group 1: alanine (Ala or A), glycine (Gly or G), serine (Ser or S), threonine (Thr or T); Group 2: aspartic acid (Asp or D), glutamic acid (Glu or Z); Group 3: asparagine (Asn or N), glutamine (Gln or Q); Group 4: arginine (Arg or R), lysine (Lys or K), histidine (His or H); Group 5: isoleucine (Ile or I), leucine (Leu or L), methionine (Met or M), valine (Val or V); and group 6: phenylalanine (Phe or F), tyrosine (Tyr or Y), tryptophan (Trp or W). Additionally or alternatively, amino acids may be grouped into conservative substitution groups based on similar function, chemical structure, or composition (e.g., acidic, basic, aliphatic, aromatic, or sulfur-containing). For example, an aliphatic group might include, for purposes of substitution, Gly, Ala, Val, Leu, and Ile. Other conservative substitution groups include: sulfur-containing: Met and cysteine (Cys or C); acidic: Asp, Glu, Asn, and Gln; small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro, and Gly; polar, negatively charged residues and their amides: Asp, Asn, Glu, and Gln; polar, positively charged residues: His, Arg, and Lys; large aliphatic, nonpolar residues: Met, Leu, Ile, Val, and Cys; and large aromatic residues: Phe, Tyr, and Trp. Further information can be found in Creighton (1984) Proteins, W.H. Freeman and Company.

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

"Nucleic acid molecule" or "polynucleotide" or "polynucleic acid" refers to a polymeric compound comprising covalently linked nucleotides that may consist of natural subunits (e.g., purine or pyrimidine bases) or non-natural subunits (e.g., a morpholine ring). Purine bases include adenine, guanine, hypoxanthine, and xanthine, and pyrimidine bases include uracil, thymine, and cytosine. Nucleic acid molecules include polyribonucleic acid (RNA), which includes mRNA (messenger RNA), microRNA, siRNA (small interfering RNA), viral genomic RNA, and synthetic RNA, and polydeoxyribonucleic acid (DNA), which includes cDNA (complementary DNA), genomic DNA, and synthetic DNA, each of which may be single-stranded or double-stranded. In the case of a single-stranded chain, a nucleic acid molecule may be a coding strand or a non-coding (antisense) strand. A nucleic acid molecule encoding an amino acid sequence includes all nucleotide sequences encoding the same amino acid sequence. Some nucleotide sequence variants may also include an intron(s) to the extent that the intron(s) will be removed by co- or post-transcriptional mechanisms. In other words, different nucleotide sequences may encode the same amino acid sequence as a result of redundancy or degeneracy in the genetic code or by splicing.

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

"Percent sequence identity" refers to the relationship between two or more sequences as determined by sequence comparison. Preferred methods for determining sequence identity are designed to ensure the best match between the sequences being compared. For example, sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in one or both of the first and second amino acid or nucleotide sequences for optimal alignment). Additionally, non-homologous sequences may be ignored for comparison purposes. The percent sequence identity reported herein is calculated over the length of the reference sequence unless otherwise stated. Methods for determining sequence identity and similarity can be found in publicly available computer programs. Sequence alignment and percent identity calculations can be performed using a BLAST program (e.g., BLAST 2.0, BLASTP, BLASTN, or BLASTX). The mathematical algorithm used in the BLAST programs can be found in Altschul et al., Nucleic Acids Res. 25:3389-3402, 1997. In the context of this document, it should be understood that for analysis using analytical software, the results of the analysis are based on the "default values" of the referenced program. "Default values" means any set of values or metrics that are initially loaded with the software when it is first run.

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

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

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

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

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

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

In some embodiments, a nucleic acid molecule or portion thereof native to a host cell will be considered heterologous to the host cell if it has been altered or mutated, or a nucleic acid molecule native to a host cell may be considered heterologous if it has been altered by a heterologous expression control sequence or has been altered by an endogenous expression control sequence not normally associated with a nucleic acid molecule native to the host cell. Additionally, the term "heterologous" may refer to a biological activity that is different, altered, or not endogenous to the host cell. As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as individual nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding an antibody or antigen-binding fragment (or other polypeptide), or any combination thereof.

As used herein, the term "endogenous" or "native" refers to a polynucleotide, gene, protein, compound, molecule, or activity that is normally present in a host cell or subject.

In the context of this document, "expression" in this context refers to the process by which a polypeptide is produced based on the coding sequence of a nucleic acid molecule, such as a gene. The method may include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post-translational modification, or any combination thereof. The expressed nucleic acid molecule is typically operably linked to an expression control sequence (e.g., a promoter).

The term "operably linked" refers to the association of two or more nucleic acid molecules on a single nucleic acid fragment such that the function of one is dependent on the other. For example, a promoter is operably linked to a coding sequence if it is capable of regulating the expression of said coding sequence (i.e., the coding sequence is under the transcriptional control of said promoter). "Unlinked" means that the associated genetic elements are not closely related to each other and the function of one does not affect the other.

As described herein, more than one heterologous nucleic acid molecule can be introduced into a host cell as individual nucleic acid molecules, as a plurality of individually controlled genes, as a polycistronic nucleic acid molecule, as a single nucleic acid molecule encoding a protein (e.g., an antibody heavy chain), or any combination thereof. When two or more heterologous nucleic acid molecules are introduced into a host cell, it is understood that the two or more heterologous nucleic acid molecules can be introduced as a single nucleic acid molecule (e.g., on a single vector), on separate vectors, integrated into the host chromosome at a single site or multiple sites, or any combination thereof. The number of said heterologous nucleic acid molecules or protein activities refers to the number of encoding nucleic acid molecules or the number of protein activities, and not to the number of individual nucleic acid molecules introduced into the host cell.

The term "construct" refers to any polynucleotide that comprises a recombinant nucleic acid molecule (or, where the context clearly indicates, a fusion protein of the present invention). The construct (polynucleotide) may be present in a vector (e.g., a bacterial vector, a viral vector) or may be integrated into a genome. A "vector" is a nucleic acid molecule that is capable of transporting another nucleic acid molecule. Vectors may be, for example, plasmids, cosmids, viruses, an RNA vector, or a linear or circular DNA or RNA molecule, which may include chromosomal, non-chromosomal, semi-synthetic, or synthetic nucleic acid molecules. Vectors of the present invention also include transposon systems (e.g., Sleeping Beauty, see, e.g., Geurts et al., Mol. Ther. 8:108, 2003: Mátés et al., Nat. Genet. 41:753, 2009). Exemplary vectors are those capable of autonomous replication (episomal vector), capable of delivering a polynucleotide into the genome of a cell (e.g., a viral vector), or capable of expressing nucleic acid molecules to which they are linked (expression vectors).

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

The term "introduced" in the context of inserting a nucleic acid molecule into a cell means "transfection," "transformation," or "transduction," and includes reference to the incorporation of a nucleic acid molecule into a eukaryotic or prokaryotic cell, where the nucleic acid molecule may be incorporated into the cell's genome (e.g., chromosomes, plasmids, plastids, or mitochondrial DNA), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA).

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

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

"Retroviruses" are viruses that have an RNA genome that is reverse transcribed into DNA using the enzyme reverse transcriptase, and the reverse transcribed DNA is then incorporated into the host cell's genome. "Gammaretrovirus" refers to a genus of retroviruses (Retroviridae). Examples of gammaretroviruses include murine stem cell virus, murine leukemia virus, feline leukemia virus, feline sarcoma virus, and avian reticuloendotheliosis viruses.

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

In some embodiments, the viral vector may be a gammaretrovirus, such as vectors derived from Moloney murine leukemia virus (MLV). In other embodiments, the viral vector may be a more complex vector derived from a retrovirus, such as a vector derived from a lentivirus. Vectors derived from HIV-1 fall into this category. Other examples include lentiviral vectors derived from HIV-2, FIV (feline immunodeficiency virus), equine infectious anemia virus, SIV, and Maedi-Visna virus (ovine lentivirus). Methods of using retroviral and lentiviral viral vectors and packaging cells to transduce mammalian host cells with viral particles containing transgenes are known in the art and have been previously described, for example, in: U.S. Patent 8,119,772; Walchli et al., PLoS One 6:327930, 2011; Zhao et al., J. Immunol. 174:4415, 2005; Engels et al., Hum. Gene Ther. 14:1155, 2003; Frecha et al., Mol. Ther.18:1748, 2010; and Verhoeyen et al., Methods Mol. Biol. 506:97, 2009. Retroviral and lentiviral vector constructs and expression systems are also commercially available. Other viral vectors can also be used to deliver polynucleotides, including DNA viral vectors, including, for example, adenovirus-based vectors and adeno-associated virus (AAV)-based vectors; vectors derived from herpes simplex viruses (HSV), including amplicon vectors, replication-defective HSV, and attenuated HSV (Krisky et al., Gene Ther. 5:1517, 1998).

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

When the viral vector genome contains multiple polynucleotides to be expressed in the host cell as separate transcripts, the viral vector may also contain additional sequences between two (or more) transcripts to allow bicistronic or multicistronic expression. Examples of such sequences used in viral vectors include internal ribosome entry sites (IRES), furin cleavage sites, viral peptide 2A, or any combination thereof.

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

In this context, the term "host" refers to a cell or microorganism intended to be genetically modified with a heterologous nucleic acid molecule to produce a polypeptide of interest (e.g., an antibody of the present invention).

A host cell may include any single cell or cell culture that can receive the vector or incorporation of nucleic acids or expression proteins. The term also encompasses progeny of the host cell, whether genetically or phenotypically the same or different. Suitable host cells may depend on the vector and may include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells. These cells may be induced to incorporate the vector or other substance by a viral vector, transformation by calcium phosphate precipitation, DEAE-dextran, electroporation, microinjection, or other means. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual 2d ed. (Cold Spring Harbor Laboratory, 1989).

In the context of SARS-CoV-2 infection, "host" refers to a cell or subject (e.g., a human) infected with SARS-CoV-2.

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

The term "epitope" or "antigenic epitope" includes any molecule, structure, amino acid sequence, or protein determinant that is recognized and specifically bound by a cognate binding molecule such as an immunoglobulin or other binding molecule, domain, or protein. Epitopic determinants typically comprise chemically active surface groups of molecules such as amino acid or sugar side chains and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. If the antigen is or comprises a peptide or protein, the epitope may be composed of consecutive amino acids (e.g., a linear epitope) or may be composed of amino acids from different parts or regions of the protein that are brought into proximity by protein folding (e.g., a discontinuous or conformational epitope) or non-contiguous amino acids that are in close proximity regardless of protein folding.

Antibodies and antigen-binding fragments

In one aspect, the present invention relates to an isolated antibody or antigen-binding fragment thereof that comprises a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3 and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, and is capable of binding to the surface glycoprotein (S) of SARS-CoV-2. In some embodiments, the antibody or antigen-binding fragment is capable of binding to the surface glycoprotein (S) of SARS-CoV-2 expressed on the surface of a host cell and/or on a SARS-CoV-2 virion.

In some embodiments, an antibody or antigen-binding fragment of the present invention associates with or combines with a surface epitope or antigen of a SARS-CoV-2 glycoprotein comprising said epitope, while not substantially associating with or combining with any other molecules or components in the sample.

In some embodiments, an antibody or antigen-binding fragment of the present invention binds or associates (e.g., binds to) a surface epitope of a SARS-CoV-2 glycoprotein, and may also bind or associate with an epitope from another coronavirus (e.g., SARS CoV) present in a sample but not substantially associated with or associated with any other molecules or components in the sample. In other words, in some embodiments, an antibody or antigen-binding fragment of the present invention is cross-reactive with SARS-CoV-2 and one or more additional coronaviruses.

In some embodiments, an antibody or antigen-binding fragment of the present invention specifically binds to a SARS-CoV-2 surface glycoprotein. As used herein, "specifically binds" refers to the association or association of the antibody or antigen-binding fragment with the antigen with an affinity or K _a (i.e., the equilibrium association constant of a particular binding interaction with units of 1/M) of 10 ⁵ M ^-1 or greater (which is equal to the ratio of the rate of association [K _on ] to the rate of dissociation [K _off ] for that association reaction), while not significantly associating or associating with any other molecules or components in the sample. Alternatively, the affinity can be defined as the equilibrium dissociation constant (K _d ) of a particular binding interaction, expressed in units of M (e.g., from 10 ^-5 M to 10 ^-13 M). Antibodies can be classified as "high-affinity" antibodies or as "low-affinity" antibodies. "High-affinity" antibodies refer to those antibodies that have a K _a of at least 10 ⁷ M ^-1 , at least 10 ⁸ M ^-1 , at least 10 ⁹ M ^-1 , at least 10 ¹⁰ M ^-1 , at least 10 ¹¹ M ^-1 , at least 10 ¹² M ^-1 , or at least 10 ¹³ M ^-1 . "Low-affinity" antibodies refer to those antibodies that have a K _a of up to 10 ⁷ M ^-1 , up to 10 ⁶ M ^-1 , up to 10 ⁵ M ^-1 . Alternatively, affinity can be defined as the equilibrium dissociation constant (K _d ) of a particular binding interaction, expressed in units of M (e.g., 10 ^-5 M to 10 ^-13 M).

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

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

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

In some embodiments, an antibody or antigen-binding fragment of the present invention binds to the SARS-CoV-2 S protein as measured by biolayer interferometry. In some embodiments, an antibody or antigen-binding fragment of the present invention binds to the SARS-CoV-2 S protein with a K _D of less than about 4.5×10 ^-9 M, less than about 5×10 ^-9 M, less than about 1×10 ^-10 M, less than about 5×10 ^-10 M, less than about 1×10 ^-11 M, less than about 5×10 ^-11 M, less than about 1×10 ^-12 M, or less than about 5×10 ^-12 M. In some embodiments, an antibody or antigen-binding fragment of the present invention binds to the RBD of the SARS-CoV-2 S protein with a K _D of less than about 4.5×10 ^-9 M, less than about 5×10 ^-9 M, less than about 1×10 ^-10 M, less than about 5×10 ^-10 M, less than about 1×10 ^-11 M, less than about 5×10 ^-11 M, less than about 1×10 ^-12 M, or less than about 5×10 ^-12 M. In some embodiments, an antibody or antigen-binding fragment of the present invention binds to the SARS-CoV-2 S protein (e.g., a glycosylated or deglycosylated S protein RBD) with a K _D , k _a , and/or k _d as shown in Table 8, Table 9, or Table 10 herein.

In particular embodiments, the antibody or antigen-binding fragment thereof is capable of binding to the glycosylated S protein RBD with a K _D of about 0.35 nM, about 0.36 nM, about 0.37 nM, about 0.38 nM, about 0.39 nM, about 0.40 nM, about 0.41 nM, about 0.42 nM, about 0.43 nM, about 0.44 nM, about 0.45 nM, about 0.46 nM, about 0.47 nM, about 0.48 nM, about 0.49 nM, about 0.50 nM, about 0.51 nM, or about 1.7 nM, optionally as measured by surface plasmon resonance, and/or with a _ka of about 8.5e4 1/Ms, about 8.6e4 1/Ms, about 8.7e4 1/Ms, about 8.8e4 1/Ms, about 8.9e4 1/Ms, about 9.0e4 1/Ms, about 9.1e4 1/Ms, about 9.2e4 1/Ms, about 9.3e4 1/Ms, about 9.4e4 1/Ms, about 9.5e4 1/Ms, about 9.6e4 1/Ms, about 9.7e4 1/Ms, about 9.8e4 1/Ms, about 9.9e4 1/Ms or about 1.0e5 1/Ms, optionally when measured by surface plasmon resonance, and/or with a k _d of about 1.6e-4 1/s, about 3.3e-5 1/s, about 3.4e-5 1/s, about 3.5e-5 1/s, about 3.6e-5 1/s, about 3.7e-5 1/s, about 3.8e-5 1/s, about 3.9e-5 1/s, about 3.9e-5 1/s, about 4.0e-5 1/s, about 4.1e-5 1/s, about 4.2e-5 1/s, about 4.3e-5 1/s, about 4.4e-5 1/s, about 4.5e-5 1/s, about 4.6e-5 1/s, about 4.7e-5 1/s, about 4.8e-5 1/s, about 4.9e-5 1/s, about 5.0e-5 1/s approx. 5.1e-5 1/s, about 5.2e-5 1/s, about 5.3e-5 1/s, about 5.4e-5 1/s, about 5.5e-5 1/s, about 5.6e-5 1/s, about 5.7e-5 1/s, about 5.8e-5 1/s, about 5.9e-5 1/s, about 6.0e-5 1/s, about 6.1e-5 1/s, about 6.2e-5 1/s, about 6.3e-5 1/s, about 6.4e-5 1/s or about 6.5e-5 1/s, optionally as measured by surface plasmon resonance.

In some embodiments, the antibody or antigen-binding fragment is capable of binding to a deglycosylated S protein RBD with a K _D of about 0.95, about 0.96 nM, about 0.97 nM, about 0.98 nM, about 0.99 nM, about 1.0 nM, about 1.1 nM, about 1.2 nM, about 1.3 nM, about 1.4 nM, about 1.5 nM, or about 1.6 nM, optionally as measured by surface plasmon resonance, and/or with a _ka of about (1/s) about 2.5e5, about 2.6e5, about 2.7e5, about 2.8e5, about 2.9e5, about 3.0e5, about 3.1e5, optionally as measured by surface plasmon resonance, and/or with a k _d (1/s) of about 2.8e-4, about 2.9e-4, about 3.0e-4, about 3.1e-4, about 3.2e-4, about 3.3e-4, about 3.4e-4, about 3.5e-4, about 3.6e-4, about 3.7e-4, about 3.8e-4, about 3.9e-4, about 4.0e-4, about 4.1e-4, about 4.2e-4, about 4.3e-4, about 4.4e-4, about 4.5e-4, about 4.6e-4 about 4.7e-4, about 4.8e-4, about 4.9e-4, or about 5.0e-4, optionally as measured by surface plasmon resonance resonance.

In some embodiments, to detect binding to the RBD, surface plasmon resonance comprises use conducted using a sensor chip with covalently immobilized anti-human Fc (e.g., from GE). The buffer can be 10 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) at pH 7.4, 150 mM NaCl, 3 mM EDTA (ethylenediaminetetraacetic acid), and 0.05% P20 detergent. SPR can be carried out at 25 °C. Antibodies can be diluted from the supernatant fluid to about 2 μg/mL. RBD concentrations can be 0.8 nM, 3.1 nM, 12.5 nM, 50 nM, and/or 200 nM.

In some embodiments, the antibody or antigen-binding fragment is capable of binding to the receptor binding domain (RBD) of the SARS-CoV-2 surface glycoprotein when the RBD is glycosylated and/or deglycosylated, wherein the binding is determined using surface plasmon resonance (SPR), wherein optionally: (1) SPR is performed using a Biacore T200 instrument using a single-cycle kinetic approach, further optionally with a 3-minute injection period and a 20-minute dissociation period; (2) the antibody or antigen-binding fragment thereof is captured on the surface; (3) the RBD is present at a concentration of 0.8 nM, 3.1 nM, 12.5 nM, 50 nM, or 200 nM; (4) the antibody or antigen-binding fragment binds to the glycosylated RBD with a KD of about 2.0 nM, about 1.9 nM, about 1.8 nM, about 1.7 nM, about 1.6 nM, about 1.5 nM, about 1.4 nM, about 1.3 nM, about 1.2 nM, about 1.1 nM, about 1.0 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, or about 0.4 nM, or with a KD of 0.4 ± 0.05 nM, or with a KD of 0.45 ± 0.05 nM, or with a KD of 0.5 ± 0.05 nM, or with KD 0.6±0.05 nM, or with KD 0.7±0.05 nM, or with KD 1.7±0.05 nM; and/or (5) the antibody or antigen-binding fragment binds to the deglycosylated RBD with a KD of about 37.0 nM, about 8.0 nM, about 2.0 nM, about 1.9 nM, about 1.8 nM, about 1.7 nM, about 1.6 nM, about 1.5 nM, about 1.4 nM, about 1.3 nM, about 1.2 nM, about 1.1 nM, about 1.0 nM, or about 0.9 nM, or with a KD of 37.0 ± 0.05 nM, or with a KD of 8.0 ± 0.05 nM, or with a KD of 1.0 ± 0.05 nM, or with a KD of 0.9 ± 0.05 nM, or with a KD of 1.3 ± 0.05 nM, or with KD 1.8±0.05 nM, or with KD 1.7±0.05 nM.

In some embodiments, an antibody of the present invention is capable of neutralizing infection with SARS-CoV-2. As used herein, a "neutralizing antibody" is an antibody that can neutralize, i.e. prevent, inhibit, reduce, delay, or interfere with the ability of a pathogen to initiate and/or prolong infection in a host. Neutralization can be quantitatively assessed, for example, by assessing SARS-CoV-2 RNA levels in a sample (n, e.g., lung), assessing the viral load of SARS-CoV-2 in a sample (n, e.g., lung), assessing the histopathology of a sample (n, e.g., lung), or the like. The terms "neutralizing antibody" and "antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. In any of the embodiments disclosed herein, the antibody or antigen-binding fragment is capable of preventing and/or neutralizing SARS-CoV-2 infection in an in vitro infection model and/or in an in vivo animal model of infection (e.g., using a Syrian hamster model of intranasal SARS-CoV-2 infection) and/or in humans. In some embodiments, the antibody or antigen-binding fragment of the present invention is capable of neutralizing SARS-CoV-2 infection with an IC90 (the concentration required to inhibit viral replication by 90%) of about 9 μg/mL. In some embodiments, the antibody or antigen-binding fragment of the present invention is capable of neutralizing SARS-CoV-2 infection with an IC50 of about 16 to about 20 μg/mL. In some embodiments, the antibody or antigen-binding fragment is capable of neutralizing SARS-CoV-2 infection or a virus pseudotyped with SARS-CoV-2 with an IC50 of about 0.3 to about 0.4 μg/mL. In some embodiments, the antibody or antigen-binding fragment, or a composition comprising two or more antibodies or antigen-binding fragments of the present invention, is capable of neutralizing SARS-CoV-2 infection or a virus pseudotyped with SARS-CoV-2 with an IC50 of about 0.07 to about 0.08 μg/mL.

In some embodiments, the antibody or antigen-binding fragment (i) recognizes an epitope within the ACE2 receptor binding motif (RBM, SEQ ID NO:167) of SARS-CoV-2; (ii) is capable of blocking the interaction between SARS-CoV-2 and ACE2; (ii) is capable of binding to the S protein of SARS-CoV-2 with greater avidity than the S protein of SARS coronavirus; (iv) is capable of staining about 30%, about 35%, about 40%, about 50%, about 55%, about 56%, about 57%, about 58%, about 59%, about 60%, or more of target cells expressing the SARS-CoV-2 surface glycoprotein in a sample containing about 50,000 target cells (e.g., ExpiCHO cells) in approximately 100 μL when the antibody or antigen-binding fragment is present at a concentration of 10 μg/mL (e.g., staining as determined by flow cytometric ELISA); (v) recognizes an epitope that is conserved in the ACE2 RBM of SARS-CoV-2 and in the ACE2 RBM of SARS coronavirus; (vi) is cross-reactive against SARS-CoV-2 and SARS coronavirus; (vii) recognizes an epitope on the SARS-CoV-2 surface glycoprotein that is absent from the ACE2 RBM; or (viii) any combination of (i)-(vii).

In some embodiments, the antibody or antigen-binding fragment thereof is capable of inhibiting the interaction between: (i) SARS-CoV-2 and human DC-SIGN; (ii) SARS-CoV-2 and human L-SIGN; (iii) SARS-CoV-2 and human SIGLEC-1; or (iv) any combination of (i)-(iii). As described herein, DC-SIGN, L-SIGN, and SIGLEC-1 may be involved in SARS-CoV-2 infection in roles that include binding receptors. Inhibition of the interaction between SARS-CoV-2 and DC-SIGN, L-SIGN, and/or SIGLEC-1 may, in some contexts, neutralize SARS-CoV-2 infection.

In some embodiments, the antibody or antigen-binding fragment thereof is capable of binding to the surface glycoprotein of: (i) SARS-CoV-2 Wuhan-Hu-1 (SEQ ID NO: 165); (ii) SARS-CoV-2 B.1.1.7; (iii) SARS-CoV-2 B.1.351; (iv) SARS-CoV-2 comprising any one or more of the following substitution mutations relative to SEQ ID NO: 165: N501Y; S477N; N439K; L452R; E484K; Y453F; A520S; K417N; K417V; S494P; N501T; S477R; V367F; P384L; A522S; A522V; V382L; P330S; T478I; S477I; P479S; or (v) any combination of (i)-(iv).

Terms understood by those skilled in the art, each of which has the meaning acquired in the art, unless expressly defined otherwise herein. For example, the term "antibody" refers to an intact antibody comprising at least two heavy (H) chains and two light (L) chains linked together by disulfide bonds, as well as any antigen-binding portion or fragment of the intact antibody that has or retains the ability to bind to an antigen target molecule recognized by the intact antibody, such as an scFv, Fab, or Fab'2 fragment. Thus, the term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and functional (antigen-binding) antibody fragments thereof, including antigen-binding fragments (Fab), F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG fragments (rIgG), single-chain antibody fragments, including single-chain variable fragments (scFv), and single-domain antibody fragments (sd) (e.g., sdAb, sdFv, nanobody). The term encompasses genetically engineered and/or otherwise modified forms of immunoglobulins such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies and heteroconjugate antibodies, multispecific, such as bispecific antibodies, diabodies, triabodies, tetrabodies, tandem dia-scFv and tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional fragments thereof. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass, including IgG and its subclasses (IgG1, IgG2, IgG3, IgG4), IgM, IgE, IgA and IgD.

The terms "V _L " or "VL" and "V _H " or "VH" refer to the variable binding region of an antibody light chain and an antibody heavy chain, respectively. In some embodiments, the VL is of the kappa (κ) class (also "VK" herein). In some embodiments, the VL is of the lambda (λ) class. Variable binding regions include discrete, well-defined subregions known as "complementarity determining regions" (CDRs) and "framework regions" (FRs). The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR" and refer to the amino acid sequences within the variable regions of an antibody that generally collectively confer the antigen specificity and/or binding affinity of the antibody, wherein consecutive CDRs (i.e., CDR1 and CDR2, CDR2 and CDR3) are separated from one another in the primary structure by the framework region. Within each variable region, there are three CDRs (HCDR1, HCDR2, HCDR3; LCDR1, LCDR2, LCDR3; also referred to as CDRH and CDRL, respectively). In some embodiments, the VH antibody comprises four FRs and three CDRs as follows: FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4; and the VL antibody contains four FRs and three CDRs as follows: FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4. Typically, VH and VL together form the antigen-binding site through their respective CDRs.

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

The numbering of the CDRs and framework regions may be according to any known method or scheme, such as the Kabat, Chothia, EU, IMGT, and AHo numbering schemes (see, e.g., Kabat et al., "Sequences of Proteins of Immunological Interest, US Dept. Health and Social Services, Public Health Service National Institutes of Health, 1991, 5th ed.; Chothia and Lesk, J. Mol. Biol. 196:901–917 (1987)); Lefranc et al., Dev. Comp. Immunol. 27:55, 2003; Honegger and Plückthun, J. Mol. Bio. 309:657–670 (2001)). Equivalent residue positions can be annotated and compared across molecules using the Antigen Receptor Numbering and Classification Tool (ANARCI) (2016, Bioinformatics 15:298-300). Accordingly, the identification of a CDR of an exemplary variable domain (VH or VL) sequence provided herein according to one numbering scheme does not exclude an antibody comprising a CDR of the same variable domain as defined using a different numbering scheme. In some embodiments, there is provided an antibody or antigen-binding fragment that comprises a CDR from a VH sequence according to any of SEQ ID NOs: 113, 1, 9-15, 23, 24, 27, 28-46, 55, 63, 79, 87, 95, 103, 105, 114-120, 129-146, 155, 172, 176-178, 194, 196, 198, 200, 202, 239, and 267, and from a VL sequence according to any of SEQ ID NOs: 168, 5, 47-50, 59, 67, 71-72, 75, 76, 83, 91, 99, 109, 147-150, 159, 182, 190, 234, and 243, as determined using any known CDR numbering method, including the Kabat, Chothia, EU, IMGT, Martin (improved Chothia), Contact, and AHo numbering methods. In some embodiments, the CDRs correspond to the IMGT numbering method. In some embodiments, the CDRs correspond to the antibody numbering method developed by the Chemical Computing Group (CCG); for example, using the Molecular Operating Environment (MOE) software (www.chemcomp.com).

In some embodiments, there is provided an antibody or antigen-binding fragment that comprises a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein: (i) CDRH1 comprises or consists of an amino acid sequence according to any of SEQ ID NOs: 106, 2, 56, 64, 80, 88, 96, 156, 179, 195, or 240, or a sequence variant thereof comprising one, two, or three acid substitutions, one or more of which is optionally a conservative substitution and/or is a germline encoded amino acid substitution; (ii) CDRH2 comprises or consists of an amino acid sequence according to any of SEQ ID NOs:121, 3, 16-22, 57, 65, 81, 89, 97, 107, 122-126, 157, 180, 197, 199 or 241, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which is optionally a conservative substitution and/or a germline encoded amino acid substitution; (iii) CDRH3 comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 108, 4, 25, 26, 58, 66, 82, 90, 98, 104, 127, 128, 158, 181, 201, 203 or 242, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which optionally is a conservative substitution and/or is a germline encoded amino acid substitution; (iv) CDRL1 comprises or consists of an amino acid sequence according to any one of SEQ ID NOs:169, 6, 51-54, 60, 68, 73, 74, 84, 92, 100, 110, 160, 183, 235 or 244, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which substitutions are optionally a conservative substitution and/or are a germline encoded amino acid substitution; (v) CDRL2 comprises or consists of an amino acid sequence according to any of SEQ ID NOs:170, 7, 61, 69, 85, 93, 101, 111, 161, 184, 236 or 245, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which optionally is a conservative substitution and/or is a germline encoded amino acid substitution; and/or (vi) CDRL3 comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 171, 8, 62, 70, 77, 78, 86, 94, 102, 112, 151, 152, 153, 154, 162, 185, 237 or 246, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which is optionally a conservative substitution and/or a germline encoded amino acid substitution, wherein the antibody or antigen-binding fragment is capable of binding to a SARS-CoV-2 surface glycoprotein expressed on the surface of a host cell, on a virion, or both.

In some embodiments, the antibody or antigen-binding fragment comprises VH and VL amino acid sequences that are encoded by:

(i) a VH1-18 gene and a VK3-20 gene, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH1-18 and VK3-20, respectively;

(ii) the VH3-7 allele and the VL3-25 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH3-7 and VL3-25, respectively;

(iii) the VH3-23 allele and the VK1-5 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH3-23 and VK1-5, respectively;

(iv) the VH3-13 allele and the VK1-39 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH3-13 and VK1-39, respectively;

(v) the VH1-18 allele and the VK3-11 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH1-18 and VK3-11, respectively; or

(vi) the VH1-69 allele and the VL2-23 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH1-69 and VL2-23, respectively.

In any of the embodiments disclosed herein, the antibody or antigen-binding fragment is capable of preventing and/or neutralizing SARS-CoV-2 infection in an in vitro infection model and/or in an in vivo animal infection model and/or in humans.

In any of the embodiments disclosed herein, the antibody or antigen-binding fragment thereof comprises the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 according to SEQ ID NO: (i) 2-4 and 6-8 or 235-237, respectively; (ii) 2, any of 16-22, 4 and 6-8 or 235-237, respectively; (iii) 2, 3, any of 25-26 and 6-8 or 235-237, respectively; (iv) 2-4, 51, 7, and 8, respectively; (v) 2-4, 52, 7, or 236 and 8 or 237, respectively; (vi) 2-4, 53, 7, or 236 and 8 or 237, respectively; (vii) 2-5, 54, 7 or 236 and 8 or 237, respectively; (viii) 56-58 and 60-62, respectively; (ix) 64-66 and 68-70, respectively; (x) 64-66, 73 or 74, 69 and 70, respectively; (xi) 64-66, 68-69 and 77 or 78, respectively; (xii) 80-82 and 84-86, respectively; (xiii) 88-90 and 92-94, respectively; (xiv) 96-98 and 101-102, respectively; (xv) 96, 97, 104 and 100-102, respectively; (xvi) 106-108 and 110-112 or 169-171, respectively; (xvii) 106, any of 121-126, 108 and 110-112, respectively; (xviii) 106, 107, 127 or 128 and 110-112, respectively; (xix) 106-108, 110, 111 and 151, respectively; (xx) 106-108, 110, 111 and 152, respectively; (xxi) 106-108, 110, 111 and 153, respectively; (xxii) 106-108, 110, 111 and 154, respectively; (xxiii) 106, 107, or any of 121-126, 108, or 127, or 128 and 169-171, respectively; (xxiv) 156-158 and 160-162, respectively; (xxv) 106, 123, 127, and 169-171, respectively; (xxvi) 2, 17, 25, 6, or 235, or any of 51-54, 7, or 236, and 8 or 237, respectively; (xxvii) 2, 20, 25, 6, or 235, or any of 51-54, 7, or 236, and 8 or 237, respectively; (xxviii) 179-181 and 183-185, respectively; (xxix) 195, 180, 181 and 183-185, respectively; (xxx) 195, 197, 181 and 183-185, respectively; (xxxi) 195, 199, 181 and 183-185, respectively; (xxxii) 195, 197, 201 and 183-185, respectively; (xxxiii) 195, 197, 203 and 183-185, respectively; (xxxiv) 195, 199, 201 and 183-185, respectively; (xxxv) 195, 199, 203 and 183-185, respectively; (xxxvi) 179, 180, 181 and 183-185, respectively; (xxxvii) 179, 197, 181 and 183-185, respectively; (xxxviii) 179, 199, 181 and 183-185, respectively; (xxxix) 179, 197, 201 and 183-185, respectively; (xxxx) 179, 197, 203 and 183-185, respectively; (xxxxi) 179, 199, 201 and 183-185, respectively; (xxxxii) 179, 199, 203 and 183-185, respectively; (xxxxiii) 179, 180, 201 and 183-185, respectively; (xxxxiv) 179, 180, 203 and 183-185, respectively; and (xxxxv) 240-242 and 244-246, respectively.

In some embodiments, an antibody or antigen-binding fragment of the present invention comprises the amino acid sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NOs: 80-82 and 84-86, respectively. In additional embodiments, an antibody or antigen-binding fragment of the present invention binds to the S protein of SARS-CoV-2 with a K _D of less than about 4.5×10 ^-9 M, less than about 5×10 ^-9 M, less than about 1×10 ^-10 M, less than about 5×10 ^-10 M, less than about 1×10 ^-11 M, less than about 5×10 ^-11 M, less than about 1×10 ^-12 M, or less than about 5×10 ^-12 M. In additional embodiments, an antibody or antigen-binding fragment of the present invention is capable of neutralizing SARS-CoV-2 infection and/or neutralizing infection of a target cell with a virus pseudotyped with SARS-CoV-2 with an IC50 of about 16 to about 20 μg/mL.

In some embodiments, an antibody or antigen-binding fragment of the present invention comprises the amino acid sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 set forth in SEQ ID NOs: 106-108 and 169-171 or 106, 121, 108, and 169-171, respectively. In additional embodiments, an antibody or antigen-binding fragment of the present invention binds to the S protein of SARS-CoV-2 with a K _D of less than about 4.5×10 ^-9 M, less than about 5×10 ^-9 M, less than about 1×10 ^-10 M, less than about 5×10 ^-10 M, less than about 1×10 ^-11 M, less than about 5×10 ^-11 M, less than about 1×10 ^-12 M, or less than about 5×10 ^-12 M. In additional embodiments, an antibody or antigen-binding fragment of the present invention is capable of neutralizing SARS-CoV-2 infection and/or neutralizing infection of a target cell with a virus pseudotyped with SARS-CoV-2 with an IC50 of about 0.3 to about 0.4 μg/mL.

In some embodiments, an antibody or antigen-binding fragment thereof of the present invention comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3, wherein each CDR is independently selected from a corresponding CDR of SARS-CoV-2 S300 mAb, SARS-CoV-2 S300-v1 mAb, SARS-CoV-2 S300-v1.1 mAb, SARS-CoV-2 S300-v1.2 mAb, SARS-CoV-2 S300-v1.3 mAb, SARS-CoV-2 S300-v1.4 mAb, SARS-CoV-2 S300-v1.5 mAb, SARS-CoV-2 S300-v1.6 mAb, SARS-CoV-2 S300-v1.7 mAb, or SARS-CoV-2 S300-v1.8 mAb SARS-CoV-2 S300-v1.9 mAb, SARS-CoV-2 S300-v2 mAb, SARS-CoV-2 S300-v2.1 mAb, SARS-CoV-2 S300-v2.2 mAb, SARS-CoV-2 S300-v2.3 mAb, SARS-CoV-2 S300-v2.4 mAb, SARS-CoV-2 S300-v2.5 mAb, SARS-CoV-2 S300-v2.6 mAb, SARS-CoV-2 S300-v2.7 mAb, SARS-CoV-2 S300-v2.8 mAb, SARS-CoV-2 S300-v2.9 mAb, SARS-CoV-2 S300-v2.10, SARS-CoV-2 S300-v2.11, SARS-CoV-2 S300-v3 mAb, SARS-CoV-2 S300-v3.1 mAb, SARS-CoV-2 S300-v3.2 mAb, SARS-CoV-2 S300-v3.3 mAb, SARS-CoV-2 S300-v3.4 mAb, SARS-CoV-2 S300-v3.5 mAb, SARS-CoV-2 S300-v3.6 mAb, SARS-CoV-2 S300-v3.7 mAb, SARS-CoV-2 S300-v3.8 mAb, SARS-CoV-2 S300-v3.9 mAb, SARS-CoV-2 S300-v10 mAb, SARS-CoV-2 S300-v11 mAb, SARS-CoV-2 S300-v12 mAb, SARS-CoV-2 S300-v13 mAb, SARS-S300-v14 mAb, SARS-CoV-2 S302 mAb, SARS-CoV-2 S303 mAb, SARS-CoV-2 S303-v1 mAb, SARS-CoV-2 S303-v2 mAb, SARS-CoV-2 S303-v3 mAb, SARS-CoV-2 S303-v4 mAb, SARS-CoV-2 S303-v5 mAb, SARS-CoV-2 S304 mAb, SARS-CoV-2 S306 mAb, SARS-CoV-2 S307 mAb, SARS-CoV-2 S308 mAb, SARS-CoV-2 S308-v1 mAb, SARS-CoV-2 S308-v2 mAb, SARS-CoV-2 S309 mAb, SARS-CoV-2 S309-v1 mAb, SARS-CoV-2 S309-v1.1 mAb, SARS-CoV-2 S309-v1.2 mAb, SARS-CoV-2 S309-v1.3 mAb, SARS-CoV-2 S309-v1.4 mAb, SARS-CoV-2 S309-v1.5 mAb, SARS-CoV-2 S309-v1.6 mAb, SARS-CoV-2 S309-v1.7 mAb, SARS-CoV-2 S309-v1.8 mAb, SARS-CoV-2 S309-v2 mAb, SARS-CoV-2 S309-v2.1 mAb, SARS-CoV-2 S309-v2.2 mAb, SARS-CoV-2 S309-v2.3 mAb, SARS-CoV-2 S309-v2.4 mAb, SARS-CoV-2 S309-v2.5 mAb, SARS-CoV-2 S309-v2.6 mAb, SARS-CoV-2 S309-v2.7 mAb, SARS-CoV-2 S309-v2.8 mAb, SARS-CoV-2 309-v2.9 mAb, SARS-CoV-2 S309-v3 mAb, SARS-CoV-2 S309-v3.1 mAb, SARS-CoV-2 S309-v3.2 mAb, SARS-CoV-2 S309-v3.3 mAb, SARS-CoV-2 S309-v3.4 mAb, SARS-CoV-2 S309-v3.5 mAb, SARS-CoV-2 S309-v3.6 mAb, SARS-CoV-2 S309-v3.7 mAb, SARS-CoV-2 S309-v3.8 mAb, SARS-CoV-2 S309-v9 mAb, SARS-CoV-2 S309-v10 mAb, SARS-CoV-2 S309-v11 mAb, SARS-CoV-2 S309-v12 mAb, SARS-CoV-2 S309-v13 mAb, SARS-CoV-2 S310 mAb, SARS-CoV-2 S311 mAb, SARS-CoV-2 S312 mAb, SARS-CoV-2 S315-v1 mAb, SARS-CoV-2 S315-v2 mAb, SARS-CoV-2 S315-v3 mAb, SARS-CoV-2 S315-v4 mAb, SARS-CoV-2 S315-v5 mAb, SARS-CoV-2 S315-v6 mAb, or SARS-CoV-2 S315-v7 mAb, as listed in Table 2. That is, all combinations of CDRs from SARS-CoV-2 mAbs and their variant sequences presented in Table 2 are considered.

Exemplary antibodies of the present invention include the S309 antibody and engineered variants thereof. In particular embodiments, the antibody or antigen-binding fragment comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 selected from any of the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 (respectively) set forth in Table 1.

In some embodiments, the antibody or antigen-binding fragment comprises: CDRH1, CDRH2, and CDRH3 of a VH amino acid sequence shown in any of SEQ ID NOs: 105, 113, 114, 115, 116, 117, 118, 119, 120, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 172, and 267; and CDRL1, CDRL2 and CDRL3 as set forth in SEQ ID NO:168 (i.e., according to any CDR numbering or definition method known in the art, such as IMGT, Kabat, Chothia, AHo, North, Contact, CCG, EU or Martin (improved Chothia)).

In further embodiments, the antibody or antigen-binding fragment comprises a VH having at least 85% identity (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the amino acid sequence of a VH set forth in Table 1 and/or a VL having at least 85% identity (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) to the amino acid sequence of a VL set forth in Table 1. In further embodiments, the antibody or antigen-binding fragment comprises a VH having at least 90% identity to the amino acid sequence of a VH, presented in Table 1 and/or a VL having at least 90% identity to an amino acid sequence of a VL presented in Table 1. In further embodiments, the antibody or antigen-binding fragment comprises a VH having at least 95% identity to an amino acid sequence of a VH presented in Table 1 and/or a VL having at least 95% identity to an amino acid sequence of a VL presented in Table 1. In further embodiments, the antibody or antigen-binding fragment comprises a VH having at least 99% identity to an amino acid sequence of a VH presented in Table 1 and/or a VL having at least 99% identity to an amino acid sequence of a VL presented in Table 1. In some embodiments, the antibody or antigen-binding fragment comprises a VH amino acid sequence selected from the VH amino acid sequences presented in Table 1 and a VL amino acid sequence selected from the VL amino acid sequence presented in Table 1.

Table 1. CDR (IMGT) and amino acid sequences of the variable region of some S309 antibodies CDRH1 GYPFTSYG (SEQ ID NO:106) CDRH2 ISTYNGNT (SEQ ID NO:107); ISTYQGNT (SEQ ID NO: 121); ISTYNSNT (SEQ ID NO:122); ISTYNANT (SEQ ID NO:123); ISTYNQNT (SEQ ID NO:124); ISTYLGNT (SEQ ID NO:125); ISTYTGNT (SEQ ID NO:126) CDRH3 ARDYTRGAWFGESLIGGFDN (SEQ ID NO:108);
ARDYTRGAFFGESLIGGFDN (SEQ ID NO:127);
ARDYTRGAYFGESLIGGFDN (SEQ ID NO:128) VH QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:105)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYQGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:113)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYNSNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:114)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYNANT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:115)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYNQNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:116)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYLGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:117)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYTGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:118)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAFFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:119)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGW ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAYFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:120)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:129)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYQGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:130)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYNSNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:131)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYNANT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:132)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYNQNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:133)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYLGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:134)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYTGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:135)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAFFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:136)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAYFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:137)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:138)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYQGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:139)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYNSNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:140)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYNANT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:141)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYNQNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:142)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYLGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:143)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYTGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAWFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:144)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAFFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:145)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGY ISTYNGNT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAYFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:146)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGF ISTYNANT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAFFGESLIGGFDN WGQGTLVTVSS (SEQ ID NO:172)
QVQLVQSGAEVKKPGASVKVSCKAS GYPFTSYG ISWVRQAPGQGLEWMGX ₁ ISTYX ₂ X ₃ NT NYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYC ARDYTRGAX ₄ FGESLIGGFDN WGQGTLVTVSS
Where X ₁ is W, F or Y; X ₂ is N, Q, L or T; X ₃ is G, S, A or Q; X ₄ is W, F or Y CDRL1 QTVSSTS (SEQ ID NO:169) CDRL2 GAS (SEQ ID NO:170) CDRL3 QQHDTSLT (SEQ ID NO:171) VL EIVLTQSPGTLLSLSPGERATLSCRAS QTVSSTS LAWYQQKPGQAPRLLIY GAS SRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYC QQHDTSLT FGGGTKVEIK (SEQ ID NO:168)

In particular embodiments, the antibody or antigen-binding fragment comprises CDRH1, CDRH2, and CDRH3 according to SEQ ID NOs: 106, 107, or 121, or 122, or 123, or 124, or 125, or 126, and 108, or 127, or 128, respectively, and CDRL1, CDRL2, and CDRL3 according to SEQ ID NOs: 169-171, respectively. In some embodiments, said antibody or antigen-binding fragment comprises the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2, and CDRL3 set forth in: (a) SEQ ID NOs: 106, 121, 108, 169, 170, and 171, respectively; (b) SEQ ID NOs: 106, 121, 127, 169, 170 and 171, respectively; (c) SEQ ID NOs: 106, 121, 128, 169, 170 and 171, respectively;

(d) SEQ ID NOs: 106, 107, 108, 169, 170 and 171, respectively; (e) SEQ ID NOs: 106, 107, 127, 169, 170 and 171, respectively; (f) SEQ ID NOs: 106, 107, 128, 169, 170 and 171, respectively; (g) SEQ ID NOs: 106, 122, 108, 169, 170 and 171, respectively; (h) SEQ ID NOs: 106, 122, 127, 169, 170 and 171, respectively;

(i) SEQ ID NOs: 106, 122, 128, 169, 170 and 171, respectively; (j) SEQ ID NOs: 106, 123, 108, 169, 170 and 171, respectively; (k) SEQ ID NOs: 106, 123, 127, 169, 170 and 171, respectively; (l) SEQ ID NOs: 106, 123, 128, 169, 170 and 171, respectively;

(m) SEQ ID NOs: 106, 124, 108, 169, 170 and 171, respectively; (n) SEQ ID NOs: 106, 124, 127, 169, 170 and 171, respectively; (o) SEQ ID NOs: 106, 124, 128, 169, 170 and 171, respectively; (p) SEQ ID NOs: 106, 125, 108, 169, 170 and 171, respectively; (q) SEQ ID NOs: 106, 125, 127, 169, 170 and 171, respectively;

(r) SEQ ID NOs: 106, 125, 128, 169, 170, and 171, respectively; (s) SEQ ID NOs: 106, 126, 108, 169, 170, and 171, respectively; (t) SEQ ID NOs: 106, 126, 127, 169, 170, and 171, respectively; or (u) SEQ ID NOs: 106, 126, 128, 169, 170, and 171, respectively.

In further embodiments, VH comprises or consists of the amino acid sequence set forth in any of SEQ ID NOs: 105, 113, 114, 115, 116, 117, 118, 119, 120, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 172, and 267, and VL comprises or consists of the amino acid sequence set forth in SEQ ID NO: 168.

The term "CL" refers to an "immunoglobulin light chain constant region" or "light chain constant region", i.e., a constant region from a light chain of an antibody. The term "CH" refers to an "immunoglobulin heavy chain constant region" or "heavy chain constant region", which is further divided depending on the antibody isotype into CH1, CH2, and CH3 (IgA, IgD, IgG) or CH1, CH2, CH3, and CH4 domains (IgE, IgM). The Fc region of an antibody heavy chain is described further herein. In any of the embodiments disclosed herein, an antibody or antigen-binding fragment of the present invention comprises any one or more of CL, CH1, CH2, and CH3. In some embodiments, CL comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:174 or SEQ ID NO:193. In some embodiments, CH1-CH2-CH3 comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:173 or SEQ ID NO:175 or SEQ ID NO:265 or SEQ ID NO:266. It should be understood that, for example, production in a mammalian cell line may remove one or more C-terminal lysines of the heavy chain of the antibody (see, for example, Liu et al. mAb 6(5):1145-1154(2014)). Accordingly, an antibody or antigen-binding fragment of the present invention may comprise a heavy chain, CH1-CH3, CH3, or Fc polypeptide, wherein the C-terminal lysine residue is present or absent; in other words, they encompass embodiments in which the C-terminal residue of the heavy chain, CH1-CH3, or Fc polypeptide is not lysine, and embodiments wherein lysine is the C-terminal residue. In some embodiments, the composition comprises a plurality of antibodies and/or antigen-binding fragments of the present invention, wherein one or more antibodies or antigen-binding fragments do not comprise a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein one or more antibodies or antigen-binding fragments comprise a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc polypeptide.

A "Fab" (antigen-binding fragment) is the portion of an antibody that binds to antigens and includes the variable region and CH1 of the heavy chain linked to the light chain by an interchain disulfide bond. Each Fab fragment is monovalent with respect to antigen binding, that is, it has a single antigen-binding site. Pepsin treatment of the antibody produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments that have bivalent antigen-binding activity and are still capable of antigen cross-linking. Both Fab and F(ab')2 are examples of "antigen-binding fragments". Fab' fragments differ from Fab fragments by the presence of several additional residues at the carboxyl terminus of the CH1 domain, including one or more cysteines from the hinge region of the antibody. Fab'-SH herein refers to Fab' in which the cysteine residue(s) of the constant domains bear a free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments having hinge cysteine residues between them. Other chemical combinations of antibody fragments are also known.

Fab fragments can be joined, for example, by a peptide linker, to form a single-chain Fab, also referred to herein as "scFab". In these embodiments, the interchain disulfide bond that is present in a native Fab can be absent, and the linker serves in whole or in part to link or connect the Fab fragments in a single polypeptide chain. A Fab fragment derived from a heavy chain (e.g., comprising, consisting of, or consisting essentially of VH + CH1 or "Fd") and a Fab fragment derived from a light chain (e.g., comprising, consisting of, or consisting essentially of VL + CL) can be linked in any arrangement to form an scFab. For example, the scFab may be arranged in the N-terminal to C-terminal direction according to (heavy chain Fab fragment - linker - light chain Fab fragment) or (light chain Fab fragment - linker - heavy chain Fab fragment). Peptide linkers and exemplary linker sequences for use in scFab are discussed in more detail herein.

The scFab may comprise any combination of VH and VL sequences or any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 sequences described herein. In some embodiments, the scFab comprises a VH sequence as set forth in SEQ ID NO: 105 or SEQ ID NO: 113 and a VL sequence as set forth in SEQ ID NO: 168. In some embodiments, the scFab comprises a CDRH1 sequence as set forth in SEQ ID NO: 106, a CDRH2 sequence as set forth in SEQ ID NO: 107 or 121, a CDRH3 sequence as set forth in SEQ ID NO: 108, a CDRL1 sequence as set forth in SEQ ID NO: 169, a CDRL2 sequence as set forth in SEQ ID NO: 170, and a CDRL3 sequence as set forth in SEQ ID NO: 171. In some embodiments, the scFab comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence presented in any of SEQ ID NOs: 218-219 or 226-227.

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

"Single chain Fv," also abbreviated "sFv" or "scFv," are antibody fragments that comprise the V _H and V _L domains of an antibody linked into a single polypeptide chain. In some embodiments, an scFv polypeptide comprises a polypeptide linker positioned between and linking the V _H and V _L domains, which allows the scFv to maintain or form a desired structure for antigen binding. Such a peptide linker can be incorporated into a fusion polypeptide using standard techniques well known in the art. Additionally or alternatively, the Fv can have a disulfide bond formed between and stabilizing the V H and V L. For a review of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, infra. In some embodiments, said antibody or antigen-binding fragment thereof comprises an scFv comprising a VH domain, a VL domain, and a peptide linker linking the VH domain to a VL domain. In particular embodiments, the scFv comprises a VH domain linked to the VL domain by a peptide linker, which may be in a VH-linker-VL orientation or a VL-linker-VH orientation. Any scFv of the present invention may be designed such that the C-terminal VL domain is linked by a short peptide sequence to the N-terminus of the VH domain or vice versa (i.e., (N)VL(C)-linker-(N)VH(C) or (N)VH(C)-linker-(N)VL(C). Alternatively, in some embodiments, the linker may be linked to the N-terminal portion or end of the VH domain, the VL domain, or both.

Peptide linker sequences can be selected based on, for example: (1) their ability to adopt a flexible extended conformation; (2) their inability or lack of ability to adopt a secondary structure that can interact with functional epitopes on the first and second polypeptides and/or on the target molecule; and/or (3) the absence or relative absence of hydrophobic or charged residues that can interact with the polypeptides and/or the target molecule. Other considerations regarding linker design (e.g., length) can include the conformation or range of conformations in which VH and VL can form a functional antigen-binding site. In some embodiments, peptide linker sequences comprise, for example, Gly, Asn, and Ser residues. Other closely related neutral amino acids, such as Thr and Ala, can also be used in the linker sequence. Other amino acid sequences that can be used as a linker include those described in Maratea et al., Gene 40:39 46 (1985); Murphy et al., Proc. Natl. Acad. Sci. USA 83:8258 8262 (1986); U.S. Patent No. 4,935,233; and U.S. Patent No. 4,751,180. Other illustrative and non-limiting examples of linkers may include, for example, Glu-Gly-Lys-Ser-Ser-Gly-Ser-Gly-Ser-Glu-Ser-Lys-Val-Asp (SEQ ID NO: 215) (Chaudhary et al., Proc. Natl. Acad. Sci. USA 87:1066-1070 (1990)) and Lys-Glu-Ser-Gly-Ser-Val-Ser-Ser-Glu-Gln-Leu-Ala-Gln-Phe-Arg-Ser-Leu-Asp (SEQ ID NO: 216) (Bird et al., Science 242:423-426 (1988)) and the pentamer Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 217), when present in one iterations or repeats from 1 to 5 or more times or more; See, for example, SEQ ID NO: 213. Any suitable linker can be used, and will typically be about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 15, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100 amino acids in length, or less than about 200 amino acids in length, and will preferably comprise a flexible structure (can provide flexibility and room for conformational movement between two regions, domains, motifs, fragments or modules connected by the linker) and will preferably be biologically inert and/or have a low risk of immunogenicity in humans. Exemplary linkers include those that comprise or consist of an amino acid sequence set forth in any one or more of SEQ ID NOs: 206-217. In some embodiments, a linker comprises or consists of an amino acid sequence having at least 75% (i.e., at least about 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 206-217.

The scFv may be constructed using any combination of VH and VL sequences or any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 sequences described herein. In some embodiments, the scFv comprises a VH sequence as set forth in SEQ ID NO: 105 or SEQ ID NO: 113 and a VL sequence as set forth in SEQ ID NO: 168. In some embodiments, the scFab comprises a CDRH1 sequence as set forth in SEQ ID NO: 106, a CDRH2 sequence as set forth in SEQ ID NO: 107 or 121, a CDRH3 sequence as set forth in SEQ ID NO: 108, a CDRL1 sequence as set forth in SEQ ID NO: 169, a CDRL2 sequence as set forth in SEQ ID NO: 170, and a CDRL3 sequence as set forth in SEQ ID NO: 171. In some embodiments, the scFv may comprise an amino acid sequence as set forth in SEQ ID NO: 220-221 or SEQ ID NO: 228-229.

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

During antibody development, DNA at the variation (V), junction (J), and diversity (D) gene loci may be rearranged and insertions and/or deletions of nucleotides in the coding sequence may occur. Somatic mutations may be encoded by the resulting sequence and may be identified by reference to the corresponding known germline sequence. In some contexts, somatic mutations that are not critical for a desired property of the antibody (e.g., binding to a SARS-CoV-2 antigen) or that confer an undesirable property on the antibody (e.g., increased risk of immunogenicity in a subject to whom the antibody is administered), or both, may be replaced with the corresponding germline-encoded amino acid or another amino acid such that the desired property of the antibody is improved or maintained and the undesirable property of the antibody is reduced or neutralized. Thus, in some embodiments, an antibody or antigen-binding fragment of the present invention comprises at least one more germline-encoded amino acid in the variable region compared to the parent antibody or antigen-binding fragment, provided that the parent antibody or antigen-binding fragment comprises one or more somatic mutations. The amino acid sequences of the variable region and CDRs of exemplary SARS-CoV-2 antibodies of the present invention are provided in Table 2 herein.

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

Also provided herein are variant antibodies that comprise one or more amino acid changes in a variable region (e.g., VH, VL, framework region, or CDR) compared to the antibody disclosed herein (the "parent"), wherein the variant antibody is capable of binding to a SARS-CoV-2 antigen.

In some embodiments, the VH comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence of any one of SEQ ID NOs: 1, 9-15, 23, 24, 27, 28-46, 55, 63, 79, 87, 95, 103, 105, 113-120, 129-146, 155, 172, 176-178, 194, 196, 198, 200, 202, and 239, wherein the variation is optionally limited to one or more framework regions and/or the variation contains one or more germline encoded amino acid substitutions; and/or (ii) the VL comprises or consists of an amino acid sequence having at least 85% (i.e., 85%, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) identity to the amino acid sequence according to any of SEQ ID NOs: 5, 47-50, 59, 67, 71-72, 75, 76, 83, 91, 99, 109, 147-150, 159, 168, 182, 190, 234, and 243, wherein the variation is optionally limited to one or more framework regions and/or the variation comprises one or more germline encoded amino acid substitutions.

In some embodiments, the antibody or antigen-binding fragment comprises VH and VL amino acid sequences that are encoded by or are the same as VH and VL amino acid sequences that are encoded by:

(i) a VH1-18 gene and a VK3-20 gene, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH1-18 and VK3-20, respectively;

(ii) the VH3-7 allele and the VL3-25 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH3-7 and VL3-25, respectively;

(iii) the VH3-23 allele and the VK1-5 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH3-23 and VK1-5, respectively;

(iv) the VH3-13 allele and the VK1-39 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH3-13 and VK1-39, respectively;

(v) the VH1-18 allele and the VK3-11 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH1-18 and VK3-11, respectively; or

(vi) the VH1-69 allele and the VL2-23 allele, respectively, or which are encoded by a polynucleotide having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to VH1-69 and VL2-23, respectively.

In some embodiments, VH comprises or consists of any VH amino acid sequence shown in Table 2 and VL comprises or consists of any VL amino acid sequence shown in Table 2. In particular embodiments, VH and VL comprise or consist of the amino acid sequences of SEQ ID NO: (i) 1 and 5 or 234, respectively; (ii) any of 9-15 and 5 or 234, respectively; (iii) 23 or 24 and 5 or 234, respectively; (iv) 27 and 5 or 234, respectively; (v) any of 28-46 and 5 or 234, respectively; (vi) 1 and any of 47-50, respectively; (vii) any of 9-15 and any of 47-50, respectively; (viii) 23 or 24 and any of 47-50, respectively; (ix) 27 and any of 47 to 50, respectively; (x) any of 28 to 46 and any of 47 to 50, respectively; (xi) 55 and 59, respectively; (xii) 63 and 67, respectively; (xiii) 63 and 71 or 72, respectively; (xiv) 63 and 75 or 76, respectively; (xv) 79 and 83, respectively; (xvi) 87 and 91, respectively; (xvii) 95 and 99, respectively; (xviii) 103 and 99, respectively; (xiv) 105 and 109 or 168, respectively; (xx) any of 113 to 120 and 109 or 168, respectively; (xxi) 129 and 109 or 168, respectively; (xxii) any of 130 to 146 and 109 or 168, respectively; (xxiii) 105 and any of 147 to 150, respectively; (xxiv) any of 113 to 120 and any of 147 to 150, respectively; (xxv) any of 130 to 146 and any of 147 to 150, respectively; (xxvi) 155 and 159, respectively; (xxvii) 172 and 168, respectively; (xxviii) 176 or 177 and 5 or any of 47 to 50, respectively; (xxix) 178 and 182 or 190, respectively (i.e. 178 and 182, respectively, or 178 and 190, respectively); (xxx) 194 and 182, respectively; (xxxi) 196 and 182, respectively; (xxxii) 198 and 182, respectively; (xxxiii) 200 and 182, respectively; (xxxiv) 202 and 182, respectively; or (xxxv) 239 and 243, respectively.

In some embodiments, an antigen or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:79 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:83. In further embodiments, an antigen or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 79 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 83, and binds to the SARS-CoV-2 S protein with a K _D of less than about 4.5 x 10 ^-9 M, less than about 5 x 10 ^-9 M, less than about 1 x 10 ^-10 M, less than about 5 x 10 ^-10 M, less than about 1 x 10 ^-11 M, less than about 5 x 10 ^-11 M, less than about 1 x 10 ^-12 M, or less than about 5 x 10 ^-12 M. In further embodiments, an antigen or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO: 79 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO: 83 and capable of neutralizing SARS-CoV-2 infection or SARS-CoV-2 pseudotyped virus with an IC50 of about 16 to about 20 μg/mL.

In some embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:105 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168. In further embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:105 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168, and binds to the SARS-CoV-2 S protein with a K _D of less than about 4.5×10 ^-9 M, less than about 5×10 ^-9 M, less than about 1×10 ^-10 M, less than about 5×10 ^-10 M, less than about 1×10 ^-11 M, less than about 5×10 ^-11 M, less than about 1×10 ^-12 M, or less than about 5×10 ^-12 M. In further embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:105 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168 and is capable of neutralizing SARS-CoV-2 infection or SARS-CoV-2 pseudotyped virus with an IC50 of about 0.3 to about 0.4 μg/mL.

In some embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:105 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168, and binds to the SARS-CoV-2 RBD S protein with an EC50 (half-maximal effective concentration) of about 11 to about 25 ng/mL. In some embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:113 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168, and binds to the SARS-CoV-2 RBD S protein with an EC50 of about 9 to about 23 ng/mL. In some embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:129 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168, and binds to the SARS-CoV-2 RBD S protein with an EC50 of about 8 to about 22 ng/ml. In some embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:119 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168, and binds to the SARS-CoV-2 RBD S protein with an EC50 of about 8 to about 22 ng/ml. In some embodiments, an antibody or antigen-binding fragment of the present invention comprises a VH comprising or consisting of the amino acid sequence of SEQ ID NO:172 and a VL comprising or consisting of the amino acid sequence of SEQ ID NO:168, and binds to the S protein of the RBD of SARS-CoV-2 with an EC50 of about 7 to about 19 ng/mL.

In some embodiments, an antibody or antigen-binding fragment of the present invention is monospecific (e.g., binds to a single epitope) or is polyspecific (e.g., binds to multiple epitopes and/or target molecules). Antibodies and antigen-binding fragments can be constructed in a variety of formats. Exemplary antibody formats are described in Spiess et al., Mol. Immunol. 67(2):95 (2015), and in Brinkmann and Kontermann, mAb 9(2): 182–212 (2017), the formats and methods of making them are incorporated herein by reference and include, for example, bispecific T cell activators (BiTEs), DARTs, knob-in-hole (KIH) assemblies, scFv-CH3-KIH assemblies, KIH common light chain antibodies, TandAbs, triplebodies, TriBi minibodies, Fab-scFv, scFv-CH-CL-scFv, F(ab')2-scFv2, tetravalent HCabs, intrabodies, CrossMabs, dual-acting Fabs (DAFs) (two-in-one or four-in-one), DutaMabs, DT-IGG, charge pairs, Fab-arm Exchange, SEEDbodies, Triomabs, LZ-Y assemblies, Fcabs, κλ-bodies or orthobodies of Fabs, DVDgs (e.g., U.S. Patent No. 8,258,268, the formats of which are incorporated herein by reference in their entireties), IgFv- IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, Zybody, and DVI-IgG, also referred to as FIT-Ig (e.g., PCT Publication No. WO 2015/103072, the formats of which are incorporated herein by reference in their entireties), so-called WuxiBody formats (e.g., PCT Publication No. WO 2019/057122, the formats of which are incorporated herein by reference in their entireties) and so-called Ig In-Elbow-Insert (IEI-Ig) formats; e.g., PCT Publication Nos. WO 2019/024979 and WO 2019/025391, the formats of which are incorporated herein by reference in their entireties).

In some embodiments, said antibody or antigen-binding fragment comprises two or more VH domains, two or more VL domains, or both (i.e., two or more VH domains and two or more VL domains). In particular embodiments, the antigen-binding fragment comprises the format (from N-terminus to C-terminus) VH-linker-VL-linker-VH-linker-VL, wherein the two VH sequences may be the same or different, and the two VL sequences may be the same or different. Such linked scFvs may include any combination of VH and VL domains intended to bind to a given target, and in formats comprising two or more VH and/or two or more VL, one, two or more different epitheliopes or antigens may be linked. It should be understood that formats comprising multiple antigen-binding domains may include VH and/or VL sequences in any combination or orientation. For example, the antigen-binding fragment may comprise the format VL-linker-VH-linker-VL-linker-VH, VH-linker-VL-linker-VL-linker-VH, or VL-linker-VH-linker-VH-linker-VL.

The monospecific or multispecific antibodies or antigen-binding fragments of the present invention may comprise any combination of VH and VL sequences and/or any combination of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 sequences described herein. In some embodiments, the antibody or antigen-binding fragment comprises a VH sequence as set forth in SEQ ID NO: 105 or SEQ ID NO: 113 and a VL sequence as set forth in SEQ ID NO: 168. In some embodiments, the antibody or antigen-binding fragment comprises a CDRH1 sequence as set forth in SEQ ID NO: 106, a CDRH2 sequence as set forth in SEQ ID NO: 107 or 121, a CDRH3 sequence as set forth in SEQ ID NO: 108, a CDRL1 sequence as set forth in SEQ ID NO: 169, a CDRL2 sequence as set forth in SEQ ID NO: 170, and a CDRL3 sequence as set forth in SEQ ID NO: 171. In some embodiments, the antibody or antigen-binding fragment comprises an amino acid sequence as set forth in any of SEQ ID NOs: 222-225 or 230-233. A bispecific or multispecific antibody or antigen-binding fragment may, in some embodiments, comprise one, two or more antigen-binding domains (e.g., VH and VL) as described herein. Two or more binding domains may be present that bind to the same or a different epitope of SARS-CoV-2, and a bispecific or multispecific antibody or antigen-binding fragment provided herein may, in some embodiments, comprise an additional SARS-CoV-2 binding domain and/or may comprise a binding domain that binds to a different antigen or pathogen altogether.

In any of the embodiments disclosed herein, the antibody or antigen-binding fragment may be multispecific; for example, bispecific, trispecific, or the like.

In some embodiments, the antibody or antigen-binding fragment thereof comprises: (i) a first VH and a first VL; and (ii) a second VH and a second VL, wherein the first VH and the second VH are different and each independently comprise an amino acid sequence having at least 85% (i.e., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 1, 9-15, 23, 24, 27-46, 55, 63, 79, 87, 95, 103, 105, 113-120, 129-146, 155, 172, 176-178, 194, 196, 198, 200, 202 and 239, and wherein the first VL and the second VL are different and each independently comprise an amino acid sequence having at least 85% (i.e. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) identity to the amino acid sequence set forth in any one of SEQ ID NOs: 5, 47-50, 59, 67, 71, 72, 75, 76, 83, 91, 99, 109, 147-150, 159, 168, 182, 190, 234 and 243, and wherein the first VH and the first VL together form a first antigen-binding site, and where the second VH and the second VL together form a second antigen-binding site.

In some embodiments, an antibody or antigen-binding fragment thereof comprises an Fc polypeptide or fragment thereof. "Fc" comprises the carboxy-terminal portions (i.e., the IgG CH2 and CH3 domains) of both antibody H chains held together by disulfides. "Effector functions" of an antibody refer to biological activities attributable to the Fc region (either a native sequence Fc region or an amino acid sequence variant Fc region) of the antibody, and vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., the B cell receptor); and B cell activation. As discussed herein, modifications (e.g., amino acid substitutions) can be made in the Fc domain to modify (e.g., improve, decrease, or remove) one or more functional groups of the Fc-containing polypeptide (e.g., an antibody of the present invention). Such functions include, for example, binding to an Fc receptor (FcR), modulation of antibody half-life (e.g., by binding to FcRn), ADCC function, protein A binding, protein G binding, and complement binding. Amino acid modifications that modify (e.g., enhance, decrease, or remove) Fc functions include, for example, TT250Q/M428L, M252Y/S254T/T256E, H433K/N434F, M428L/N434S, E233P/L234V/L235A/G236 + A327G/A330S/P331S, E333A, S239D/A330L/I332E, P257I/Q311, K326W/E333S, S239D/I332E/G236A, N297Q, K322A, S228P, L235E + E318A/K320A/K322A, L234A/L235A (also referred to herein as “LALA”) and the L234A/L235A/P329G mutations, which are summarized and annotated in “Engineered Fc Regions” published by InvenGo (2011) and available online at invivogenPreview.com/DFre/review-Eng-Egene-Fegogen-in-in.pdf.utm_source=review&utm_medium=pdf&utm_campaign=review&utm_content=Engineered-Fc-Regions, and are incorporated herein by reference. Unless the context otherwise requires, Fc amino acid residues are numbered herein according to the EU numbering system.

For example, to activate the complement cascade, the C1q protein complex can bind to at least two IgG1 molecules or one IgM molecule when attached to the antigen target (Ward, E. S., and Ghetie, V., Ther. Immunol. 2 (1995) 77-94). Burton, D. R., reported (Mol. Immunol. 22 (1985) 161-206) that the region of the heavy chain containing amino acid residues at position 318 to 337 is involved in complement fixation. Duncan, A. R., and Winter, G. (Nature 332 (1988) 738-740), using site-directed mutagenesis, reported that Glu318, Lys320, and Lys322 form the binding site for C1q. The role of residues Glu318, Lys320, and Lys 322 in C1q binding was confirmed by the ability of a short synthetic peptide containing these residues to inhibit complement-mediated lysis.

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

Cross-linking of receptors by the Fc domain of native IgG antibodies (FcγR) triggers a wide range of effector functions including phagocytosis, antibody-dependent cellular cytotoxicity and release of inflammatory mediators, as well as immune complex clearance and regulation of antibody production. This paper reviews the Fc fragments that mediate receptor cross-linking (e.g., FcγR). In humans, three classes of FcγR have been characterized to date, which are: (i) FcγRI (CD64), which binds monomeric IgG with high affinity and is expressed on macrophages, monocytes, neutrophils and eosinophils; (ii) FcγRII (CD32), which binds complex IgG with intermediate to low affinity, is widely expressed particularly on leukocytes, is thought to be a central player in antibody-mediated immunity, and which can be divided into FcγRIIA, FcγRIIB, and FcγRIIC, which have different functions in the immune system but bind with similar low affinity to IgG-Fc, and the ectodomains of these receptors are highly homologous; and (iii) FcγRIII (CD16), which binds IgG with intermediate to low affinity and has been found in two forms: FcγRIIIA, which has been found on NK cells, macrophages, eosinophils, and some monocytes and T cells, and is thought to mediate ADCC, and FcγRIIIB, which is highly expressed on neutrophils.

FcγRIIA is found on many cells involved in killing (e.g., macrophages, monocytes, neutrophils) and appears to be able to activate the killing process. FcγRIIB appears to play a role in inhibitory processes and is found on B cells, macrophages, and on mast cells and eosinophils. Importantly, 75% of all FcγRIIB is found in the liver (Ganesan, L. P., et al., 2012: "FcγRIIb on liver sinusoidal endothelium clears small immune complexes," Journal of Immunology 189: 4981-4988). FcγRIIB is abundantly expressed on the liver sinusoidal endothelium, called LSEC, and Kupffer cells in the liver and LSEC are the major site of small immune complex clearance (Ganesan, L. P. et al., 2012: FcγRIIb on liver sinusoidal endothelium clears small immune complexes. Journal of Immunology 189: 4981-4988).

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

On B cells, FcγRIIB may function to suppress further immunoglobulin production and isotype switching to, for example, the IgE class. On macrophages, FcγRIIB is thought to inhibit phagocytosis mediated through FcγRIIA. On eosinophils and mast cells, the B form may help suppress activation of these cells by binding IgE to a distinct receptor.

With respect to FcγRI binding, modification of native IgG at least one of E233-G236, P238, D265, N297, A327, and P329 reduces binding to FcγRI. IgG2 residues at positions 233-236 substituted into the corresponding positions of IgG1 and IgG4 reduce binding of IgG1 and IgG4 to FcγRI by ^103- fold and abolish the human monocytic response to antibody-sensitized red blood cells (Armour, KL, et al. Eur. J. Immunol. 29 (1999) 2613-2624).

With respect to FcγRII binding, a decrease in binding to FcγRIIA was found, for example, for the IgG mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, R292, and K414.

The two allelic forms of human FcγRIIA are the "H131" variant, which binds to IgG1 Fc with high affinity, and the "R131" variant, which binds to IgG1 Fc with low affinity. See, e.g., Bruhns et al., Blood 113:3716–3725 (2009).

With respect to FcγRIII binding, a decrease in binding to FcγRIIIA was found, for example, for a mutation of at least one of E233-G236, P238, D265, N297, A327, P329, D270, Q295, A327, S239, E269, E293, Y296, V303, A327, K338, and D376. Mapping of binding sites on human IgG1 for Fc receptors, the above-mentioned mutation sites, and methods for measuring binding to FcγRI and FcγRIIA are described in Shields, R. L., et al., J. Biol. Chem. 276 (2001) 6591-6604.

The two allelic forms of human FcγRIIIA are the "F158" variant, which binds to IgG1 Fc with low affinity, and the "V158" variant, which binds to IgG1 Fc with high affinity. See, e.g., Bruhns et al., Blood 113:3716–3725 (2009).

With respect to binding to FcγRII, two regions of native IgG Fc appear to be involved in the interaction between FcγRII and IgG, namely (i) the lower hinge site of IgG Fc, specifically amino acid residues L, L, G, G (234-237, EU numbering), and (ii) the adjacent region of the CH2 domain of IgG Fc, specifically the loop and chains in the upper CH2 domain adjacent to the lower hinge region, such as in the P331 region (Wines, B.D., et al., J. Immunol. 2000; 164: 5313-5318). Furthermore, FcγRI appears to bind to the same site on IgG Fc, whereas FcRn and protein A bind to a different site on IgG Fc, which appears to be at the CH2-CH3 interface (Wines, B.D., et al., J. Immunol. 2000; 164: 5313–5318).

Also contemplated are mutations that increase the binding affinity of an Fc polypeptide or fragment thereof of the present invention for (i.e., one or more) Fcγ receptors (e.g., compared to a reference Fc polypeptide or fragment thereof that does not contain the mutation(s); e.g., a wild-type Fc polypeptide or fragment thereof (e.g., of the same isotype as the Fc polypeptide or fragment thereof that contains the mutation or mutations) or an Fc polypeptide or fragment thereof that is otherwise identical or substantially identical to the Fc polypeptide or fragment thereof that contains the mutation or mutations). See, e.g., Delillo and Ravetch, Cell 161(5):1035-1045 (2015) and Ahmed et al., J. Struc. Biol. 194(1):78 (2016), the Fc mutations and methodologies of which are incorporated herein by reference.

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

In some embodiments, the Fc polypeptide or fragment thereof may comprise or consist of at least a portion of an Fc polypeptide or fragment thereof that participates in binding to an FcRn binding site. In some embodiments, the Fc polypeptide or fragment thereof comprises one or more amino acid modifications that improve the binding affinity for (e.g., enhance binding to) FcRn (e.g., at about pH 6.0) and thus, in some embodiments, prolong the in vivo half-life of a molecule comprising the Fc polypeptide or fragment thereof (e.g., compared to a reference (e.g., wild-type) Fc polypeptide or fragment thereof or an antibody that is otherwise the same but lacks the modification(s)). In some embodiments, the Fc polypeptide or fragment thereof comprises or is derived from an IgG Fc and the half-life prolonging mutation comprises any one or more of: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I Q311I; D376V; T307A; E380A (EU numbering). In some embodiments, the half-life prolonging mutation comprises M428L/N434S (also referred to herein as "MLNS" or "LS"). In some embodiments, the half-life prolonging mutation comprises M252Y/S254T/T256E. In some embodiments, the half-life prolonging mutation comprises T250Q/M428L. In some embodiments, the half-life prolonging mutation comprises P257I/Q311I. In some embodiments, the half-life prolonging mutation comprises P257I/N434H. In some embodiments, the half-life extending mutation comprises D376V/N434H. In some embodiments, the half-life extending mutation comprises T307A/E380A/N434A.

In some embodiments, the antibody or antigen-binding fragment comprises an Fc region that comprises the M428L/N434S substitutions. In some embodiments, the antibody or antigen-binding fragment comprises an Fc polypeptide or fragment thereof that comprises the G236A/A330L/I332E mutation substitutions. In some embodiments, the antibody or antigen-binding fragment comprises (e.g., an IgG) Fc region that comprises the G236A mutation, the A330L mutation, and the I332E (GAALIE) mutation and lacks the S239D mutation (e.g., comprises a native S at position 239). In particular embodiments, the antibody or antigen-binding fragment comprises an Fc polypeptide or fragment thereof that comprises the M428L/N434S and G236A/A330L/I332E substitution mutations, and optionally lacks S239D. In some embodiments, the antibody or antigen-binding fragment thereof comprises an Fc polypeptide or fragment thereof that comprises the substitution type mutations: M428L/N434S and G236A/S239D/A330L/I332E.

In some embodiments, the antibody or antigen-binding fragment comprises a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G, and/or the antibody or antigen-binding fragment is partially or completely aglycosylated and/or partially or completely afucosylated. Host cell lines and methods for producing partially or completely aglycosylated or partially or completely afucosylated antibodies and antigen-binding fragments are known (see, e.g., PCT Publication No. WO 2016/181357; Suzuki et al. Clin. Cancer Res. 13(6):1875-82 (2007); Huang et al. MAbs 6:1-12 (2018)).

In some embodiments, the antibody or antigen-binding fragment is capable of inducing continued protection in vivo in a subject even if detectable levels of the antibody or antigen-binding fragment cannot be detected in the subject (i.e., if the antibody or antigen-binding fragment has been removed from the subject after administration). Such protection is referred to herein as a vaccine effect. Without being limited by theory, it is believed that dendritic cells can internalize antibody-antigen complexes and thereafter induce or promote an endogenous immune response against the antigen. In some embodiments, the antibody or antigen-binding fragment comprises one or more modifications, such as, for example, Fc mutations comprising G236A, A330L, and I332E, that are capable of activating dendritic cells that can induce, for example, T cell immunity to the antigen.

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

In any of the embodiments disclosed herein, the antibody or antigen-binding fragment may be monoclonal. The term "monoclonal antibody" (mAb) as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies in the population are identical except for possible naturally occurring mutations that may be present in small amounts. Monoclonal antibodies are highly specific because they are directed against a single region of a determinant. Furthermore, compared to polyclonal antibody preparations containing different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope of an antigen. In addition to specificity, monoclonal antibodies have the advantage that they can be synthesized in a form uncontaminated by other antibodies. The modifier "monoclonal" should not be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies suitable for use in the present invention can be produced using the hybridoma technique first described by Kohler et al., Nature 256:495 (1975), or can be produced using recombinant DNA techniques in bacterial, eukaryotic animal, or plant cells (see, e.g., U.S. Patent No. 4,816,567). Monoclonal antibodies can also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol., 222:581-597 (1991), for example. Monoclonal antibodies can also be produced using the methods described in PCT Publication No. WO 2004/076677A2.

The antibodies and antigen-binding fragments of the present invention include "chimeric antibodies" in which a portion of the heavy and/or light chain is identical to or homologous with corresponding sequences of antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to or homologous with corresponding sequences of antibodies derived from another species or belonging to a different antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see U.S. Pat. Nos. 4,816,567; 5,530,101; and 7,498,415; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). For example, chimeric antibodies can contain human and non-human moieties. In addition, chimeric antibodies may contain residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further improve the characteristics of the antibodies. For more information, see Jones et al., Nature 321:522–525 (1986); Riechmann et al., Nature 332:323–329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593–596 (1992). Chimeric antibodies also include primatized and humanized antibodies.

A "humanized antibody" is generally considered to be a human antibody that has one or more amino acid residues introduced into it from a source that is not human. These non-human amino acid residues are typically taken from the variable domain. Humanization can be accomplished by the method of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the non-human variable sequences with the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent Nos. 4,816,567; 5,530,101; and 7,498,415), wherein substantially less than an intact human variable domain is replaced by the corresponding sequence from a non-human species. In some cases, a "humanized" antibody is an antibody that is produced by a non-human or animal cell and contains human sequences, such as H and _C domains.

A "human antibody" is an antibody that contains only sequences that are present in an antibody produced by a human. However, as used herein, human antibodies may contain residues or modifications that are not found in a naturally occurring human antibody (e.g., an antibody isolated from a human), including those modifications and sequence variations that are described herein. These are typically performed to further improve or enhance the performance of the antibody. In some cases, human antibodies are produced by transgenic animals. For example, see U.S. Patent Nos. 5,770,429; 6,596,541; and 7,049,426.

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

Polynucleotides, vectors and host cells

In another aspect, the present invention provides isolated polynucleotides that encode any of the antibodies or antigen-binding fragments thereof described herein, or a portion thereof (e.g., a CDR, VH, VL, heavy chain, or light chain). In some embodiments, the polynucleotide is codon-optimized for expression in a host cell. Once the coding sequence is known or identified, codon optimization can be performed using known techniques and tools, such as the GenScript® OptimiumGene™ tool or Gen Synthesis by GeneArt® (ThermoFisher); see also Scholten et al., Clin. Immunol. 119:135, 2006). Codon-optimized sequences include those that are partially codon-optimized (i.e., one or more codons are optimized for expression in a host cell) and those that are fully codon-optimized.

It should also be understood that polynucleotides encoding the antibodies and antigen-binding fragments of the present invention may have different nucleotide sequences while still encoding the same antibody or antigen-binding fragment due to, for example, degeneracy of the genetic code, splicing, etc.

In some embodiments, the polynucleotide comprises a polynucleotide having at least 50% (i.e., 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) identity to a polynucleotide sequence according to any one or more of SEQ ID NOs: 186-189, 191-192, 238, 247, 248-250, 254-255, and 257-262.

It should be understood that in some embodiments, a polynucleotide encoding an antibody or antigen-binding fragment is comprised in a polynucleotide that comprises other sequences and/or features, for example, of expression of the antibody or antigen-binding fragment in a host cell. Exemplary features include a promoter sequence, a polyadenylation sequence, a sequence that encodes a signal peptide (e.g., located at the N-terminus of an expressed antibody heavy chain or light chain), or the like. Accordingly, in some embodiments, the polynucleotide further comprises a polynucleotide sequence that has at least 50% identity to, comprising, or consisting of a polynucleotide sequence set forth in any of SEQ ID NOs:251-253 and 263. In some embodiments, the polynucleotide comprises a sequence that encodes a signal peptide (also referred to as a leader sequence) that has at least 90% identity to, comprising, or consisting of the amino acid sequence set forth in SEQ ID NO:256 or SEQ ID NO:264.

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

Also provided are vectors, wherein the vectors comprise or include a polynucleotide as described herein (e.g., a polynucleotide encoding an antibody or antigen-binding fragment that binds to SARS-CoV-2). A vector can comprise any one or more of the vectors described herein. In particular embodiments, provided is a vector that comprises a DNA plasmid construct encoding an antibody or antigen-binding fragment, or a portion thereof (e.g., a so-called "DMAb"; see, e.g., Muthumani et al., J Infect Dis. 214(3):369-378 (2016); Muthumani et al., Hum Vaccin Immunother 9:2253-2262 (2013)); Flingai et al., Sci Rep. 5:12616 (2015); and Elliott et al., NPJ Vaccines 18 (2017), wherein the DNA constructs encoding the antibody and related uses, including administration of the same, are incorporated herein by reference.). In some embodiments, the DNA plasmid construct comprises a single open reading frame encoding a heavy chain and a light chain (or VH and VL) of an antibody or antigen-binding fragment, wherein the heavy chain encoding sequence and the light chain encoding sequence are optionally separated by a polynucleotide encoding a protease cleavage site and/or a polynucleotide encoding a self-cleaving peptide. In some embodiments, the replacement components of the antibody or antigen-binding fragment are encoded by a polynucleotide contained in a single plasmid. In other embodiments, the replacement components of the antibody or antigen-binding fragment are encoded by a polynucleotide contained in two or more plasmids (e.g., a first plasmid comprises a polynucleotide encoding a heavy chain, VH or VH+CH, and a second plasmid comprises a polynucleotide encoding a related light chain, VL or VL+CL). In some embodiments, a single plasmid comprises a polynucleotide encoding a heavy chain and/or a light chain of two or more antibodies or antigen-binding fragments of the present invention. An exemplary expression vector is pVax1, available from Invitrogen®. A DNA plasmid of the present invention can be delivered to a subject, for example, by electroporation (e.g., intramuscular electroporation) or with an appropriate formulation (e.g., hyaluronidase). In some embodiments, a vector of the present invention comprises a nucleotide sequence encoding a signal peptide. The signal peptide may or may not be present (e.g., may be enzymatically cleaved from) on the mature antibody or antigen-binding fragment. In some embodiments, the signal peptide is encoded by the nucleotide sequence set forth in SEQ ID NO: 252 or SEQ ID NO: 263, and/or the signal peptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 256 or SEQ ID NO: 264. In some embodiments, a vector of the present invention comprises a polyadenylation signal sequence. In some embodiments, the polyadenylation signal sequence comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 253.

In some embodiments, a vector of the present invention comprises a CMV promoter. In some embodiments, the promoter comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 251.

In a further aspect, the present invention also relates to a host cell expressing an antibody or antigen-binding fragment according to the present invention; or comprising or including a vector or polynucleotide according to the present invention.

Examples of such cells include, but are not limited to, eukaryotic cells, such as yeast cells, animal cells, insect cells, plant cells; and prokaryotic cells, including E. coli. In some embodiments, the cells are mammalian cells. In some such embodiments, the cells are a mammalian cell line such as CHO cells (e.g., DHFR-CHO cells (Urlaub et al., PNAS 77:4216 (1980)), human embryonic kidney cells (e.g., HEK293T cells), PER.C6 cells, Y0 cells, Sp2/0. NS0 cells, human liver cells, e.g., Hepa RG cells, myeloma cells, or hybridoma cells. Other examples of mammalian host cell lines include mouse Sertoli cells (e.g., TM4 cells); SV40-transformed monkey kidney line CV1 (COS-7); baby hamster kidney (BHK) cells; African green monkey kidney (VERO-76) cells; monkey kidney (CV1) cells; human cervical carcinoma (HELA) cells; human lung (W138) cells; human liver cells (Hep G2); kidney cells dog (MDCK); Buffalo rat liver cells (BRL 3A); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Mammalian host cell lines suitable for antibody production also include those described in, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J.), pp. 255–268 (2003).

In some embodiments, the host cell is a prokaryotic cell, such as E. coli. Expression of peptides in prokaryotic cells, such as E. coli, is well known (see, e.g., Pluckthun, A. Bio/Technology 9:545-551 (1991). For example, antibodies can be produced in bacteria, particularly where glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. Nos. 5,648,237; 5,789,199; and 5,840,523.

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

In addition, the host cells of the present invention can be stably or transiently transfected with a vector of the present invention, for example, to express an antibody or antigen-binding fragment thereof of the present invention. In such embodiments, the cells can be stably transfected with a vector as described herein. Alternatively, the cells can be transiently transfected with a vector of the present invention encoding an antibody or antigen-binding fragment as described herein. In any of the embodiments disclosed herein, the polynucleotide can be heterologous to the host cell.

Accordingly, the present invention also provides recombinant host cells that heterologously express an antibody or antigen-binding fragment of the present invention. For example, the cell can be a species that is different from the species from which the antibody was derived in whole or in part (e.g., CHO cells expressing a human antibody or an engineered human antibody). In some embodiments, the cell type of the host cell does not naturally express the antibody or antigen-binding fragment. In addition, the host cell can introduce a post-translational modification (PTM; e.g., glycosylation or fucosylation) to the antibody or antigen-binding fragment that is not present in the native state of the antibody or antigen-binding fragment (or in the native state of the original antibody from which the antibody or antigen-binding fragment was engineered or derived). Such a PTM can result in a functional difference (e.g., reduced immunogenicity). Accordingly, an antibody or antigen-binding fragment of the present invention produced by a host cell as described herein may comprise one or more post-translational modifications that differ from the antibody (or parent antibody) in its native state (e.g., a human antibody produced by a CHO cell may comprise more than one post-translational modification that differs from the antibody when isolated from the human and/or is produced by a native human B cell or plasma cell).

Insect cells suitable for expressing the binding protein of the present invention are known in the art and include, for example, Spodoptera frugipera Sf9 cells, Trichoplusia ni BTI-TN5B1-4 cells, and Spodoptera frugipera SfSWT01“Mimic™” cells. See, for example, Palmberger et al., J. Biotechnol. 153(3-4):160-166 (2011). Numerous strains of baculoviruses have been identified that can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Eukaryotic microorganisms such as filamentous fungi or yeast are also suitable hosts for cloning or expression of protein-encoding vectors, and include fungi and yeast strains with "humanized" glycosylation pathways, resulting in antibodies with a partially or fully human glycosylation profile. See Gerngross, Nat. Biotech.22:1409-1414 (2004); Li et al., Nat. Biotech.24:210-215 (2006).

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

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

In a related aspect, the present invention provides methods for producing an antibody or antigen-binding fragment, the methods comprising culturing a host cell of the present invention under conditions and for a time sufficient to produce the antibody or antigen-binding fragment. Methods suitable for isolating and purifying recombinantly produced antibodies, for example, can comprise obtaining supernatants from suitable host cell/vector systems that secrete the recombinant antibody into a culture medium, and then concentrating the medium using a commercially available filter. After concentration, the concentrate can be applied to a single suitable purification matrix or to a series of suitable matrices, such as an affinity matrix or an ion exchange resin. One or more reversed-phase HPLC (high performance liquid chromatography) steps can be used to further purify the recombinant polypeptide. These purification methods can also be used in the isolation of an immunogen from its natural environment. The methods for large-scale production of one or more isolated/recombinant antibodies described herein include a batch culture of cells that is monitored and controlled to maintain appropriate culture conditions. Purification of soluble antibodies can be carried out in accordance with the methods described herein and known in the art, and which comply with laws and recommendations of domestic and foreign regulatory agencies.

Compositions

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

In some embodiments, the composition comprises a plurality of antibodies and/or antigen-binding fragments of the present invention, wherein one or more antibodies or antigen-binding fragments do not comprise a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc polypeptide, and wherein the one or more antibodies or antigen-binding fragments comprise a lysine residue at the C-terminus of the heavy chain, CH1-CH3, or Fc polypeptide. In some embodiments, the composition comprises an antibody or antigen-binding fragment thereof that comprises an amino acid sequence set forth in SEQ ID NO:173, SEQ ID NO:175, SEQ ID NO:265, or SEQ ID NO:266.

In some embodiments, the composition comprises two or more different antibodies or antigen-binding fragments of the present invention. In some embodiments, the antibodies or antigen-binding fragments for use in combination each independently have one or more of the following characteristics: neutralize naturally occurring variants of SARS-CoV-2; do not compete with each other for binding to the spike protein; bind different epitopes of the spike protein; have reduced development of resistance to SARS-CoV-2; in combination, have reduced development of resistance to SARS-CoV-2; effectively neutralize live SARS-CoV-2 virus; exhibit additive or synergistic effects in neutralizing live SARS-CoV-2 virus when used in combination; exhibit effector functions; are protective in the relevant animal infection model(s); and are capable of being produced in sufficient quantities for large-scale production.

In some embodiments, the composition comprises a first antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 79 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 83; and a second antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 105, and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 168. In some embodiments, the composition comprises a first antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 80-82, respectively, and CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 84-86, respectively, and a second antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2 and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 106-108, respectively, and CDRL1 and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 169-171, respectively. In further embodiments, the composition is capable of neutralizing SARS-CoV-2 infection or a virus pseudotyped by SARS-CoV-2 with an IC50 of about 0.07 to about 0.08 μg/mL. In some embodiments, the composition comprises a first antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 178 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 182 or SEQ ID NO: 190; and a second antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 105 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 168. In some embodiments, the composition comprises a first antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3 and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2 and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 179-181, respectively, and CDRL1, CDRL2 and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 183-185, respectively, and a second antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2 and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 106-108, respectively, and CDRL1, CDRL2 and CDRL3 comprise the amino acid sequences set forth in SEQ ID NOs: 169-171, respectively.

In some embodiments, the composition comprises a first vector comprising a first plasmid and a second vector comprising a second plasmid, wherein the first plasmid comprises a polynucleotide encoding a heavy chain, VH or VH+CH, and the second plasmid comprises a polynucleotide encoding a related light chain, VL or VL+CL, of an antibody or antigen-binding fragment thereof. In some embodiments, the composition comprises a polynucleotide (e.g., mRNA) linked to a suitable delivery vehicle or carrier. Exemplary carriers or vehicles for administration to a human subject include a lipid or lipid delivery vehicle such as a liposome, a solid lipid nanoparticle, an oleaginous suspension, a submicron lipid emulsion, a lipid microbubble, a reverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, or a lipid nanoparticle (LNP) or nanoscale platform (see, e.g., Li et al. Wilery Interdiscip Rev. Nanomed Nanobiotechnol. 11(2):e1530 (2019)). Principles, reagents, and methodologies for designing suitable mRNA and formulating mRNA-LNPs and delivering them are described, e.g., in Pardi et al. (J Control Release 217345-351(2015)); Thess et al. (Mol Ther 23: 1456-1464 (2015)); Thran et al. (EMBO Mol Med 9(10):1434–1448 (2017); Kose et al. Sci. Immunol. 4 eaaw6647 (2019); and Sabnis et al. (Mol. Ther. 26: 1509–1519 (2018)), which include capping, codon optimization, nucleoside modification, mRNA purification, incorporation of mRNA into stable lipid nanoparticles (e.g., ionizable cationic lipid/phosphatidylcholine/cholesterol/PEG lipid; ionizable lipid:distearoyl PC:cholesterol:polyethylene glycol lipid (PC)) and subcutaneous, intramuscular, intradermal, intravenous, intraperitoneal, and intratracheal administration, are included in this document by reference.

Methods and applications

The present document also provides methods for using an antibody or antigen-binding fragment, nucleic acid, vector, cell, or composition of the present invention in diagnosing SARS-CoV-2 (e.g., in a human subject or in a sample obtained from a human subject).

The diagnostic methods (e.g., in vitro, ex vivo) may comprise contacting an antibody, an antibody fragment (e.g., an antigen-binding fragment) with a sample. Such samples may be isolated from a subject, such as an isolated tissue sample taken, for example, from the nasal passages, sinus cavities, salivary glands, lungs, liver, pancreas, kidneys, ear, eye, placenta, digestive tract, heart, ovaries, pituitary gland, adrenal glands, thyroid gland, brain, skin or blood. The diagnostic methods may also comprise detecting the antigen/antibody complex, in particular, after contacting the antibody or antibody fragment with the sample. Such a detection step may be performed in a laminar flow hood, i.e., without any contact with the human or animal body. Examples of detection methods are well known to those skilled in the art and include, for example, ELISA (enzyme-linked immunosorbent assay), including direct, indirect and sandwich ELISA.

Also provided herein are methods of treating a subject using an antibody or antigen-binding fragment of the present invention, or a composition comprising the same, wherein the subject is believed to have or is at risk of infection with SARS-CoV-2. "Treatment," "treatment," or "alleviation" refers to medical treatment of a disease, disorder, or condition of a subject (e.g., a human or a non-human mammal such as a primate, horse, cat, dog, goat, mouse, or rat). Typically, a suitable dose or regimen comprising an antibody or composition of the present invention is administered in an amount sufficient to produce a therapeutic or prophylactic effect. A therapeutic or prophylactic/preventative effect includes improved clinical outcome; reduction or alleviation of symptoms associated with a disease; reduction in the incidence of symptoms; improved quality of life; longer disease-free status; reduction in the extent of the disease, stabilization of the disease state; delay or prevention of disease progression; remission; survival; prolonged survival; or any combination thereof. In some embodiments, the therapeutic, prophylactic/preventive effect includes reducing or preventing hospitalization during treatment of SARS-CoV-2 infection (i.e., in a statistically significant manner). In some embodiments, the therapeutic or prophylactic/preventive effect includes reducing the length of hospitalization during treatment of SARS-CoV-2 infection (i.e., in a statistically significant manner). In some embodiments, the therapeutic or prophylactic/preventive effect includes reducing or eliminating the need for respiratory intervention, such as intubation and/or use of a respiratory device. In some embodiments, the therapeutic or prophylactic/preventive effect includes reversal of late-stage disease pathology and/or reduction in mortality.

"A therapeutically effective amount" or "effective quantity" of an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition of the present invention refers to an amount of the composition or molecule sufficient to produce a therapeutic effect, including improved clinical outcome; reduction or alleviation of symptoms associated with a disease; decreased incidence of symptoms; improved quality of life; prolonged disease-free status; decreased severity of disease, stabilization of disease status; delay in disease progression; remission; survival; or prolonged survival in a statistically significant manner. When referring to a single active ingredient administered alone, a therapeutically effective amount refers to the effects of that ingredient or a cell expressing that ingredient alone. When referring to a combination, a therapeutically effective amount refers to the combined amounts of the active ingredients or the combined auxiliary active ingredient with the cell expressing the active ingredient, which results in a therapeutic effect, regardless of whether administered serially, sequentially, or simultaneously. The combination may comprise, for example, two different antibodies that specifically bind a SARS-CoV-2 antigen, which in some embodiments may be the same or a different Wuhan coronavirus antigen, and/or may comprise the same or different epitopes.

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

Subjects that can be treated according to the present invention are generally human subjects and other primates, such as monkeys and apes, for the purposes of veterinary medicine. Other model organisms, such as mice and rats, can also be treated according to the present invention. In any of the above embodiments, the subject can be a human subject. Subjects can be male or female and can be of any suitable age, including infants, juveniles, adolescents, adults and elderly subjects.

A number of criteria are considered to contribute to a high risk of severe symptoms or death associated with SARS CoV-2 coronavirus infection. These include, but are not limited to, age, occupation, general health, pre-existing health conditions, and lifestyle habits. In some embodiments, a subject receiving treatment in accordance with the present invention has one or more risk factors.

In some embodiments, the human subject receiving treatment in accordance with the present invention is an infant, a child, an adolescent, a middle-aged adult, or an elderly person. In some embodiments, the human subject receiving treatment in accordance with the present invention is under 1 year of age, or is between 1 and 5 years of age, or is between 5 and 125 years of age (e.g., 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 125 years of age, including any and all ages on or between the listed ages). In some embodiments, the human subject treated according to the present invention is 0-19 years old, 20-44 years old, 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years or older. Middle-aged and especially elderly individuals are considered to be at particular risk. In particular embodiments, the human subject is 45-54 years old, 55-64 years old, 65-74 years old, 75-84 years old, or 85 years or older. In some embodiments, the human subject is a male. In some embodiments, the human subject is a female.

In some embodiments, the human subject receiving treatment in accordance with the present invention is a resident of a nursing home or long-term care facility, is a hospice worker, is a healthcare worker or health care worker, is an emergency medical technician, is a family member or other person in close contact with a subject diagnosed with or suspected of having a SARS-CoV-2 infection, is overweight or clinically obese, is or has been a smoker, has or has had chronic obstructive pulmonary disease (COPD), is asthmatic (e.g., has moderate to severe asthma), has an autoimmune disease or condition (e.g., diabetes), and/or has a weakened or depleted immune system (e.g., due to AIDS/HIV infection (AIDS - immunodeficiency infection syndrome, HIV - human immunodeficiency virus), cancer such as blood cancer, lymphodepleting therapy such as chemotherapy, bone marrow or organ transplant, or genetic immune condition), has chronic liver disease, has cardiovascular disease, lung disease, or heart defect, works or otherwise spends time in close proximity to others, such as in a factory, delivery center, hospital, etc.

In some embodiments, a subject receiving treatment in accordance with the present invention has received a SARS-CoV-2 vaccine and the vaccine has been found to be ineffective, such as as a result of post-vaccination infection or symptoms in the subject, as a result of a clinical diagnosis, or scientific or regulatory criteria.

In some embodiments, the treatment was administered as a post-exposure prophylaxis. In some embodiments, the treatment was administered to a subject with a mild or moderate disease that may be in an outpatient setting. In some embodiments, the treatment was administered to a subject with a moderate or severe disease, such as requiring hospitalization.

Thus, typical routes of administration of the compositions described herein include, but are not limited to, oral, topical, transdermal, inhalation, parenteral, sublingual, buccal, rectal, vaginal, and intranasal. The term "parenteral" as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injections, or infusion techniques. In some embodiments, administration comprises administration by a route selected from oral, intravenous, parenteral, intragastric, intrapleural, intrapulmonary, intrarectal, intradermal, intraperitoneal, intratumoral, subcutaneous, topical, transdermal, intracranial, intrathecal, intranasal, and intramuscular administration. In particular embodiments, the method comprises orally administering to a subject an antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition.

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

The composition may be in the form of a solid or a liquid. In some embodiments, the carrier(s) are solid particles, so that the compositions are, for example, in the form of a tablet or powder. The carrier(s) may be liquid(s), so that the compositions are, for example, an oral oil, an injection liquid, or an aerosol that can be used, for example, in inhalation administration. When the pharmaceutical composition is intended for oral administration, it is preferably in either a solid or a liquid form, where semi-solid, semi-liquid, suspension and gel forms are included in the forms considered herein as solid or liquid.

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

The composition may be in the form of a liquid, such as an elixir, syrup, solution, emulsion or suspension. The liquid may be intended for oral administration or for delivery by injection, as two examples. If intended for oral administration, preferred compositions contain, in addition to the present compounds, one or more sweeteners, preservatives, coloring agents/dyes and flavor enhancers. In a composition intended for administration by injection, one or more of a surfactant, a preservative, a wetting agent, a dispersing agent, a suspending agent, a buffer, a stabilizer and an isotonic agent may be included.

Liquid pharmaceutical compositions, whether solutions, suspensions or other similar forms, can include one or more of the following adjuvants: sterile diluents such as water for injection, physiological saline, preferably physiological saline, Ringer's solution, isotonic sodium chloride, fixed oils such as synthetic mono- or diglycerides which can serve as a solvent or suspending medium, polyethylene glycols, glycerol, propylene glycol or other solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for achieving tonicity such as sodium chloride or dextrose. The parenteral preparation may be included in ampoules, disposable syringes or a glass or plastic multi-dose vial. Saline is the preferred adjuvant. The injectable pharmaceutical composition is preferably sterile.

A liquid composition intended for parenteral or oral administration should contain an amount of the antibody or antigen-binding fragment as described herein so that a suitable dosage is obtained. Typically, this amount is at least 0.01% of the antibody or antigen-binding fragment in the composition. When intended for oral administration, this amount can vary from 0.1 to about 70% by weight of the composition. Some oral pharmaceutical compositions contain from about 4% to about 75% of the antibody or antigen-binding fragment. In some embodiments, the pharmaceutical compositions and preparations of the present invention are prepared such that a parenteral dosage unit contains from 0.01 to 10% by weight of the antibody or antigen-binding fragment before dilution.

The composition may be intended for topical administration, in which case the carrier may suitably comprise a solution, emulsion, ointment or gel base. The base may, for example, comprise one or more of the following: petrolatum, lanolin, polyethylene glycols, beeswax, mineral oil, diluents such as water and alcohol, and emulsifiers and stabilizers. Thickening agents may be present in the composition for topical administration. If the composition is intended for transdermal administration, it may include a transdermal patch or an iontophoresis device. The pharmaceutical composition may be intended for rectal administration, in the form of, for example, a suppository that will dissolve in the rectum and release the drug. The composition for rectal administration may comprise an oleaginous base as a suitable non-irritating excipient. Such bases include, but are not limited to, lanolin, cocoa butter and polyethylene glycol.

The composition may contain various substances that modify the physical form of the solid or liquid dosage unit. For example, the composition may include materials that form a coating shell around the active ingredients. The substances that form the coating shell are usually inert and can be selected, for example, from sugar, shellac and other enteric coating agents. Alternatively, the active ingredients can be enclosed in a gelatin capsule. The composition in solid or liquid form may contain an agent that binds to an antibody or antigen-binding fragment according to the present description and thereby facilitates delivery of the compound. Suitable agents that can act in this capacity include monoclonal or polyclonal antibodies, one or more proteins or a liposome. The composition may consist essentially of dosage units that can be administered as an aerosol. The term "aerosol" is used to designate various systems, ranging from systems of a colloidal nature to systems consisting of pressurized packages. The delivery may be by liquefied or compressed gas or by means of a suitable pump system that doses the active ingredients. Aerosols may be delivered in single-phase, two-phase or three-phase systems for delivering the active ingredient(s). The aerosol delivery includes the necessary container, activators, valves, subcontainers, etc., which together may form a kit. A person skilled in the art can determine the preferred aerosols without undue experimentation.

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

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

In general, a suitable dose and treatment regimen provides the composition(s) in an amount sufficient to provide a therapeutic and/or prophylactic effect (such as those described herein), including an improved clinical outcome (e.g., a reduction in the incidence, duration, or severity of diarrhea or associated dehydration, or inflammation, or a longer disease-free and/or overall survival, or a reduction in the severity of symptoms). For prophylactic use, the dose should be sufficient to prevent, delay the onset, or reduce the severity of the disease associated with the disease or disorder. The prophylactic effect of the compositions administered according to the methods described herein can be determined by performing preclinical (including in vitro and in vivo animal studies) and clinical studies and analyzing the data obtained therefrom using appropriate statistical, biological, and clinical methods and techniques, all of which can be readily performed by one of skill in the art.

The compositions are administered in an effective amount (e.g., to treat SARS-CoV-2 infection) that will vary depending on a variety of factors, including the activity of the particular compound being administered; the metabolic stability and duration of action of the compound; the age, body weight, general health, sex, and diet of the subject; the mode and time of administration; the rate of elimination; the drug combination; the severity of the particular disorder or condition; and the subject being treated. In some embodiments, upon subsequent administration of the medicaments in accordance with the formulations and methods of the present invention, the subjects will experience a reduction in one or more symptoms associated with the disease or disorder being treated by from about 10% to about 99%, compared to subjects receiving a placebo or other suitable control subjects.

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

In some embodiments, the method comprises administering the antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition to the subject in an amount of 2, 3, 4, 5, 6, 7, 8, 9, 10 times or more.

In some embodiments, the method comprises administering the antibody, antigen-binding fragment, or composition to the subject multiple times, wherein the second or sequential administration is performed about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 24, about 48, about 74, about 96 hours or more after the first or previous administration, respectively.

In some embodiments, the method comprises administering the antibody, antigen-binding fragment, polynucleotide, vector, host cell, or composition at least once prior to infecting the subject with SARS-CoV-2.

Compositions comprising an antibody, antigen-binding fragment, polynucleotide, vector, host cell or composition of the present invention can also be administered simultaneously, before or after the administration of one or more other therapeutic agents. Such combination therapy can include the administration of a single pharmaceutical dosage form that contains a compound of the invention and one or more additional active agents, as well as the administration of compositions comprising an antibody or antigen-binding fragment of the invention and each active agent in its own separate dosage form. For example, an antibody or antigen-binding fragment thereof, as described herein, and another active agent can be administered to a patient together in a single oral dosage composition, such as a tablet or capsule, or each agent administered in separate oral dosage forms. Likewise, an antibody or antigen-binding fragment, as described herein, and another active agent can be administered to a subject together in a single parenteral dosage composition, such as a saline solution or other physiologically acceptable solution, or each agent can be administered in separate parenteral dosage forms. Where separate dosage forms are used, the compositions comprising the antibody or antigen-binding fragment and one or more additional active agents may be administered at substantially the same time, i.e., simultaneously, or at different times, i.e., sequentially and in any order; combination therapy is intended to include all of these regimens.

In some embodiments, the present invention provides a combination therapy that comprises one or more SARS-CoV-2 antibodies (or one or more nucleic acids, host cells, vectors or compositions) of the present invention and one or more anti-inflammatory agents and/or one or more antiviral agents. In particular embodiments, the one or more anti-inflammatory agents comprises a corticosteroid, such as, for example, dexamethasone, prednisone, or the like. In some embodiments, the one or more anti-inflammatory agents comprises a cytokine antagonist, such as, for example, an antibody that binds to IL6 (IL-interleukin) (such as siltuximab), or to IL-6R (IL-6 receptor) (such as tocilizumab), or to IL-1β, IL-7, IL-8, IL-9, IL-10, FGF (fibroblast growth factor), G-CSF (granulocyte colony-stimulating factor), GM-CSF (granulocyte-macrophage colony-stimulating factor), IFN-γ (interferon γ), IP-10 (interferon-γ-inducible protein 10), MCP-1 (monocyte chemoattractant protein), MIP-1A (MIP - macrophage inflammatory protein), MIP1-B, PDGR (platelet-derived growth factor), TNF-α (tumor necrosis factor α) or VEGF (vascular endothelial growth factor). In some embodiments, anti-inflammatory agents such as ruxolitinib and/or anakinra are used. In some embodiments, the one or more antiviral agents comprise nucleotide analogs or nucleotide analog prodrugs such as, for example, remdesivir, sofosbuvir, acyclovir and zidovudine. In particular embodiments, the antiviral agent comprises lopinavir, ritonavir, favipiravir or any combination thereof. In some embodiments, the combination therapy comprises leronlimab. Anti-inflammatory agents for use in the combination therapy of the present invention also include non-steroidal anti-inflammatory drugs (NSAIDs). It should be understood that in such combination therapy, one or more antibodies (or one or more nucleic acids, host cells, vectors or compositions) and one or more anti-inflammatory agents and/or one or more antiviral agents may be administered in any order and in any sequence or together.

In some embodiments, the antibody (or one or more nucleic acids, host cells, vectors, or compositions) was administered to a subject that had previously received one or more anti-inflammatory agents and/or one or more antiviral agents. In some embodiments, one or more anti-inflammatory agents and/or one or more antiviral agents were administered to a subject that had previously received the antibody (or one or more nucleic acids, host cells, vectors, or compositions).

In some embodiments, the present invention provides a combination therapy that comprises two or more SARS-CoV-2 antibodies of the present invention. The method may comprise administering a first antibody to a subject that has received a second antibody, or may comprise administering two or more antibodies together. For example, in particular embodiments, a method is provided that comprises administering to a subject (a) a first antibody or antigen-binding fragment when the subject has received a second antibody or antigen-binding fragment; (b) a second antibody or antigen-binding fragment when the subject has received a first antibody or antigen-binding fragment; or (c) a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment.

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

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

In some embodiments, the present invention provides an antibody, antigen-binding fragment, or composition for use in a method for making or preparing a medicament for treating SARS-CoV-2 infection in a subject.

The present invention also relates to the following embodiments.

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

(i) CDRH1 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs:106, 2, 56, 64, 80, 88, 96, 156, 179, 195 or 240, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which is optionally a conservative substitution and/or is a germline encoded amino acid substitution;

(ii) CDRH2 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs:121, 3, 16-22, 57, 65, 81, 89, 97, 107, 122-126, 157, 180, 197, 199 or 241, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which optionally is a conservative substitution and/or is a germline encoded amino acid substitution;

(iii) CDRH3 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 108, 4, 25, 26, 58, 66, 82, 90, 98, 104, 127, 128, 158, 181, 201, 203 or 242, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which optionally is a conservative substitution and/or is a germline encoded amino acid substitution;

(iv) CDRL1 comprises or consists of an amino acid sequence as set forth in any one of SEQ ID NOs:169, 6, 51-54, 60, 68, 73, 74, 84, 92, 100, 110, 160, 183, 235 or 244, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which is optionally a conservative substitution and/or is a germline encoded amino acid substitution;

(v) CDRL2 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs:170, 7, 61, 69, 85, 93, 101, 111, 161, 184, 236 or 245, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which optionally is a conservative substitution and/or is a germline encoded amino acid substitution; and/or

(vi) CDRL3 comprises or consists of the amino acid sequence set forth in any one of SEQ ID NOs: 171, 8, 62, 70, 77, 78, 86, 94, 102, 112, 151-154, 162, 185, 237 or 246, or a sequence variant thereof comprising one, two or three amino acid substitutions, one or more of which is optionally a conservative substitution and/or is a germline encoded amino acid substitution,

wherein the antibody or antigen-binding fragment is capable of binding to the surface glycoprotein (S) of SARS-CoV-2 expressed on the cell surface of a host cell, on the SARS-CoV-2 virion, or both.

Embodiment 2. An antibody or antigen-binding fragment thereof according to embodiment 1, capable of neutralizing SARS-CoV-2 infection in an in vitro infection model and/or in an in vivo animal infection model and/or in humans.

Embodiment 3. An antibody or antigen-binding fragment according to any one of embodiments 1 or 2, comprising the amino acid sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 presented in SEQ ID NO:

(i) 106, 121, 108, 169, 170 and 171, respectively;

(ii) 2-4 and 6-8 or 235-237, respectively;

(iii) 2, any of 16-22, 4 and 6-8 or 235-237, respectively;

(iv) 2, 3, any of 25-26 and 6-8 or 235-237, respectively;

(v) 2-4, 51, 7 or 236 and 8 or 237, respectively;

(vi) 2-4, 52, 7 or 236 and 8 or 237, respectively;

(vii) 2-4, 53, 7 or 236 and 8 or 237, respectively;

(viii) 2-5, 54, 7 or 236 and 8 or 237, respectively;

(ix) 56-58 and 60-62 respectively;

(x) 64-66 and 68-70 respectively;

(xi) 64-66, 73 or 74, 69 and 70 respectively;

(xii) 64-66, 68, 69 and 77 or 78 respectively;

(xiii) 80-82 and 84-86 respectively;

(xiv) 88-90 and 92-94, respectively;

(xv) 96-98 and 101-102 respectively;

(xvi) 96, 97, 104 and 100-102, respectively;

(xvii) 106-108 and 169-171, respectively;

(xviii) 106, any of 121-126, 108 and 169-171, respectively;

(xix) 106, 107, 127 or 128 and 169-171, respectively;

(xx) 106, 107, or any of 121-126, 108, and 169-171, respectively;

(xxi) 156-158 and 160-162, respectively;

(xxii) 106, 123, 127 and 169-171, respectively;

(xxiii) 2, 17, 25, 6 or 235 or any of 51-54, 7 or 236 and 8 or 237, respectively;

(xxiv) 2, 20, 25, 6 or 235 or any of 51-54, 7 or 236 and 8 or 237, respectively;

(xxv) 179-181 and 183-185, respectively;

(xxvi) 195, 180, 181 and 183-185, respectively;

(xxvii) 195, 197, 181 and 183-185 respectively;

(xxviii) 195, 199, 181 and 183-185 respectively;

(xxiv) 195, 197, 201 and 183-185, respectively;

(xxx) 195, 197, 203 and 183-185 respectively;

(xxxi) 195, 199, 201 and 183-185, respectively;

(xxxii) 195, 199, 203 and 183-185 respectively;

(xxxiii) 179, 180, 181 and 183-185, respectively;

(xxxiv) 179, 197, 181 and 183-185, respectively;

(xxxv) 179, 199, 181 and 183-185, respectively;

(xxxvi) 179, 197, 201 and 183-185, respectively;

(xxxvii) 179, 197, 203 and 183-185, respectively;

(xxxviii) 179, 199, 201 and 183-185, respectively;

(xxxix) 179, 199, 203 and 183-185, respectively;

(xxxx) 179, 180, 201 and 183-185, respectively;

(xxxxi) 179, 180, 203 and 183-185, respectively; or

(xxxxii) 240-242 and 244-246, respectively.

Embodiment 4. An antibody or antigen-binding fragment according to any one of embodiments 1-3, comprising:

(i) the amino acid sequence of CDRH1 shown in SEQ ID NO:106;

(ii) the CDRH2 amino acid sequence shown in SEQ ID NO:121, SEQ ID NO:107, SEQ ID NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, or SEQ ID NO:126;

(iii) the CDRH3 amino acid sequence shown in SEQ ID NO:108, SEQ ID NO:127 or SEQ ID NO:128;

(iv) the amino acid sequence of CDRL1 shown in SEQ ID NO:169;

(v) the amino acid sequence of CDRL2 as set forth in SEQ ID NO:170; and

(vi) the amino acid sequence of CDRL3 as set forth in SEQ ID NO:171,

wherein, optionally, the antibody or antigen-binding fragment comprises the amino acid sequences CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in:

(a) SEQ ID NO:106, 121, 108, 169, 170 and 171, respectively;

(b) SEQ ID NO:106, 121, 127, 169, 170 and 171, respectively;

(c) SEQ ID NO:106, 121, 128, 169, 170 and 171, respectively;

(d) SEQ ID NO:106, 107, 108, 169, 170 and 171, respectively;

(e) SEQ ID NO:106, 107, 127, 169, 170 and 171, respectively;

(f) SEQ ID NO:106, 107, 128, 169, 170 and 171, respectively;

(g) SEQ ID NO:106, 122, 108, 169, 170 and 171, respectively;

(h) SEQ ID NO:106, 122, 127, 169, 170 and 171, respectively;

(i) SEQ ID NO:106, 122, 128, 169, 170 and 171, respectively;

(j) SEQ ID NO:106, 123, 108, 169, 170 and 171, respectively;

(k) SEQ ID NO:106, 123, 127, 169, 170 and 171, respectively;

(l) SEQ ID NO:106, 123, 128, 169, 170 and 171, respectively;

(m) SEQ ID NO:106, 124, 108, 169, 170 and 171, respectively;

(n) SEQ ID NO:106, 124, 127, 169, 170 and 171, respectively;

(o) SEQ ID NO:106, 124, 128, 169, 170 and 171, respectively;

(p) SEQ ID NO:106, 125, 108, 169, 170 and 171, respectively;

(q) SEQ ID NO:106, 125, 127, 169, 170 and 171, respectively;

(r) SEQ ID NO:106, 125, 128, 169, 170 and 171, respectively;

(s) SEQ ID NO:106, 126, 108, 169, 170 and 171, respectively;

(t) SEQ ID NO:106, 126, 127, 169, 170 and 171, respectively; or

(u) SEQ ID NO:106, 126, 128, 169, 170 and 171, respectively.

Embodiment 5. An antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising the amino acid sequence of CDRH1 presented in SEQ ID NO:106, the amino acid sequence of CDRH2 presented in SEQ ID NO:121, and the amino acid sequence of CDRH3 presented in SEQ ID NO:108, and a light chain variable domain (VL) comprising the amino acid sequence of CDRL1 presented in SEQ ID NO:169, the amino acid sequence of CDRL2 presented in SEQ ID NO:170, and the amino acid sequence of CDRL3 presented in SEQ ID NO:171,

wherein the antibody or antigen-binding fragment is capable of binding to the surface glycoprotein (S) of SARS-CoV-2 expressed on the cell surface of a host cell, on the virion, or on both.

Embodiment 6. An antibody or antigen-binding fragment thereof according to embodiment 5, capable of neutralizing SARS-CoV-2 infection in an in vitro infection model and/or in an in vivo animal infection model and/or in humans.

Embodiment 7. An antibody or antigen-binding fragment according to any one of embodiments 1-6, comprising:

(i) a VH comprising or consisting of an amino acid sequence having at least 85% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 113, 1, 9-15, 23, 24, 27, 28-46, 55, 63, 79, 87, 95, 103, 105, 114-120, 129-146, 155, 172, 176-178, 194, 196, 198, 200, 202, 239 and 267, wherein the variation, when present, relative to the reference VH amino acid sequence is optionally limited to one or more framework regions and/or the variation comprises one or more germline encoded amino acid substitutions; and/or

(ii) a VL comprising or consisting of an amino acid sequence having at least 85% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 168, 5, 47-50, 59, 67, 71-72, 75, 76, 83, 91, 99, 109, 147-150, 159, 182, 190, 234 and 243, wherein the change, when compared to the VH reference amino acid sequence, if present, is optionally limited to one or more framework regions and/or the change comprises one or more germline encoded amino acid substitutions.

Embodiment 8. The antibody or antigen-binding fragment of any one of embodiments 1-7, wherein VH comprises or consists of any VH amino acid sequence shown in Table 2, and wherein VL comprises or consists of any VL amino acid sequence shown in Table 2, wherein, optionally, VH and VL comprise or consist of the amino acid sequences shown in SEQ ID NO:

(i) 113 and 168, respectively;

(ii) 1 and 5 or 234, respectively;

(iii) any of 9-15 and 5 or 234, respectively;

(iv) 23 or 24 and 5 or 234, respectively;

(v) 27 and 5 or 234, respectively;

(vi) any of 28-46 and 5 or 234, respectively;

(vii) 1 and any of 47-50, respectively;

(viii) any of 9-15 and any of 47-50, respectively;

(ix) 23 or 24 and any of 47-50, respectively;

(x) 27 and any of 47-50, respectively;

(xi) any of 28-46 and any of 47-50, respectively;

(xii) 55 and 59, respectively;

(xiii) 63 and 67, respectively;

(xiv) 63 and 71 or 72, respectively;

(xv) 63 and 75 or 76, respectively;

(xvi) 79 and 83, respectively;

(xvii) 87 and 91, respectively;

(xviii) 95 and 99, respectively;

(xix) 103 and 99, respectively;

(xx) 105 and 168, respectively;

(xxi) any of 114-120 or 267 and 168, respectively;

(xxii) 129 and 168, respectively;

(xxiii) any of 130-146 and 168, respectively;

(xxiv) 105 and any of 147-150, respectively;

(xxv) any of 113-120 and any of 147-150, respectively;

(xxvi) any of 130-146 and any of 147-150, respectively;

(xxvii) 155 and 159, respectively;

(xxviii) 172 and 168, respectively;

(xxix) 176 or 177 and 5 or 234 or any of 47-50, respectively;

(xxx) 178 and 182 or 190, respectively;

(xxxi) 194 and 182 respectively;

(xxxii) 196 and 182, respectively;

(xxxiii) 198 and 182, respectively;

(xxxiv) 200 and 182, respectively;

(xxxv) 202 and 182, respectively; or

(xxxvi) 239 and 243, respectively.

Embodiment 9. An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises or consists of the amino acid sequence presented in SEQ ID NO:113, and VL comprises or consists of the amino acid sequence presented in SEQ ID NO:168.

Embodiment 10. An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises or consists of the amino acid sequence presented in any of SEQ ID NOs: 105, 114-120, 129-146, 172, and 267, and VL comprises or consists of the amino acid sequence presented in SEQ ID NO: 168.

Embodiment 11. An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises or consists of the amino acid sequence presented in SEQ ID NO:79, and VL comprises or consists of the amino acid sequence presented in SEQ ID NO:83.

Embodiment 12. An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:80-82, respectively, and CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:84-86, respectively,

wherein the antibody or antigen-binding fragment is capable of binding to the SARS-CoV-2 surface glycoprotein (S) expressed on the cell surface of a host cell, on the SARS-CoV-2 virion, or both.

Embodiment 13. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises or consists of the amino acid sequence presented in SEQ ID NO:105, and VL comprises or consists of the amino acid sequence presented in SEQ ID NO:168.

Embodiment 14. An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 106-108, respectively, and CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 169-171, respectively,

wherein the antibody or antigen-binding fragment is capable of binding to the SARS-CoV-2 surface glycoprotein (S) expressed on the cell surface of a host cell, on the SARS-CoV-2 virion, or both.

Embodiment 15. An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises or consists of the amino acid sequence presented in SEQ ID NO:178, and VL comprises or consists of the amino acid sequence presented in SEQ ID NO:182 or SEQ ID NO:190.

Embodiment 16. An antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:179-181, respectively, and CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:183-185, respectively,

wherein the antibody or antigen-binding fragment is capable of binding to the SARS-CoV-2 surface glycoprotein (S) expressed on the cell surface of a host cell, on the SARS-CoV-2 virion, or both.

Embodiment 17. An antibody or antigen-binding fragment according to any one of embodiments 1-16, which:

(i) recognizes an epitope within the receptor binding motif (RBM) of ACE2 (SEQ ID NO:167) of SARS-CoV-2;

(ii) is capable of blocking the interaction between SARS-CoV-2 (e.g., SARS-CoV-2 RBM) and ACE2;

(ii) is able to bind to the SARS-CoV-2 S protein with greater avidity than to the SARS coronavirus S protein;

(iii) recognizes an epitope that is conserved in the ACE2 RBM of SARS-CoV-2 and in the ACE2 RBM of SARS coronavirus;

(vi) has cross-reactivity against SARS-CoV-2 and SARS coronavirus;

(vii) recognizes an epitope on the SARS-CoV-2 surface glycoprotein that is not part of the ACE2 RBM,

or

(viii) any combination of (i) to (vii).

Embodiment 18. An antibody or antigen-binding fragment of any one of embodiments 1-17, capable of inhibiting the interaction between SARS-CoV-2 and any one or more of DC-SIGN, L-SIGN, and SIGLEC-1.

Embodiment 19. The antibody or antigen-binding fragment of any one of embodiments 1-18, capable of inhibiting the interaction between SARS-CoV-2 and any one or more of: DC-SIGN; L-SIGN; SIGLEC-1; CD22; CD33; CLEC4M, SIGLEC-16; SIGLEC-15; SIGLEC-14; SIGLEC-12; SIGLEC-11; SIGLEC-10; SIGLEC-9; SIGLEC-8; SIGLEC-7; SIGLEC-6; SIGLEC-5, or any combination thereof.

Embodiment 20. The antibody or antigen-binding fragment of any one of embodiments 1-19, which is of the IgG, IgA, IgM, IgE, or IgD isotype.

Embodiment 21. The antibody or antigen-binding fragment of any one of embodiments 1-20, which is an IgG isotype selected from IgG1, IgG2, IgG3, and IgG4.

Embodiment 22. An antibody or antigen-binding fragment of any one of embodiments 1-21, which is human, humanized, or chimeric.

Embodiment 23. The antibody or antigen-binding fragment of any one of embodiments 1-22, wherein the antibody or antigen-binding fragment comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, Fab', F(ab')2, Fv, scFv, or scFab.

Embodiment 24. The antibody or antigen-binding fragment of embodiment 23, wherein the scFab comprises:

(i) the amino acid sequence shown in any one of SEQ ID NOs:218-219 and 226-227;

(ii) a VL comprising the amino acid sequence set forth in SEQ ID NO:168 and a VH comprising the amino acid sequence set forth in SEQ ID NO:105 or SEQ ID NO:113; or

(iii) CDRH1 comprising the amino acid sequence presented in SEQ ID NO: 106, CDRH2 comprising the amino acid sequence presented in SEQ ID NO: 107 or 121, CDRH3 comprising the amino acid sequence presented in SEQ ID NO: 108, CDRL1 comprising the amino acid sequence presented in SEQ ID NO: 169, CDRL2 comprising the amino acid sequence presented in SEQ ID NO: 170, and CDRL3 comprising the amino acid sequence presented in SEQ ID NO: 171.

Embodiment 25. The antibody or antigen-binding fragment of embodiment 23, wherein the scFv comprises:

(i) the amino acid sequence shown in any of SEQ ID NOs:220-221 or 228-229;

(ii) a VL comprising the amino acid sequence set forth in SEQ ID NO:168 and a VH comprising the amino acid sequence set forth in SEQ ID NO:105 or SEQ ID NO:113; or

(iii) CDRH1 comprising the amino acid sequence presented in SEQ ID NO:106, CDRH2 comprising the amino acid sequence presented in SEQ ID NO:107 or 121, CDRH3 comprising the amino acid sequence presented in SEQ ID NO:108, CDRL1 comprising the amino acid sequence presented in SEQ ID NO:169, CDRL2 comprising the amino acid sequence presented in SEQ ID NO:170, and CDRL3 comprising the amino acid sequence presented in SEQ ID NO:171.

Embodiment 26. The antibody or antigen-binding fragment of embodiment 25, wherein the scFv comprises more than one VH domain and more than one VL domain.

Embodiment 27. The antibody or antigen-binding fragment of embodiment 26, wherein the scFv comprises:

(i) the amino acid sequence shown in any one of SEQ ID NOs:222-225 or SEQ ID NOs:230-233;

(ii) two VL domains, each comprising the amino acid sequence set forth in SEQ ID NO:168, and two VH domains, each comprising the amino acid sequence set forth in SEQ ID NO:105 or SEQ ID NO:113; or

(iii) two VL domains, each comprising a CDRL1 comprising the amino acid sequence presented in SEQ ID NO: 169, a CDRL2 comprising the amino acid sequence presented in SEQ ID NO: 170, and a CDRL3 comprising the amino acid sequence presented in SEQ ID NO: 171, and two VH domains, each comprising a CDRH1 comprising the amino acid sequence presented in SEQ ID NO: 106, a CDRH2 comprising the amino acid sequence presented in SEQ ID NO: 107 or 121, a CDRH3 comprising the amino acid sequence presented in SEQ ID NO: 108.

Embodiment 28. The antibody or antigen-binding fragment of any one of embodiments 1-27, wherein the antibody or antigen-binding fragment is a multispecific antibody or antigen-binding fragment.

Embodiment 29. The antibody or antigen-binding fragment of embodiment 28, wherein the antibody or antigen-binding fragment is a bispecific antibody or antigen-binding fragment.

Embodiment 30. An antibody or antigen-binding fragment according to embodiment 28 or 29, comprising:

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

(ii) the second VH and the second VL,

wherein the first VH and the second VH are different and each independently comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 113, 1, 9-15, 23, 24, 27-46, 55, 63, 79, 87, 95, 103, 105, 114-120, 129-146, 155, 172, 176-178, 194, 196, 198, 200, 202, 239 and 267,

wherein the first VL and the second VL are different and each independently comprises an amino acid sequence having at least 85% identity to the amino acid sequence set forth in any one of SEQ ID NOs: 168, 5, 47-50, 59, 67, 71, 72, 75, 76, 83, 91, 99, 109, 147-150, 159, 182, 190, 234 and 243;

and wherein the first VH and the first VL together form a first antigen-binding site, and wherein the second VH and the second VL together form a second antigen-binding site.

Embodiment 31. The antibody or antigen-binding fragment of any one of embodiments 1-30, wherein the antibody or antigen-binding fragment further comprises an Fc polypeptide or fragment thereof.

Embodiment 32. The antibody or antigen-binding fragment of embodiment 31, wherein the Fc polypeptide or fragment thereof comprises:

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

(ii) a mutation that enhances binding to FcγR compared to a reference Fc polypeptide that does not contain the mutation.

Embodiment 33. The antibody or antigen-binding fragment of embodiment 32, wherein the mutation that enhances binding to FcRn comprises: M428L; N434S; N434H; N434A; N434S; M252Y; S254T; T256E; T250Q; P257I; Q311I; D376V; T307A; E380A, or any combination thereof.

Embodiment 34. The antibody or antigen-binding fragment of embodiment 32 or 33, wherein the mutation that enhances binding to FcRn comprises:

(i) M428L/N434S;

(ii) M252Y/S254T/T256E;

(iii) T250Q/M428L;

(iv) P257I/Q311I;

(v) P257I/N434H;

(vi) D376V/N434H;

(vii) T307A/E380A/N434A or

(viii) any combination of (i) to (vii).

Embodiment 35. The antibody or antigen-binding fragment of any one of embodiments 32-34, wherein the mutation that enhances binding to FcRn comprises M428L/N434S.

Embodiment 36. The antibody or antigen-binding fragment of any one of embodiments 32-35, wherein the mutation that enhances binding to FcγR comprises S239D; I332E; A330L; G236A, or any combination thereof.

Embodiment 37. The antibody or antigen-binding fragment of any one of embodiments 32-36, wherein the mutation that enhances binding to FcγR comprises:

(i) S239D/I332E;

(ii) S239D/A330L/I332E;

(iii) G236A/S239D/I332E or

(iv) G236A/A330L/I332E.

Embodiment 38. The antibody or antigen-binding fragment of any one of embodiments 1-37:

which contains a mutation that alters glycosylation, wherein the mutation that alters glycosylation comprises N297A, N297Q, or N297G; and/or

which is aglycosylated and/or afucosylated.

Embodiment 39. The antibody or antigen-binding fragment of any one of embodiments 31-38, wherein the Fc polypeptide comprises an L234A mutation and an L235A mutation.

Embodiment 40. The antibody or antigen-binding fragment of any one of embodiments 1-39, wherein the antibody or antigen-binding fragment binds to the S protein of SARS-CoV-2 as measured by biolayer interferometry.

Embodiment 41. The antibody or antigen-binding fragment of embodiment 40, wherein the antibody or antigen-binding fragment binds to the SARS-CoV-2 S protein with a KD (dissociation constant) of less than about 4.5×10 ^-9 M, such as less than 4.5×10 ^-9 M.

Embodiment 42. The antibody or antigen-binding fragment of embodiment 40 or 41, wherein the antibody or antigen-binding fragment binds to the SARS-CoV-2 S protein with a KD of less than about 1.0×10 ^-10 M, such as less than 1.0×10 ^-10 M.

Embodiment 43. The antibody or antigen-binding fragment of any one of embodiments 40-42, wherein the antibody or antigen-binding fragment binds to the SARS-CoV-2 S protein with a KD of less than about 1.0×10 ^-11 M, such as less than 1.0×10 ^-11 M.

Embodiment 44. The antibody or antigen-binding fragment of any one of embodiments 40-43, wherein the antibody or antigen-binding fragment binds to the SARS-CoV-2 S protein with a KD of less than about 1×10 ^-12 M, such as less than 1×10 ^-12 M.

Embodiment 45. The antibody or antigen-binding fragment of any one of embodiments 1-44, wherein the antibody or antigen-binding fragment is capable of neutralizing SARS-CoV-2 infection and/or neutralizing target cell infection with an IC50 of about 16 to about 20 μg/mL.

Embodiment 46. The antibody or antigen-binding fragment of any one of embodiments 1-45, wherein the antibody or antigen-binding fragment is capable of neutralizing SARS-CoV-2 infection and/or neutralizing target cell infection with an IC50 of about 0.3 to about 0.4 μg/mL or about 3 to about 4 nM.

Embodiment 47. The antibody or antigen-binding fragment of any one of embodiments 1-46, wherein the antibody or antigen-binding fragment is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) against a target cell infected with SARS-CoV-2.

Embodiment 48. The antibody or antigen-binding fragment of any one of embodiments 40-47, wherein the Fab of the antibody or antigen-binding fragment is capable of binding to the S protein of SARS-CoV-2 with a KD of 2.0×10-9 or less, 1.9× ^10-9 or less, or 1.8× ^10-9 or less.

Embodiment 49. The antibody or antigen-binding fragment of any one of embodiments 1-48, wherein the antibody or antigen-binding fragment is capable of neutralizing SARS-CoV-2 infection and does not compete with human ACE2 for binding to the SARS-CoV-2 S protein,

wherein, optionally, neutralizing comprises neutralizing the infection in an in vitro infection model.

Embodiment 50. The antibody or antigen-binding fragment of any one of embodiments 1-49, wherein the antibody or antigen-binding fragment is capable of neutralizing SARS-CoV-2 infection with an IC50 of 3.0 nM, 3.1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, or 4.0 nM.

Embodiment 51. The antibody or antigen-binding fragment of any one of embodiments 47-50, wherein inducing ADCC comprises activating a natural killer cell that comprises a V158 FcγRIIIa variant, a natural killer cell that comprises an F158 FcγRIIIa variant, or both.

Embodiment 52. The antibody or antigen-binding fragment of any one of embodiments 47-51, wherein the ADCP comprises engagement of FcγRIIa and/or FcγRIIIa expressed on the surface of a phagocytic cell, such as a monocyte, macrophage, or dendritic cell.

Embodiment 53. An antibody or antigen-binding fragment thereof that competes for binding to a SARS-CoV-2 surface glycoprotein with the antibody or antigen-binding fragment of any one of embodiments 1-52, wherein, optionally, the antibody or antigen-binding fragment is capable of inhibiting the interaction between SARS-CoV-2 and any one or more of DC-SIGN, L-SIGN, and SIGLEC-1.

Embodiment 54. An antibody or antigen-binding fragment thereof that competes for binding to a SARS-CoV-2 surface glycoprotein with antibody S309 (VH SEQ ID NO:105; VL SEQ ID NO:168) and/or antibody S303 (VH SEQ ID NO:63; VL SEQ ID NO:67), wherein, optionally, the antibody or antigen-binding fragment is capable of inhibiting the interaction between SARS-CoV-2 and any one or more of DC-SIGN, L-SIGN, and SIGLEC-1.

Embodiment 55. An antibody or antigen-binding fragment thereof that competes for binding to the SARS-CoV-2 surface glycoprotein with antibody S304 (VH SEQ ID NO:79; VL SEQ ID NO:81) and/or antibody S315 (VH SEQ ID NO:178; VL SEQ ID NO:182).

Embodiment 56. An antibody or antigen-binding fragment of any one of embodiments 1-55, capable of binding to a SARS-CoV-2 surface glycoprotein, wherein the SARS-CoV-2 surface glycoprotein is contained in a prefusion trimer.

Embodiment 57. The antibody or antigen-binding fragment of any one of embodiments 1-56, capable of binding to the receptor binding domain (RBD) of the surface glycoprotein of SARS-CoV-2 when the RBD is glycosylated and/or deglycosylated, wherein the binding is detected using surface plasmon resonance (SPR), wherein optionally:

(1) SPR is performed using a Biacore T200 instrument using a single cycle kinetic approach, optionally with a 3-minute infusion period and a 20-minute dissociation period;

(2) the antibody or antigen-binding fragment is captured on the surface;

(3) RBD is present at a concentration of 0.8 nM, 3.1 nM, 12.5 nM, 50 nM, or 200 nM;

(4) the antibody or antigen-binding fragment binds to the glycosylated RBD with a KD of about 2.0 nM, about 1.9 nM, about 1.8 nM, about 1.7 nM, about 1.6 nM, about 1.5 nM, about 1.4 nM, about 1.3 nM, about 1.2 nM, about 1.1 nM, about 1.0 nM, about 0.9 nM, about 0.8 nM, about 0.7 nM, about 0.6 nM, about 0.5 nM, about 0.4 nM, or about 0.3 nM, or with a KD of 0.4 ± 0.05 nM, or with a KD of 0.45 ± 0.05 nM, or with a KD of 0.5±0.05 nM, or with a KD of 0.6±0.05 nM, or with a KD of 0.7±0.05 nM, or with a KD of 1.7±0.05 nM; and/or

(5) the antibody or antigen-binding fragment binds to the deglycosylated RBD with a KD of about 37.0 nM, about 8.0 nM, about 2.0 nM, about 1.9 nM, about 1.8 nM, about 1.7 nM, about 1.6 nM, about 1.5 nM, about 1.4 nM, about 1.3 nM, about 1.2 nM, about 1.1 nM, about 1.0 nM, or about 0.9 nM, or with a KD of 37.0 ± 0.05 nM, or with a KD of 8.0 ± 0.05 nM, or with a KD of 1.0 ± 0.05 nM, or with a KD of 0.9 ± 0.05 nM, or with a KD of 1.3±0.05 nM, or with a KD of 1.8±0.05 nM, or with a KD of 1.7±0.05 nM.

Embodiment 58. An antibody or antigen-binding fragment of any one of embodiments 1-57, capable of neutralizing SARS-CoV-2 infection in a human lung cell, wherein, optionally, the human lung cell comprises a Calu-3 cell (a human lung cancer cell line), wherein, further optionally, the antibody or antigen-binding fragment has a neutralization IC50 of about 97 ng/mL.

Embodiment 59. An antibody or antigen-binding fragment of any one of embodiments 1-58, capable of binding to human complement component C1q, wherein optionally binding to C1q is detected by biolayer interferometry (BLI), such as using an Octet instrument.

Embodiment 60. An antibody or antigen-binding fragment of any one of embodiments 1-59, capable of inhibiting cell fusion mediated by a SARS-CoV-2 surface glycoprotein.

Embodiment 61. The antibody or antigen-binding fragment of any one of embodiments 1-60, which does not cause antibody-mediated enhancement of SARS-CoV-2 replication in a peripheral blood mononuclear cell (PBMC) or dendritic cell obtained from a human donor.

Embodiment 62. An antibody or antigen-binding fragment according to any one of embodiments 1-61, comprising:

(i) CH1-CH3 comprising or consisting of the amino acid sequence presented in SEQ ID NO:173 or 175; and/or

(ii) a CL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:174 or SEQ ID NO:193.

Embodiment 63. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising the amino acid sequence of the complementarity determining region (CDR) H1 set forth in SEQ ID NO:106, the amino acid sequence of CDRH2 set forth in SEQ ID NO:121, and the amino acid sequence of CDRH3 set forth in SEQ ID NO:108, and a light chain variable domain (VL) comprising the amino acid sequence of CDRL1 set forth in SEQ ID NO:169, the amino acid sequence of CDRL2 set forth in SEQ ID NO:170, and the amino acid sequence of CDRL3 set forth in SEQ ID NO:171,

wherein the antibody or antigen-binding fragment is capable of binding to the SARS-CoV-2 surface glycoprotein (S) expressed on the cell surface of a host cell, on the SARS-CoV-2 virion, or both.

Embodiment 64. An isolated antibody or antigen-binding fragment of any one of embodiments 1-63, capable of binding to a surface glycoprotein (S):

(i) SARS-CoV-2 Wuhan-Hu-1 (SEQ ID NO:165);

ii) SARS-CoV-2 B.1.1.7;

iii) SARS-CoV-2 B.1.351;

(iv) SARS-CoV-2 comprising any one or more of the following substitution mutations relative to SEQ ID NO:165: N501Y; S477N; N439K; L452R; E484K; Y453F; A520S; K417N; K417V; S494P; N501T; S477R; V367F; P384L; A522S; A522V; V382L; P330S; T478I; S477I; P479S; or

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

Embodiment 65. An isolated antibody or antigen-binding fragment of embodiment 63 or 64 capable of neutralizing SARS-CoV-2 infection:

(i) in vitro infection model;

(ii) in an animal model of infection in vivo;

(iii) in a human being; or

(iv) any combination of (i) to (iii).

Embodiment 66. An isolated antibody or antigen-binding fragment of any one of embodiments 63-65, wherein:

(i) the VH comprises or consists of an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:113; and/or

(ii) VL comprises or consists of an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 67. An isolated antibody or antigen-binding fragment of any one of embodiments 63-66, wherein:

(i) the VH comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:113; and/or

(ii) VL comprises or consists of an amino acid sequence having at least 90% identity to the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 68. An isolated antibody or antigen-binding fragment of any one of embodiments 63-67, wherein:

(i) the VH comprises or consists of an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:113; and/or

(ii) VL comprises or consists of an amino acid sequence having at least 95% identity to the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 69. An isolated antibody or antigen-binding fragment of any one of embodiments 63-68, wherein:

(i) the VH comprises or consists of an amino acid sequence having at least 99% identity to the amino acid sequence set forth in SEQ ID NO:113; and/or

(ii) VL comprises or consists of an amino acid sequence having at least 99% identity to the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 70. An isolated antibody or antigen-binding fragment of any one of Embodiments 63-69, capable of inhibiting the interaction between:

(i) SARS-CoV-2 and human DC-SIGN;

(ii) SARS-CoV-2 and human L-SIGN;

(iii) SARS-CoV-2 and human SIGLEC-1; or

(iv) any combination of (i) to (iii).

Embodiment 71. The isolated antibody or antigen-binding fragment of any one of embodiments 63-70, wherein the antibody or antigen-binding fragment comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, Fab', F(ab')2, Fv, scFv, or scFab.

Embodiment 72. The isolated antibody or antigen-binding fragment of any one of embodiments 63-71, wherein the antibody or antigen-binding fragment further comprises an Fc polypeptide or fragment thereof.

Embodiment 73. An isolated antibody or antigen-binding fragment of any one of embodiments 63-72, which is of the IgG, IgA, IgM, IgE, or IgD isotype.

Embodiment 74. The isolated antibody or antigen-binding fragment of embodiment 72 or 73, wherein the Fc polypeptide or fragment thereof comprises:

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

(ii) a mutation that enhances binding to FcγR compared to a reference Fc polypeptide that does not contain the mutation.

Embodiment 75. The isolated antibody or antigen-binding fragment of embodiment 74, wherein the mutation that enhances binding to FcRn comprises:

(i) M428L/N434S;

(ii) M252Y/S254T/T256E;

(iii) T250Q/M428L;

(iv) P257I/Q311I;

(v) P257I/N434H;

(vi) D376V/N434H;

(vii) T307A/E380A/N434A or

(viii) any combination of (i) to (vii).

Embodiment 76. The isolated antibody or antigen-binding fragment of embodiment 75, wherein the mutation that enhances binding to FcRn comprises M428L/N434S.

Embodiment 77. The isolated antibody or antigen-binding fragment of any one of embodiments 74-76, wherein the mutation that enhances binding to FcγR comprises S239D, I332E, A330L, G236A, or any combination thereof.

Embodiment 78. The isolated antibody or antigen-binding fragment of embodiment 74, wherein the mutation that enhances binding to FcRn comprises:

(i) S239D/I332E;

(ii) S239D/A330L/I332E;

(iii) G236A/S239D/I332E; or

(iv) G236A/A330L/I332E.

Embodiment 79. The isolated antibody or antigen-binding fragment of any one of embodiments 63-78, further comprising CH1-CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:265 or 266.

Embodiment 80. An isolated antibody or antigen-binding fragment thereof comprising a heavy chain variable domain (VH) and a light chain variable domain (VL), wherein VH comprises or consists of the amino acid sequence presented in SEQ ID NO:113, and VL comprises or consists of the amino acid sequence presented in SEQ ID NO:168.

Embodiment 81. An isolated antibody or antigen-binding fragment of embodiment 80 capable of neutralizing SARS-CoV-2 infection:

(i) in vitro infection model;

(ii) in an animal model of infection in vivo;

(iii) in a human being; or

(iv) any combination of (i) to (iii).

Embodiment 82. An isolated antibody or antigen-binding fragment of embodiment 80 or 81, capable of inhibiting the interaction between:

i) SARS-CoV-2 and human DC-SIGN;

ii) SARS-CoV-2 and human L-SIGN;

iii) SARS-CoV-2 and human SIGLEC-1; or

(iv) any combination of (i) to (iii).

Embodiment 83. The isolated antibody or antigen-binding fragment of any one of embodiments 80-82, wherein the antibody or antigen-binding fragment further comprises an Fc polypeptide or fragment thereof.

Embodiment 84. An isolated antibody or antigen-binding fragment of any one of embodiments 80-83, which is of the IgG, IgA, IgM, IgE, or IgD isotype.

Embodiment 85. The isolated antibody or antigen-binding fragment of any one of embodiments 83 or 84, wherein the Fc polypeptide or fragment thereof comprises:

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

(ii) a mutation that enhances binding to FcγR compared to a reference Fc polypeptide that does not contain the mutation.

Embodiment 86. The isolated antibody or antigen-binding fragment of embodiment 85, wherein the mutation that enhances binding to FcRn comprises:

(i) M428L/N434S;

(ii) M252Y/S254T/T256E;

(iii) T250Q/M428L;

(iv) P257I/Q311I;

(v) P257I/N434H;

(vi) D376V/N434H;

(vii) T307A/E380A/N434A or

(viii) any combination of (i) to (vii).

Embodiment 87. The isolated antibody or antigen-binding fragment of embodiment 86, wherein the mutation that enhances binding to FcRn comprises M428L/N434S.

Embodiment 88. The isolated antibody or antigen-binding fragment of any one of embodiments 85-87, wherein the mutation that enhances binding to FcγR comprises S239D, I332E, A330L, G236A, or any combination thereof.

Embodiment 89. The isolated antibody or antigen-binding fragment of embodiment 88, wherein the mutation that enhances binding to FcγR comprises:

(i) S239D/I332E;

(ii) S239D/A330L/I332E;

(iii) G236A/S239D/I332E or

(iv) G236A/A330L/I332E.

Embodiment 90. The isolated antibody or antigen-binding fragment of any one of embodiments 80-89, further comprising CH1-CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:265 or 266.

Embodiment 91. An isolated antibody that comprises:

(i) a heavy chain comprising (i)(1) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:113 and (i)(2) a CH1-CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:173; and

(ii) a light chain comprising (ii)(1) a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 168 and (ii)(2) a CL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 174.

Embodiment 92. An isolated antibody or antigen-binding fragment thereof capable of binding to the surface glycoprotein (S) of SARS-CoV-2 and inhibiting the interaction between SARS-CoV-2 and human DC-SIGN, human L-SIGN, human SIGLEC-1, or any combination thereof.

Embodiment 93. An isolated antibody that comprises:

(i) a heavy chain comprising (i)(1) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:113 and (i)(2) a CH1-CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:175; and

(ii) a light chain comprising (ii)(1) a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 168 and (ii)(2) a CL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 174.

Embodiment 94. An isolated antibody or antigen-binding fragment thereof, comprising a heavy chain variable domain (VH) comprising the amino acid sequence of the complementarity determining region (CDR) H1 set forth in SEQ ID NO:106, the amino acid sequence of CDRH2 set forth in SEQ ID NO:121, and the amino acid sequence of CDRH3 set forth in SEQ ID NO:108, and a light chain variable domain (VL) comprising the amino acid sequence of CDRL1 set forth in SEQ ID NO:169, the amino acid sequence of CDRL2 set forth in SEQ ID NO:170, and the amino acid sequence of CDRL3 set forth in SEQ ID NO:171,

wherein the antibody or antigen-binding fragment is capable of binding to the SARS-CoV-2 surface glycoprotein (S) expressed on the cell surface of a host cell, on the SARS-CoV-2 virion, or both.

Embodiment 95. An isolated antibody or antigen-binding fragment of embodiment 94 capable of neutralizing SARS-CoV-2 infection:

(i) in vitro infection model;

(ii) in an animal model of infection in vivo;

(iii) in a human being; or

(iv) any combination of (i) to (iii).

Embodiment 96. The antibody or antigen-binding fragment of embodiment 94 or 95, wherein:

(i) the VH comprises or consists of an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:113; and/or

(ii) VL comprises or consists of an amino acid sequence having at least 85% identity to the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 97. The antibody or antigen-binding fragment of any one of embodiments 94-96, wherein VH comprises or consists of the amino acid sequence set forth in SEQ ID NO:113, and VL comprises or consists of the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 98. The antibody or antigen-binding fragment of any one of embodiments 94-97, which:

(i) is able to bind to the SARS-CoV-2 surface glycoprotein with greater avidity than to the SARS coronavirus S protein;

(ii) has cross-reactivity against SARS-CoV-2 and SARS coronavirus;

(iii) recognizes an epitope on the SARS-CoV-2 surface glycoprotein that is not part of the ACE2 RBM; or

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

Embodiment 99. The antibody or antigen-binding fragment of any one of embodiments 94-98, which is an IgG, IgA, IgM, IgE or IgD isotype, and preferably is an IgG isotype selected from IgG1, IgG2, IgG3 and IgG4.

Embodiment 100. The antibody or antigen-binding fragment of any one of embodiments 94-99:

(i) which is human, humanized or chimeric;

(ii) wherein the antibody or antigen-binding fragment comprises a human antibody, a monoclonal antibody, a purified antibody, a single chain antibody, a Fab, Fab', F(ab')2, Fv, scFv or scFab; and/or

(iii) wherein the antibody or antigen-binding fragment is a multispecific antibody or antigen-binding fragment, wherein, optionally, the antigen-binding fragment is a bispecific antibody or antigen-binding fragment.

Embodiment 101. The antibody or antigen-binding fragment of any one of embodiments 94-100, wherein the antibody or antigen-binding fragment further comprises an Fc polypeptide or fragment thereof.

Embodiment 102. The antibody or antigen-binding fragment of embodiment 101, wherein the Fc polypeptide or fragment thereof comprises:

(1) a mutation that enhances binding to FcRn compared to a reference Fc polypeptide that does not comprise the mutation, wherein optionally the mutation that enhances binding to FcRn comprises M428L, N434S, N434H, N434A, N434S, M252Y, S254T, T256E, T250Q, P257I, Q311I, D376V, T307A, E380A, or any combination thereof, wherein further optionally the mutation that enhances binding to FcRn comprises: (i) M428L/N434S; (ii) M252Y/S254T/T256E; (iii) T250Q/M428L; (iv) P257I/Q311I; (v) P257I/N434H; (vi) D376V/N434H; (vii) T307A/E380A/N434A; or (viii) any combination of (i)-(vii) and/or

(2) a mutation that enhances binding to an FcγR compared to a reference Fc polypeptide that does not comprise the mutation, wherein optionally the mutation that enhances binding to an FcγR comprises S239D, I332E, A330L, G236A, or any combination thereof, wherein further optionally the mutation that enhances binding to an FcγR comprises: (i) S239D/I332E; (ii) S239D/A330L/I332E; (iii) G236A/S239D/I332E; or (iv) G236A/A330L/I332E.

Embodiment 103. The antibody or antigen-binding fragment of embodiment 102, wherein the mutation that enhances binding to FcRn comprises M428L/N434S and/or the mutation that enhances binding to FcγR comprises G236A/A330L/I332E.

Embodiment 104. An antibody or antigen-binding fragment of any one of embodiments 94-103, comprising:

(i) CH1-CH3 having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO:173 or 175; and/or

(ii) a CL comprising an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:174.

Embodiment 105. The antibody or antigen-binding fragment of any one of embodiments 94-104, which comprises a mutation that alters glycosylation of the antibody or antigen-binding fragment, wherein the mutation that alters glycosylation of the antibody or antigen-binding fragment comprises N297A, N297Q, or N297G, and/or wherein the antibody or antigen-binding fragment is aglycosylated and/or afucosylated.

Embodiment 106. The antibody or antigen-binding fragment of any one of claims 94-105, wherein the antibody or antigen-binding fragment binds to a SARS-CoV-2 surface glycoprotein with a KD of less than about 4.5× ^10-9 M, less than about 5× ^10-9 M, less than about 1× ^10-10 M, less than about 5× ^10-10 M, less than about 1× ^10-11 M, less than about 5× ^10-11 M, or less than about 1× ^10-12 M, as measured by biolayer interferometry, wherein optionally the antibody or antigen-binding fragment binds to a SARS-CoV-2 surface glycoprotein with a KD of less than 1× ^10-12 M, as measured by biolayer interferometry (e.g., by immobilizing the antibody or antigen-binding fragment thereof on sensors and dipping the sensors into wells containing different concentrations of SARS-CoV-2 or RBD, recording antibody binding kinetics during the association phase, after which the sensors are immersed in buffer without observing SARS-CoV-2 antibody separation kinetics or RD during the dissociation phase. Protein A biosensors (Pall ForteBio) can be used to immobilize recombinant antibodies at 2.7 μg/mL for 1 min, following a 10 min hydration step with kinetic assay buffer (KB; 0.01% endotoxin-free BSA, 0.002^ Tween-20, 0.005% NaN3 in PBS). Association curves can be recorded within 5 min by incubating antibody-coated sensors with different concentrations of SARS-CoV-1 RBD (Sino Biological) or SARS-CoV-2 RBD (produced in-house in Expi-CHO cells; residues 331-550 of the spike protein from BetaCoV/Wuhan-Hu-1/2019, Cat. No. MN908947). The assayed concentration of SARS-CoV-2 or RBD can be 10 μg/mL, then serially diluted 1:2.5. Dissociation can be recorded within 9 min by moving the sensors into wells containing KB. Affinities, represented by KD values, can be calculated using the global matching model (Octet). Octet Red96 equipment (ForteBio) was used.

Embodiment 107. The antibody or antigen-binding fragment of any one of embodiments 94-106, wherein the antibody or antigen-binding fragment is capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP) against a target cell infected with SARS-CoV-2,

wherein optionally inducing ADCC comprises activation of a natural killer cell that contains an FcγRIIIa V158 variant, a natural killer cell that contains an FcγRIIIa F158 variant, or both, and/or inducing ADCP comprises engagement of FcγRIIa expressed on the surface of a phagocytic cell such as a monocyte, macrophage, or dendritic cell.

Embodiment 108. The antibody or antigen-binding fragment of any one of embodiments 94-107, wherein the antibody or antigen-binding fragment is a Fab, and the Fab is capable of binding to a SARS-CoV-2 surface glycoprotein with a KD of 2.0×10 ^-9 M or less, 1.9×10 ^-9 M or less, or 1.8×10 ^-9 M or less, as measured by biolayer interferometry.

Embodiment 109. The antibody or antigen-binding fragment of any one of embodiments 94-108, wherein the antibody or antigen-binding fragment is capable of neutralizing SARS-CoV-2 infection and does not compete with human ACE2 for binding to the SARS-CoV-2 S protein.

Embodiment 110. An antibody or antigen-binding fragment thereof that competes for binding to a SARS-CoV-2 surface glycoprotein with the antibody or antigen-binding fragment of any one of embodiments 94-109.

Embodiment 111. An antibody or antigen-binding fragment of any one of embodiments 94-110, capable of binding to a SARS-CoV-2 surface glycoprotein, wherein the SARS-CoV-2 surface glycoprotein is contained in a prefusion trimer.

Embodiment 112. The antibody or antigen-binding fragment of any one of embodiments 94-111, capable of binding to the receptor binding domain (RBD) of the surface glycoprotein of SARS-CoV-2 when the RBD is glycosylated and/or deglycosylated, wherein the binding is detected using surface plasmon resonance (SPR), wherein optionally:

(1) SPR is performed using a Biacore T200 instrument using a single cycle kinetic approach, optionally with a 3-minute infusion period and a 20-minute dissociation period;

(2) the antibody or antigen-binding fragment is captured on the surface;

(3) RBD is present at a concentration of 0.8 nM, 3.1 nM, 12.5 nM, 50 nM, or 200 nM;

(4) the antibody or antigen-binding fragment binds to the glycosylated RBD with a KD of about 0.6 nM, about 0.5 nM, about 0.4 nM, or about 0.3 nM, or with a KD of 0.3 ± 0.05 nM, or with a KD of 0.4 ± 0.05 nM, or with a KD of 0.45 ± 0.05 nM, or with a KD of 0.5 ± 0.05 nM, or with a KD of 0.6 ± 0.05 nM; and/or

(5) The antibody or antigen-binding fragment binds to the deglycosylated RBD with a KD of about 1.6 nM, about 1.5 nM, about 1.4 nM, about 1.3 nM, about 1.2 nM, about 1.1 nM, about 1.0.

Embodiment 113. The antibody or antigen-binding fragment of any one of embodiments 94-112, capable of neutralizing SARS-CoV-2 infection in a human lung cell, wherein, optionally, the human lung cell comprises a Calu-3 cell, wherein, optionally, the antibody or antigen-binding fragment has a neutralization IC50 of about 97 ng/mL.

Embodiment 114. An antibody or antigen-binding fragment of any one of embodiments 94-113, capable of binding to human complement component C1q, wherein optionally binding to C1q is detected by biolayer interferometry (BLI), such as using an Octet instrument.

Embodiment 115. An antibody or antigen-binding fragment of any one of embodiments 94-114, capable of inhibiting cell fusion mediated by a SARS-CoV-2 surface glycoprotein.

Embodiment 116. The antibody or antigen-binding fragment of any one of embodiments 94-115, which does not cause antibody-mediated enhancement of SARS-CoV-2 replication in a peripheral blood mononuclear cell (PBMC) or dendritic cell obtained from a human donor.

Embodiment 117. An antibody or antigen-binding fragment of any one of Embodiments 94-116, capable of inhibiting the interaction between:

i) SARS-CoV-2 and human DC-SIGN;

ii) SARS-CoV-2 and human L-SIGN;

iii) SARS-CoV-2 and human SIGLEC-1; or

(iv) any combination of (i) to (iii).

Embodiment 118. An antibody or antigen-binding fragment of any one of embodiments 94-117, capable of binding to a surface glycoprotein:

(i) SARS-CoV-2 Wuhan-Hu-1 (SEQ ID NO:165);

(ii) SARS-CoV-2 B.1.1.7;

(iii) SARS-CoV-2 B.1.351;

(iv) SARS-CoV-2 comprising any one or more of the following substitution mutations relative to SEQ ID NO:165: N501Y; S477N; N439K; L452R; E484K; Y453F; A520S; K417N; K417V; S494P; N501T; S477R; V367F; P384L; A522S; A522V; V382L; P330S; T478I; S477I; P479S; or

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

Embodiment 119. The antibody or antigen-binding fragment of any one of embodiments 94-118, wherein the antibody or antigen-binding fragment is capable of neutralizing SARS-CoV-2 infection with an IC50 of 3.0 nM, 3.1 nM, 3.2 nM, 3.3 nM, 3.4 nM, 3.5 nM, 3.6 nM, 3.7 nM, 3.8 nM, 3.9 nM, or 4.0 nM.

Embodiment 120. An antibody or antigen-binding fragment of any one of embodiments 94-119, comprising:

(i) a heavy chain comprising (i)(1) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:113 and (i)(2) a CH1-CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:173; and

(ii) a light chain comprising (ii)(1) a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 168 and (ii)(2) a CL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 174.

Embodiment 121. An antibody or antigen-binding fragment of any one of embodiments 94-120, comprising:

(i) a heavy chain comprising (i)(1) a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:113 and (i)(2) a CH1-CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:175; and

(ii) a light chain comprising (ii)(1) a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 168 and (ii)(2) a CL comprising or consisting of the amino acid sequence set forth in SEQ ID NO: 174.

Embodiment 122. An isolated antibody or antigen-binding fragment of any one of embodiments 94-121, comprising a CH1-CH3 comprising or consisting of the amino acid sequence set forth in SEQ ID NO:265 or 266.

Embodiment 123. An isolated polynucleotide encoding an antibody or antigen-binding fragment of any one of embodiments 1-122 or encoding a VH, heavy chain, VL and/or light chain of an antibody or antigen-binding fragment.

Embodiment 124. The isolated polynucleotide of embodiment 123, wherein the polynucleotide comprises deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), wherein the RNA optionally includes messenger RNA (mRNA).

Embodiment 125. An isolated polynucleotide of embodiment 123 or 124 that is codon optimized for expression in a host cell.

Embodiment 126. An isolated polynucleotide according to any one of embodiments 123-125, comprising a polynucleotide having at least 50% identity to a polynucleotide sequence shown in any one or more of SEQ ID NOs:186-189, 191-192, 238, 247, 248-255, and 257-262.

Embodiment 127. An isolated polynucleotide according to any one of embodiments 123-126, comprising a polynucleotide sequence as set forth in any one or more of SEQ ID NOs:249, 250, and 257-262.

Embodiment 128. A recombinant vector comprising a polynucleotide according to any one of embodiments 123-127.

Embodiment 129. A host cell comprising a polynucleotide of any one of embodiments 123-127 and/or a vector of embodiment 128, wherein the polynucleotide is heterologous to the host cell.

Embodiment 130. A human B cell comprising a polynucleotide of any one of embodiments 123-129, wherein the polynucleotide is heterologous to the human B cell and/or wherein the human B cell is immortalized.

Embodiment 131. A composition comprising

(i) an antibody or antigen-binding fragment of any one of embodiments 1-122;

(ii) a polynucleotide according to any one of embodiments 123-127;

(iii) the recombinant vector of embodiment 128;

(iv) a host cell according to the embodiment of paragraph 129; and/or

(v) A human B cell according to embodiment 130

and a pharmaceutically acceptable excipient, carrier or diluent.

Embodiment 132. The composition of embodiment 131 comprising two or more antibodies or antigen-binding fragments of any one of embodiments 1-122.

Embodiment 133. The composition of embodiment 132, comprising:

(i) a first antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:79 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:83; and

(ii) a second antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:105 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 134. The composition of embodiment 132, comprising:

(i) a first antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2 and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:80-82, respectively, and CDRL1, CDRL2 and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:84-86, respectively, and

(ii) a second antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2 and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 106-108, respectively, and CDRL1, CDRL2 and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 169-171, respectively.

Embodiment 135. The composition of embodiment 132, comprising:

(i) a first antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:178 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:182 or SEQ ID NO:190; and

(ii) a second antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:105 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:168.

Embodiment 136. The composition of embodiment 135, comprising:

(i) a first antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:179-181, respectively, and CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:183-185, respectively; and

(ii) a second antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2 and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2 and CDRL3, wherein CDRH1, CDRH2 and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 106-108, respectively, and CDRL1, CDRL2 and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs: 169-171, respectively.

Embodiment 137. The composition of embodiment 132, comprising:

(i) a first antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:178 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:182 or SEQ ID NO:190; and

(ii) a second antibody or antigen-binding fragment comprising a VH comprising or consisting of the amino acid sequence set forth in SEQ ID NO:63 and a VL comprising or consisting of the amino acid sequence set forth in SEQ ID NO:67, any of SEQ ID NOs:71-71, or any of SEQ ID NOs:75-76.

Embodiment 138. The composition of embodiment 132, comprising:

(i) a first antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:179-181, respectively, and CDRL1, CDRL2, and CDRL3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:183-185, respectively; and

(ii) a second antibody or antigen-binding fragment comprising a heavy chain variable domain (VH) comprising CDRH1, CDRH2, and CDRH3, and a light chain variable domain (VL) comprising CDRL1, CDRL2, and CDRL3, wherein CDRH1, CDRH2, and CDRH3 comprise or consist of the amino acid sequences set forth in SEQ ID NOs:64-66, respectively, CDRL1 comprises or consists of the amino acid sequences set forth in any of SEQ ID NO:68, SEQ ID NO:73, or SEQ ID NO:74, CDRL2 comprises or consists of the amino acid sequences set forth in SEQ ID NO:69, and CDRL3 comprises or consists of the amino acid sequences set forth in SEQ ID NO:70, SEQ ID NO:77, or SEQ ID NO:78.

Embodiment 139. A composition comprising (i) an antibody or antigen-binding fragment of embodiment 8 or 9 and (ii) an antibody or antigen-binding fragment of embodiment 10 or 11, wherein the composition is capable of neutralizing SARS-CoV-2 infection with an IC50 of about 0.07 to about 0.08 μg/mL.

Embodiment 140. A composition comprising a polynucleotide of any one of embodiments 123-127 encapsulated in a carrier molecule, wherein the carrier molecule optionally comprises a lipid, a carrier derived from a lipid, such as a liposome, a solid lipid nanoparticle, an oleaginous suspension, a submicron lipid emulsion, a lipid microbubble, a reverse lipid micelle, a cochlear liposome, a lipid microtubule, a lipid microcylinder, a lipid nanoparticle (LNP), or a nanoscale platform.

Embodiment 141. A method of treating a SARS-CoV-2 infection in a subject, comprising administering to said subject an effective amount of:

(i) an antibody or antigen-binding fragment of any one of embodiments 1-122;

(ii) a polynucleotide according to any one of embodiments 123-127;

(iii) the recombinant vector of embodiment 128;

(iv) a host cell according to the embodiment of paragraph 129;

(v) human B cells according to embodiment 130 and/or

(vi) compositions according to any one of embodiments 131-140.

Embodiment 142. A method of inhibiting SARS-CoV-2 infection in a subject, comprising administering to said subject an effective amount of:

(i) an antibody or antigen-binding fragment of any one of embodiments 1-122;

(ii) a polynucleotide according to any one of embodiments 123-127;

(iii) the recombinant vector of embodiment 128;

(iv) a host cell according to the embodiment of paragraph 129;

(v) human B cells according to embodiment 130 and/or

(vi) compositions according to any one of embodiments 131-140.

Embodiment 143. The antibody or antigen-binding fragment of any one of embodiments 1-122, the polynucleotide of any one of embodiments 123-127, the recombinant vector of embodiment 128, the host cell of embodiment 129, the human B cell of embodiment 130, and/or the composition of any one of embodiments 131-140, for use in a method of treating SARS-CoV-2 infection in a subject.

Embodiment 144. The antibody or antigen-binding fragment of any one of embodiments 1-122, the polynucleotide of any one of embodiments 123-127, the recombinant vector of embodiment 128, the host cell of embodiment 129, the human B cell of embodiment 130, and/or the composition of any one of embodiments 131-140, for use in a method of inhibiting SARS-CoV-2 infection in a subject.

Embodiment 145. The antibody or antigen-binding fragment of any one of embodiments 1-122, the polynucleotide of any one of embodiments 123-127, the recombinant vector of embodiment 128, the host cell of embodiment 129, the human B cell of embodiment 130, and/or the composition of any one of embodiments 131-140 in the preparation of a medicament for treating SARS-CoV-2 infection in a subject.

Embodiment 146. A method for diagnosing SARS-CoV-2 infection in vitro, comprising:

(i) contacting a sample obtained from the subject with the antibody or antigen-binding fragment of any one of embodiments 1-122, and

(ii) detection of a complex containing an antigen and an antibody or containing an antigen and an antigen-binding fragment.

Embodiment 147. The method of embodiment 146, wherein the sample comprises blood isolated from the subject.

Embodiment 148. A combination or composition comprising:

(i) an antibody or antigen-binding fragment comprising

(i)(a) the CDRH1 amino acid sequence GYPFTSYG, the CDRH2 amino acid sequence ISTYNGNT or ISTYQGNT, the CDRH3 amino acid sequence ARDYTRGAWFGESLIGGFDN; the CDRL1 or QTVSSTS amino acid sequence, the CDRL2 amino acid sequence GAS, and the CDRL3 amino acid sequence QHDTSLT; or

(i)(b) a VH amino acid sequence comprising or consisting of QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYNGNTNYAQKFQGRVTMTTDTSTTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSS

or containing or consisting of QVQLVQSGAEVKKPGASVKVSCKASGYPFTSYGISWVRQAPGQGLEWMGWISTYQGNTNYAQKFQGRVTMTTDTSTTGYMELRRLRSDDTAVYYCARDYTRGAWFGESLIGGFDNWGQGTLVTVSS,

and an amino acid sequence VL comprising or consisting of EIVLTQSPGTLSLSPGERATLSCRASQTVSSTSLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQHDTSLTFGGGTKVEIK;

And

(ii) an antibody or antigen-binding fragment comprising

(ii)(a) the VH and VL amino acid sequences shown in SEQ ID NOs:79 and 83, respectively;

(ii)(b) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs:80-82 and 84-86, respectively;

(ii)(c) the VH and VL amino acid sequences shown in SEQ ID NO: 178 or 194 or 196 or 198 or 200 or 202 and 182 or 190, respectively; or

(ii)(d) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 as set forth in SEQ ID NO:179 or 195, 180 or 197 or 199, 181, 201 or 203 and 183-185, respectively.

Embodiment 149. A method for preventing, or treating, or neutralizing a coronavirus infection in a subject, comprising administering to the subject a combination or composition of embodiment 148, wherein, optionally, the antibody or antigen-binding fragment (i) and the antibody or antigen-binding fragment (ii) are administered in parallel, simultaneously, or sequentially.

Embodiment 150. A method for preventing, or treating, or neutralizing a coronavirus infection in a subject, comprising administering to the subject who has received a first antibody or antigen-binding fragment comprising:

(a) the VH and VL amino acid sequences shown in SEQ ID NOs:79 and 83, respectively; or

(b) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs:80-82 and 84-86, respectively;

and a second antibody or antigen-binding fragment comprising:

(a) the VH amino acid sequence shown in SEQ ID NO: 105 or 113 and the VL amino acid sequence shown in SEQ ID NO: 168; or

(b) the amino acids CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs: 106-108, respectively, or SEQ ID NOs: 106, 121 and 108, respectively, and the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs: 169-171, respectively.

Embodiment 151. A method for preventing, or treating, or neutralizing a coronavirus infection in a subject, comprising administering to the subject who has received a first antibody or antigen-binding fragment comprising:

(a) the VH amino acid sequence shown in SEQ ID NO: 105 or 113 and the VL amino acid sequence shown in SEQ ID NO: 168; or

(b) the amino acids CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs: 106-108, respectively, or SEQ ID NOs: 106, 121 and 108, respectively, and the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs: 169-171, respectively;

and a second antibody or antigen-binding fragment comprising:

(a) the VH and VL amino acid sequences shown in SEQ ID NOs:79 and 83, respectively; or

(b) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs:80-82 and 84-86, respectively.

Embodiment 152. A method for preventing, or treating, or neutralizing a coronavirus infection in a subject, comprising administering to the subject who has received a first antibody or antigen-binding fragment comprising:

(a) the VH and VL amino acid sequences shown in SEQ ID NO: 178 or 194 or 196 or 198 or 200 or 202 and 182 or 190, respectively; or

(b) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 as set forth in SEQ ID NO:179, or 195, 180, or 197, or 199, 181, 201 or 203 and 183-185, respectively.

and a second antibody or antigen-binding fragment comprising:

(a) the VH amino acid sequence shown in SEQ ID NO: 105 or 113 and the VL amino acid sequence shown in SEQ ID NO: 168; or

(b) the amino acids CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs: 106-108, respectively, or SEQ ID NOs: 106, 121 and 108, respectively, and the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs: 169-171, respectively.

Embodiment 153. A method for preventing, or treating, or neutralizing a coronavirus infection in a subject, comprising administering to the subject who has received a first antibody or antigen-binding fragment comprising:

(a) the VH amino acid sequence shown in SEQ ID NO: 105 or 113 and the VL amino acid sequence shown in SEQ ID NO: 168; or

(b) the amino acids CDRH1, CDRH2 and CDRH3 shown in SEQ ID NOs: 106-108, respectively, or SEQ ID NOs: 106, 121 and 108, respectively, and the amino acid sequences CDRL1, CDRL2 and CDRL3 shown in SEQ ID NOs: 169-171, respectively;

and a second antibody or antigen-binding fragment comprising:

(a) the VH and VL amino acid sequences shown in SEQ ID NO: 178 or 194 or 196 or 198 or 200 or 202 and 182 or 190, respectively; or

(b) the amino acid sequences of CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 as set forth in SEQ ID NO: 179 or 195, 180 or 197 or 199, 181, 201 or 203 and 183-185, respectively.

Examples

Example 1

Human monoclonal antibodies that bind the SARS-CoV-2 spike protein

B cells from a donor with a previous SARS-CoV infection were sorted and immortalized with EBV (Epstein-Barr virus) and screened in 384-well plates (method described in European patent EP1597280B1, which is incorporated herein by reference).

Two weeks after immortalization, the supernatants of immortalized B cells were tested for antibody binding to the SARS-CoV-2 spike protein (“S”) using a flow cytometric method. Briefly, ExpiCHO cells were transfected with the SARS-CoV-2 S protein (BetaCoV/Wuhan-Hu-1/2019 strain) or an empty plasmid as a negative control. Fourteen monoclonal antibodies that bind SARS-CoV-2 S were identified and named SARS-CoV-2 S300 through SARS-CoV-2 S312 and SARS-CoV-2 S315, respectively. The binding data for SARS-CoV-2 S300 through SARS-CoV-2 S310 are shown in Fig. 4A and 4B (in these figures, antibodies are designated as “S300”–“S310”, respectively). Plots showing positive binding are indicated by boxes.

The amino acid sequences of the 3 heavy-chain complementarity determining regions (CDR) and light-chain (L) CDR3 of some of these antibodies, along with the percent sequence identity to the variable germline region gene (IMGT; imgt.org), are presented in Table 3.

Example 2

Antibody Binding to SARS-CoV-2 RBD Using Octet

Strepavidin biosensors (Pall ForteBio) were used to immobilize anti-Strep Tag II antibody at 3 μg/mL (clone 5A9F9, biotin, LabForce AG, Muttenz CH) after a 10 min hydration step with kinetic buffer (KB; 0.01% endotoxin-free BSA, 0.002^ Tween-20, 0.005% NaN3 in PBS). Then, SARS-CoV-2 RBD with Strep Tag II (produced in-house) was loaded for 6 min at 4 μg/mL in KB. Antibodies from B cell supernatant were allowed to associate for 1620 sec (27 min). To observe dissociation, sensors were transferred from the antibody solution to KB and antibody dissociation was monitored.

The "S303" mAb contains the S303-v1 VH and VL amino acid sequences shown in Table 2 (SEQ ID NOS:63 and 67, respectively). The "S309" mAb contains the S309-v1 VH and S309-v13 VL amino acid sequences shown in Table 2 (SEQ ID NOS:105 and 168, respectively). Alleles encoding SEQ ID NOS:109 and 147-150 from the S309 B cell were defined as non-productive; SEQ ID NOS:168 is the productive allele.

Comparison of the binding curves for mAbs S303 and S309 to SARS-CoV-2 RBD (Figs. 1A and 1B) indicates that S303 has both a higher association rate and a higher dissociation rate than S309, suggesting that S309 may bind to SARS-CoV-2 RBD with higher affinity.

Example 3

Assessing Antibody Binding to SARS-CoV-2 and SARS-CoV-1 RBDs Using Octet

Unless the context clearly indicates otherwise (e.g., that the antibodies were present in B cell supernatant or a Fab fragment of the antibody was used), the antibodies of the present invention are described in this and subsequent examples as recombinantly expressed human IgG1, in some cases with amino acid mutations in the Fc as described herein.

The binding affinity of three cross-reactive recombinant SARS-CoV/SARS-CoV-2 antibodies (S303 rIgG1, S304 rIgG1, S309 rIgG1) and two SARS-CoV-1 specific antibodies (S109 rIgG1, S230 rIgG1) was studied by biolayer interferometry (BLI) using Octet. Affinity was measured by immobilizing the antibody on sensors and dipping sensors into wells containing different concentrations of RBD.

The kinetics of antibody binding to the RBD were recorded during the association phase, after which the sensors were immersed in buffer without antibody to observe the kinetics of antibody release from the RBD during the dissociation phase. Briefly, protein A biosensors (Pall ForteBio) were used to immobilize recombinant antibodies at 2.7 μg/mL for 1 min, following a 10 min hydration step with kinetic assay buffer (KB; 0.01% endotoxin-free BSA, 0.002^ Tween-20, 0.005% NaN3 in PBS). Association curves were recorded for 5 min by incubating antibody-coated sensors with different concentrations of SARS-CoV-1 RBD (Sino Biological) or SARS-CoV-2 RBD (produced in-house in Expi-CHO cells; residues 331-550 of spike protein from BetaCoV/Wuhan-Hu-1/2019, cat. no. MN908947). The highest RBD concentration tested was 10 μg/mL, then serially diluted 1:2.5 s. Dissociation was recorded for 9 min by moving the sensors into wells containing KB. Affinities, represented by KD values, were calculated using the global matching model (Octet). Octet Red96 equipment (ForteBio) was used.

Figures 6A-6E show association and dissociation curves for antibodies using the highest concentration of RBD tested (10 μg/mL). Switching from RBD solution to buffer is indicated by the vertical dotted line. Three cross-reactive antibodies (S303 rIgG1, S304 rIgG1 (VH with SEQ ID NO:79, VL with SEQ ID NO:73), S309 rIgG1 (VH with SEQ ID NO:105, VL with SEQ ID NO:168) and two SARS-CoV-1 specific antibodies (S230 and S109) were tested. All antibodies showed strong binding to the RBD of SARS-CoV-1. S230 and S109 did not bind to the RBD of SARS-CoV-2. Binding of S303 rIgG1, S304 rIgG1 and S309 rIGg1 to the RBD of SARS-CoV-2 was in the nanomolar to subpicomolar range, with S309 rIgG1 showing the highest affinity. KD values are shown below plots in Fig. 6A-6E. KD values are estimates (KD is less than 1.0×10 ^-12 M) if antibody binding is very strong and dissociation is slow. The exact KD value for S309 rIgG1 could not be measured with this assay because dissociation was too slow.

Example 4

Neutralization of SARS-CoV-2 infection

Replication-incompetent viruses pseudotyped with the SARS-CoV-2 genome (isolate BetaCoV/Wuhan-Hu-1/2019; cat. #MN908947) were generated using methods described previously (Temperton NJ, et al. (2005) Longitudinally profiling neutralizing antibody response to SARS coronavirus with pseudotypes. Emerg Infect Dis 11(3):411–416). Briefly, HEK293T/17 cells were cotransfected with the SARS-CoV-2 S-cell S-expressing plasmid (phCMV1, Genlantis) and the viral genome complementary reporter gene vector pNL4-3. Luc+E-R+. To measure neutralization of luciferase-encoding virions pseudotyped with the SARS-CoV-2 S protein, a single-cycle infectivity assay was used as previously described (Temperton NJ, et al. (2007) A sensitive retroviral pseudotype assay for influenza H5N1-neutralizing antibodies. Influenza Other Respi Viruses 1(3):105–112.). Briefly, appropriate dilutions of virion-containing culture supernatants were preincubated at 37°C for 1 h with various concentrations of antibodies, and then virus-mAb mixtures were added to Vero E6 cells that had been seeded the day before infection. Cells were then lysed with Steady-Glo reagent (Promega, E2520) and relative luminescent units (RLU) in cell lysates were determined on a microplate reader with a luminometer (Synergy H1 Hybrid Multi-Mode Reader; Biotek). The reduction in infectivity was determined by comparing RLU in the presence and absence of antibody and expressed as percent neutralization.

Antibodies S300-v1 (VH: SEQ ID NO:1; VL: SEQ ID NO:5), S301, S302, S303-v1 (VH SEQ ID NO:63; VL SEQ ID NO:67), S304 (VH SEQ ID NO:79; VL SEQ ID NO:83), S306 (VH SEQ ID NO:87; VL SEQ ID NO:91), S307 (VH SEQ ID NO:239; VL SEQ ID NO:243), S308-v1, S309 (comprising the VH sequence of S309-v1 shown in SEQ ID NO:105 and the VL sequence of S309-v13 shown in SEQ ID NO:168) and S310 were tested for the neutralization effect (Table 4, Fig. 2A). SARS-CoV-2 S300-v1 and SARS-CoV-2 S309 antibodies neutralized SARS-CoV-2 infection (Figs. 2A and 2B).

Table 4. Percentage of infection neutralization by antibodies (titration series) Breeding S300 S301 S302 S303 S304 S306 S307 S308 S309 S310 2 93 -31 -47 6 -21 -19 36 -46 65 -4 6 94 10 6 5 25 15 5 19 11 -15 18 75 21 -16 2 -22 -9 2 32 -29 -15 54 -41 7 -60 12 7 6 32 16 14 -6

Additional neutralization assays were performed using plasma from SARS CoV-1 survivors and SARS-CoV-2 antibodies S309, S311, S312, S303-v1 (rIgG1), S304 (rIgG1), S306 (rIgG1), S310 (rIgG1), and S315 (Figs. 3A–3I). Fig. 3A shows the neutralizing activity of SARS-CoV donor plasma. Figs. 3B–3D and 3I show the neutralizing activity of supernatant from B cells producing S309, S311, S312, and S315, respectively. Figs. 3E–3H show the neutralizing activity of the recombinant antibody at different concentrations. Using this assay, supernatant containing antibody S309, S311, S312, or S315 neutralizes SARS-CoV-2 infection.

Additional neutralization assays were performed using antibodies S303, S304, S306, S309 (VH SEQ ID NO: 105; VL SEQ ID NO: 168), S310, and S315. Figure 13 shows the neutralizing activity of these antibodies at different concentrations against MLV pseudotyped with SARS-CoV-2. DBT cells stably transfected with ACE2 (DBT-ACE2) were used as target cells. Figure 34 shows the neutralizing activity against MLV pseudotyped with SARS-CoV-1 with these antibodies at different concentrations. Additional neutralization data for S304, S309, S304 + S309, S315, and S315 + S309 are shown in Figures 36 and 37.

Example 5

Neutralization of SARS-CoV-2 infection

The neutralizing activity of two SARS-CoV-1 and SARS-CoV-2 cross-neutralizing antibodies, S304 rIgG1 and S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) rIgG1, against SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pp) was assessed.

Murine leukemia virus (MLV) pseudotyped with the SARS-CoV-2 spike protein (SARS-CoV-2pp) was used. DBT cells stably transfected with ACE2 (DBT-ACE2) were used as target cells. SARS-CoV-2pp was activated with trypsin TPCK (tosylphenylalanyl chloromethyl ketone) at a concentration of 10 μg/mL. Activated SARS-CoV-2pp was added to a dilution series of antibodies (starting with a final concentration of 50 μg/mL per antibody, 3-fold dilution). Antibodies were tested at concentrations from 50 μg/mL to 0.02 μg/mL. For the S304 rIgG1 and S309 rIgG1 combination, starting concentrations were 50 μg/mL for each antibody, i.e., the total starting antibody concentration was 100 μg/mL. DBT-ACE2 cells were added to the antibody-virus mixtures and incubated for 48 h. Luminescence was measured after aspiration of the cell culture supernatant and addition of stable GLO substrate (Promega).

In this assay, S309 rIgG1 demonstrated an IC50 of 0.37 μg/mL neutralization of infection, and S304 rIgG1 demonstrated an IC50 of approximately 17 μg/mL. The combination of these two antibodies demonstrated an IC50 of 0.077 μg/mL. See Fig. 7 and Table 5.

Table 5. IC50 (μg/ml) of antibodies S309 S304 S304 + S309 IC50 0.3707 ~16.95 0,07704

Further neutralization assays were performed using the same procedure for recombinant monoclonal antibodies S309 and S315, individually and in combination. In this assay, S309 exhibited an IC50 of 1.091 μg/mL and S315 exhibited an IC50 of 25.1 μg/mL. The combination of both of these antibodies exhibited an IC50 of 0.3047 μg/mL. See Fig. 23 and Table 6.

Table 6. IC50 (μg/ml) of antibodies and antibody combinations S309 + S315 S309 S315 IC50 0.3047 1,091 25.10

Example 6

Reactivity of human monoclonal antibodies against SARS-CoV and SARS-CoV-2

The reactivity of additional human mAbs "S311" and "S312" against the S1 subunit of the spike protein and the RBD protein of SARS-CoV and SARS-CoV-2 was assessed using enzyme-linked immunosorbent assay (ELISA).

96-well plates were coated with recombinant SARS-CoV-2 spike protein S1 subunit (Sino Biological), SARS-CoV-2 RBD (Sino Biological or self-produced; residues 331-550 of spike protein from BetaCoV/Wuhan-Hu-1/2019, Cat. No. MN908947), recombinant SARS-CoV Spike S1 subunit S1 (Sino Biological), or SARS-CoV RBD (Sino Biological).

Wells were washed and blocked with PBS+1%BSA for 1 h at room temperature and then incubated with serially diluted mAbs for 1 h at room temperature. Bound mAbs were detected by incubating alkaline phosphatase-conjugated goat anti-human IgG (Southern Biotechnology: 2040-04) for 1 h at room temperature and developed with 1 mg/ml p-nitrophenyl phosphate substrate in 0.1 M glycine buffer (pH 10.4) for 30 min at room temperature. Optical density (OD) values were measured at 405 nm in an ELISA reader (Powerwave 340/96 spectrophotometer, BioTek).

The results are shown in Fig. 5A (SARS-CoV-2 S311) and Fig. 5B (SARS-CoV-2 S312).

Further assays were performed to examine the reactivity of antibody variants constructed from S300, S305, or S307 to the RBDs of SARS-CoV-2 and SARS-CoV-1 using the same procedure described above in this Example. The results are shown in Figs. 38A-38D. The "S300 V4-rIgG1" antibody, as shown in Fig. 38C, comprises a VH comprising the amino acid sequence of SEQ ID NO: 1 and a VL (Vκ) comprising the amino acid sequence of SEQ ID NO: 234. The "S307 V3-rIgG1" antibody, as shown in Fig. 38D, comprises a VH comprising the amino acid sequence of SEQ ID NO: 239 and a VL (Vκ) comprising the amino acid sequence of SEQ ID NO: 243.

Example 7

Neutralization of SARS-CoV-2 infection by S309 and S315

The neutralizing activity of recombinant antibodies S309 (VH: SEQ ID NO:105; VL SEQ ID NO:168) rIgG1-MLNS and S315 rIgG1-MLNS against SARS-CoV-2 pseudotyped viruses (SARS-CoV-2pp) was determined. These recombinant antibodies contained the M428L and N434S mutations in the Fc domain (see, e.g., Zalevsky et al., Nat. Biotechnol. 28(2):157–159 (2010); this combination of Fc mutations is also referred to as “MLNS” or “LS” throughout this disclosure, including in the figures).

Murine leukemia virus (MLV) pseudotyped with the SARS-CoV-2 spike protein (SARS-CoV-2pp) was used. DBT cells stably transfected with ACE2 (DBT-ACE2) were used as target cells. SARS-CoV-2pp was activated with trypsin TPCK at a concentration of 10 μg/ml. Activated SARS-CoV-2pp was added to a dilution series of the antibody to be tested. DBT-ACE2 cells were added to the antibody-virus mixtures and incubated for 48 h. Luminescence was measured after aspiration of the cell culture supernatant and addition of stable GLO substrate (Promega). The luciferase signal of infected cells was used to calculate the percentage of neutralization compared to the no-antibody control.

S309 rIgG1 MLNS (“S309-rIgG1-LS” in Fig. 9) exhibited an IC50 of approximately 3.9 nM for neutralizing infection, and S315 rIgG1 MLNS (“S315-rIgG1-LSv1” in Fig. 9) exhibited an IC50 of approximately 111.7 mM. See Fig. 9.

The neutralizing activity of S309-rFab was compared with that of full-length S309 rIgG1 MLNS (“S309-rIgG1-LS” in Fig. 10). Full-length S309 rIgG-LS exhibited an IC50 of 3.821 nM, while S309-rFab exhibited an IC50 of 3.532 nM. See Fig. 10.

Example 8

Antibody reactivity against SARS-CoV-1 RBD, SARS-CoV-2 RBD and ectodomains of different coronaviruses

The reactivity of monoclonal antibodies against the RBD of SARS-CoV-1 and SARS-CoV-2 and the spike proteins of SARS-CoV-1, SARS-CoV-2, OC43 and MERS coronavirus was studied using enzyme-linked immunosorbent assay (ELISA). 384-well shallow ELISA plates were coated with stabilized prefusion trimer of SARS-CoV-1, SARS-CoV-2, OC43, or MERS spike protein at a concentration of 1 μg/mL, or SARS-CoV-2 RBD (produced in-house; BetaCoV/Wuhan-Hu-1/2019 spike protein residues 331-550, Cat. No. MN908947) at a concentration of 10 μg/mL, or SARS-CoV-1 RBD (Sino Biological) at a concentration of 1 μg/mL.

Wells were washed and blocked with PBS+1% BSA for 1 hour at room temperature and then incubated with serially diluted antibodies for 1-2 hours at room temperature. Antibodies were tested over a concentration range from 5 to 0.00028 μg/mL. Plates were washed and bound antibodies were detected by incubation with alkaline phosphatase-conjugated goat anti-human IgG (Southern Biotechnology: 2040-04) for 1 hour at room temperature, followed by color development using 1 mg/mL p-nitrophenyl phosphate substrate (Sigma-Aldrich 71768) in 0.1 M glycine buffer (pH 10.4) for 30 minutes at room temperature. Optical density (OD) was measured at 405 nm in an ELISA reader (Powerwave 340/96 spectrophotometer, BioTek).

The ELISA assay results are shown in Figs. 8A-8K and 18A-18J. Recombinant antibodies, some of which carry FcγMLNS mutations, are shown with rIgG1.

Example 9

Antibody binding to the spike protein of SARS-CoV-1 and SARS-CoV-2

ExpiCHO cells were transfected with phCMV1-SARS-CoV-2-S, SARS-spike_pcDNA.3 (SARS strain), or empty phCMV1 using Expifectamine CHO Enhancer. Two days after transfection, cells were harvested for antibody immunostaining. Alexa647-labeled anti-human IgG Fc secondary antibody was used for detection. Binding of the monoclonal antibody to transfected cells was analyzed by flow cytometry using a ZE5 Cell Analyzer (Biorard) and FlowJo software (TreeStar). Positive binding was determined by differential staining of CoV-S transfectants compared to mock transfectants. The S309 antibody (VH SEQ ID NO:105; VL SEQ ID NO:168) was tested by flow cytometry at a concentration of 10 μg/mL for its ability to stain ExpiCHO cells expressing the SARS-CoV-1 or SARS-CoV-2 S protein. Stacked histograms of the flow cytometry plots demonstrate dose-dependent binding of the S309 antibody to the SARS-CoV or SARS-CoV-2 S protein. The results are shown in Fig. 11.

The binding of monoclonal antibodies S303, S304, S306, S309, S310, S315, S110, S124, S230, and S109 (all expressed as rIgG1) to SARS-CoV-1 S protein and SARS-CoV-2 S protein was measured by flow cytometry. The results are shown in Figs. 12A, 12B, 40A, and 40B. Eight of the tested antibodies exhibited EC50 values ranging from 1.4 ng/mL to 6100 ng/mL for binding to SARS-CoV-2 S protein and from 0.8 ng/mL to 254 ng/mL for binding to SARS-CoV-1 S protein.

Additional binding assays using the same procedure were performed for S309 and four engineered variants of S309 carrying different mutations in VH (N55Q, W50F, W105F, or W50F + G56A + W105F). The results are shown in Fig. 27. The EC50 values for each antibody tested in these assays are shown in Table 7; the numbers in parentheses in the “Antibody” column in Table 7 correspond to the designations in Fig. 27.

Table 7 Antibody VH SEQ ID NO: VL SEQ ID NO: EC50 (ng/ml) - experiment 1 EC50 (ng/ml) - experiment 2 S309 WT ("11") 105 168 23.1 11.5 S309 N55Q ("12") 113 168 22.3 9.6 S309 W50F ("13") 129 168 21.8 8.9 S309 W105F ("14") 119 168 21.4 8.4 S309 W50F-G56A-W105F ("15") 172 168 18.8 7.8

Additional binding assays using the same procedure were performed using antibodies S303, S304, S306, S309, S310, S315 and comparative antibodies S109, S110, S124 and S230. The results are shown in Figs. 33A and 42A (binding to SARS-CoV-2 S protein) and 33B and 42B (binding to SARS-CoV-1 S protein). MFI: mean fluorescence intensity measured by flow cytometry.

The same analysis was performed using recombinant antibodies S300 and S307. The results for antibody S300-rIgG1, which contains a VH containing the amino acid sequence of SEQ ID NO:1 and a VL (Vκ) containing the amino acid sequence of SEQ ID NO:234, are shown in Fig. 39A. The results for antibody S307-rIgG1, which contains a VH containing the amino acid sequence of SEQ ID NO:239 and a VL (Vκ) containing the amino acid sequence of SEQ ID NO:24, are shown in Fig. 39B.

Example 10

Binding of S309, S303, S304, and S315 antibodies to the RBD of SARS-CoV-2 and SARS-CoV-1

The affinity of recombinant antibodies S309, S303, S304, and S315 to the RBDs of CoV-1 and CoV-2 was tested using biolayer interferometry (BLI; Octet). Briefly, His-tagged SARS-CoV-1 or SARS-CoV-2 RBD was loaded at 3 μg/mL in kinetic buffer (KB) for 15 min onto anti-HIS (HIS2) biosensors (Molecular Devices, ForteBio). Full-length antibody association was performed in KB at 15 μg/mL for 5 min. Fab fragment association was performed in KB at 5 μg/mL for 5 min. Dissociation in KB was measured for 10 min. Affinities, represented by KD values, were calculated using the global matching model (Octet). We used Octet Red96 (ForteBio) equipment.

Figures 14A-14D show the association and dissociation curves for S309, S303, S304, and S315, respectively. Each of these antibodies binds to the RBD of SARS-CoV-2 and SARS-CoV-1 with nanomolar to subpicomolar affinities. Figures 20A and 20B show the association and dissociation curves for IgG S309 and Fab S309, respectively. In these figures, the vertical dotted line represents the transition from antibody (or Fab) solution to buffer.

Example 11

Binding of S309 IgG and S309 Fab to the SARS-CoV-2 S protein ectodomain trimer and RBD

IgG1 and Fab affinity and avidity determination: Biotinylated SARS-CoV-2 RBD (produced in-house; amino acid residues 331-550 of spike protein from BetaCoV/Wuhan-Hu-1/2019, Cat# MN908947, biotinylated EZ-Link NHS-PEG4-Biotin from ThermoFisher) and biotinylated avi-tagged SARS-CoV-2 2P S were loaded at 7.5 μg/mL in kinetic buffer (KB; 0.01% endotoxin-free BSA, 0.002% Tween-20, 0.005% NaN3 in PBS) for 8 min onto streptavidin biosensors (Molecular Devices, ForteBio). Association of IgG1 and Fab with the target was performed in KB at 100, 33, 11, 3.6, 1.2 nM for 5 min. Dissociation in KB was measured for 10 min. KD values were calculated using a 1:1 global fit model (Octet).

The results are shown in Fig. 41A and 41B. In this assay, IgG S309 bound to the SARS-CoV-2 RBD and the S-ectodomain trimer with subpicomolar and picomolar avidity, respectively. S309 Fab bound to both the SARS-CoV-2 RBD and the S-ectodomain trimer with nanomolar and subnanomolar affinity.

Example 12

Competitive binding of antibodies to the RBD of SARS-CoV-1 or SARS-CoV-2

Competitive binding of monoclonal antibody pairs to SARS-CoV-1 RBD or SARS-CoV-2 RBD was measured to identify the corresponding antibody binding sites.

Strepavidin biosensors (Pall ForteBio) were used to immobilize anti-Strep Tag II antibody at 3 μg/mL (clone 5A9F9, biotin, LabForce AG, Muttenz CH) after a 10 min hydration step with kinetic buffer (KB; 0.01% endotoxin-free BSA, 0.002^ Tween-20, 0.005% NaN3 in PBS). Then, SARS-CoV-1 or SARS-CoV-2 RBD with Strep Tag II (produced in-house) was loaded for 6 min at a concentration of 4 μg/mL in KB. The first antibody was allowed to associate for a certain period of time, and then the second antibody was allowed to associate for 7 min (420 seconds). Figure 15A shows the competition of antibody pairs for binding to the SARS-CoV-1 RBD. Figure 15B shows the competition of antibody pairs for binding to the SARS-CoV-2 RBD. The dashed vertical lines in Figs. 15A and 15B indicate the transition from the first antibody, indicated on the left of the array, to the second antibody, indicated on the top of the array. Using this and other data, four antigenic regions or sites were identified (I-IV in Figs. 15A and 15B).

Example 13

RBD Interference: Human ACE2 Binding

The ability of antibodies to interfere with the binding of RBD to human ACE2 was measured. ACE2-His (Bio-Techne AG) was loaded for 30 min at 5 μg/ml in kinetic buffer (KB) onto anti-HIS (HIS2) biosensors (Molecular Devices-ForteBio). SARS-CoV-1 rabbit RBD Fc or SARS-CoV-2 mouse RBD Fc (Sino Biological Europe GmbH) at 1 μg/ml was associated for 15 min after pre-incubation with or without antibody at 30 μg/ml for 30 min. Dissociation was monitored for 5 min. Figure 16 shows the data obtained using the S309 or S230 antibody. Figure 16 19A and 19B show data obtained using antibodies S304, S303, or S230 (Fig. 19A) or RBD and antibody S315 (Fig. 19B). The vertical dotted line in each of Figs. 16, 19A, and 19B indicates the start of RBD loading with or without antibody.

Example 14

Effector function of antibodies

Natural killer (NK) cell-mediated antibody-dependent cellular cytotoxicity (ADCC) can contribute to virus control by killing infected cells that display viral proteins on their surface. To investigate the ability of antibodies to utilize this function, ADCC was assayed in vitro using human NK cells (isolated from fresh blood of healthy donors using the MACSxpress NK Isolation Kit (Miltenyi Biotec, Cat.# 130-098-185)) as effector cells and SARS-CoV-2 S protein-transfected ExpiCHO cells as target cells. Target cells were incubated with varying amounts of antibody and 10 min later incubated with primary human NK cells as effector cells at a target:effector ratio of 9:1. Antibody-dependent cell killing was measured using an LDH (lactate dehydrogenase) release assay (LDH Cytotoxicity Detection Kit (Roche; Cat. No. 11644793001)) after 4 hours of incubation at 37°C.

Macrophage or dendritic cell-mediated antibody-dependent cellular phagocytosis (ADCP) may also contribute to virus control by clearing infected cells and potentially stimulating T cell responses by presenting viral antigen. ADCP was tested using peripheral blood mononuclear cells as phagocytes and ExpiCHO transfected with the SARS-CoV-2 S protein fluorescently labeled with the PKH67 Fluorescent Cellular Linker Kit (Sigma Aldrich, Cat.# MINI67) as target cells. Target cells were incubated with varying amounts of antibodies for 10 min followed by incubation with human PBMCs isolated from healthy donors that were fluorescently labeled with Cell Trace Violet (Invitrogen, Cat# C34557) at an effector:target ratio of 20:1. After overnight incubation at 37°C, cells were stained with anti-human CD14-APC antibody (BD Pharmingen, Cat# 561708, clone M5E2) to stain phagocytic cells. Antibody-mediated phagocytosis was determined by flow cytometry by measuring the % of monocytes that were positive for PKH67 fluorescence.

Antibodies S309 (VH SEQ ID NO:105; VL SEQ ID NO:168), S304, S306, S315, S230 and a combination of S309 and S304 were tested.

Fig. 17A shows the ADCC function of antibodies using primary NK effector cells and SARS-CoV-2 ExpiCHO S protein-expressing cells as target cells. Values show the mean ±SD of duplicate measurements. Fig. 17B shows the ADCP function of antibodies using PBMCs as phagocytic cells and PKF67-labeled ExpiCHO S protein-expressing cells as target cells. Values show the mean ±SD of duplicate measurements.

Fc S309 variants were tested for ADCC. S309-LS includes Fc mutations M428L and N434S. S309-GRLR includes Fc mutation G236R/L328R, which shows minimal binding to FcγR. S309-LS-GAALIE includes Fc mutations MLNS and GAALIE (G236A/A330L/I332E). The results are shown in Fig. 45.

Antibodies S303, S304, S306, S309, S315, and the combination of S309 and S315 were analyzed for ADCC and ADCP function. Figure 24A shows ADCC of antibodies using primary NK effector cells and SARS-CoV-S or SARS-CoV-2 ExpiCHO expressing S protein as target cells. The graph in Figure 24A illustrates the percentage of killing determined for one representative donor homozygous for high-affinity FcγRIIIa (values show mean ±SD). Figure 24B shows the area under the curve (AUC) for the responses of donors homozygous for the high-affinity FcγRIIIa variant 158V (VV) compared to donors heterozygous for 158V (FV) or homozygous for the low-affinity 158F variant (FF) (mean ± SD). Fig. 25A shows ADCP using PBMCs as phagocytic cells and PKH67-labeled SARS-CoV-2 ExpiCHO S protein-expressing cells as target cells for one representative donor. % ADCP indicates the percentage of PKH67-positive monocytes. Fig. 25B shows the area under the curve (AUC) for the responses of multiple donors.

Example 15

Antibody reactivity to cell lysate of SARS-CoV-2 infected cells

The reactivity of antibodies S304, S306, S309, and S310 against cell lysate of VeroE6 cells infected with SARS-CoV-2 was measured. Figure 21A shows the antibody reactivity measured by indirect S protein ELISA against T×100-extracted VeroE6 cell lysate infected with SARS-CoV-2. Figure 21B shows the antibody reactivity measured by indirect S protein ELISA against SDS-extracted (denatured) VeroE6 cell lysate infected with SARS-CoV-2. Figure 21C shows the reactivity of SARS-CoV-1 human convalescent serum measured by indirect S protein ELISA against T×100- or SDS-extracted VeroE6 cell lysate infected with SARS-CoV-2.

Example 16

Neutralization of SARS-CoV-2 infection by S304 and S309 antibodies alone or in combination

Neutralization of SARS-CoV-2 infection by monoclonal antibodies S304 and S309 was assessed using a SARS-CoV-2 live virus assay. The live virus neutralization assay quantifies the number of infected cells by staining the viral nucleoprotein (NP) with an NP-specific rabbit polyclonal serum. Inhibition was assessed by measuring NP expression at 24 and 45 hours post-infection. An enzyme-linked immunosorbent assay (EIA) was used to quantify the level of infection for each antibody dilution tested.

Data are shown in Fig. 22A-22D. Neutralization was performed for one hour at room temperature at the indicated antibody concentrations using Vero E6 cells in monolayer in 96-well plates. Wells were infected with 100 TCID50 virus. After 24 or 45 hours, monolayers were fixed and stained for inhibition of NP expression. S304 and S309 in combination demonstrated synergistic enhancement of neutralization.

Example 17

Obtaining the rIgG variant of S309 antibodies

Recombinant IgG1 antibodies were prepared using the VH and VL sequences of the S309 antibody. In this example, the antibodies are referred to as "S309-11", "S309-12", "S309-13", "S309-14", and "S309-15", respectively.

"S309-11" comprises the wild-type VH sequence (SEQ ID NO: 105) and the wild-type VL sequence (SEQ ID NO: 168) of S309. "S309-12" comprises the N55Q mutation in CDRH2, providing the VH variant sequence (SEQ ID NO: 113) and the wild-type VL sequence (SEQ ID NO: 168) of S309. "S309-13" comprises the W50F mutation in VH (SEQ ID NO: 129) and the wild-type VL sequence (SEQ ID NO: 168) of S309. "S309-14" comprises the W105F VH variant sequence (SEQ ID NO: 119) and the wild-type VL sequence (SEQ ID NO: 168) of S309. "S309-15" contains the W50F/G56A/W105F VH variant (SEQ ID NO: 172) and the wild-type VL sequence of S309 (SEQ ID NO: 168). Recombinant S309 antibody (S309-11) and each of the four variants S309-12 through S309-15 were produced by transient transfection and expression of a plasmid vector encoding the recombinant antibody in HD 293F cells (GenScript). The plasmid vector encoding the S309 antibodies also encodes a signal peptide as indicated in SEQ ID NO: 252. This signal peptide resulted in superior antibody production compared to the other signal peptides tested. Data not shown. Cells were harvested on day 4 and IgG expression was confirmed by Western blotting and protein A titer analysis.

Example 18

Binding of S309 rIgG and variants to the SARS-CoV-2 RBD

Binding of recombinant monoclonal antibody S309 and four S309 variants described in Example 17 (S309-12 - S309-15) to the RBD was measured using surface plasmon resonance (SPR). SPR experiments were performed using a Biacore T200 instrument using a single-cycle kinetic approach. The antibody, expressed as IgG, was captured on the surface and increasing concentrations of purified SARS-CoV-2 RBD in either the glycosylated or deglycosylated form were introduced. SPR was performed using a covalently immobilized anti-human Fc (GE) sensor chip. The buffer used was 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% P20 detergent. Assays were performed at 25°C. Recombinant antibodies were diluted from the supernatant to approximately 2 μg/mL. RBD concentrations were 0.8 nM, 3.1 nM, 12.5 nM, 50 nM, and/or 200 nM. Glycosylated RBD was generated by expression in HEK293 cells and purified using a one-step Ni affinity purification. Deglycosylated RBD was generated by expression of intrinsically produced Expi293 cells grown in the presence of kifunensine, purification using a one-step Ni affinity purification, and treatment with endoglycosidase H. Single-cycle kinetic assays were performed with 3-min injections and 20-min dissociation periods. Association and dissociation kinetics were monitored and fitted to a binding model to determine affinity. The results are shown in Figs. 30A–30F and Table 8.

Table 8 mAb supernatant Glycosylated RBD Deglycosylated RBD S309 WT or variant K _D K _a (1/Ms) K _d (1/s) K _D K _a (1/Ms) K _d (1/s) S309-11 (WT) 0.50 nM 10,0e4 5.0e-5 0.91 nM 3,0e5 2.8e-4 Replication S309-11 (WT) 0.68 nM 9.5e4 6.5e-5 0.98 nM 2.9e5 2.9e-4 S309-12 (N55Q) 0.46 nM 9,2e4 4.2e-5 1.3 nM 2.7e5 3.6e-4 S309-13 (W50F) 0.51 nM 9,9e4 5.0e-5 1.8 nM 3,0e5 5.3e-4 S309-14 (W105F) 0.38 nM 1,0e5 3.9e-5 7.9 nM 9.8e5 7.7e-3 S309-15 (W50F/G56A/W105F) 1.7 nM 9,9e4 1.6e-4 more than 10 nM calculated Kd with adjustment to steady state

Binding to deglycosylated RBD was measured in two different SPR assays using different parameters. Experiment 1 used 10-min injections and a 4-fold dilution series of RBD from 100 nM. Experiment 2 used 3-min injections and a 4-fold dilution series of RBD from 200 nM as described above. The results are shown in Table 9. The results of experiment 1 for S309-15 are also shown in Fig. 30F, top two panels.

Table 9 mAb supernatant Experiment #1: Experiment #2: S309 WT or variant K _D K _a (1/Ms) K _d (1/s) K _D K _a (1/Ms) K _d (1/s) S309-11 (WT) 0.83 nM 3,0e5 2.5e-4 0.91 nM 3,0e5 2.8e-4 Replication S309-11 (WT) 0.91 nM 3,0e5 2.7e-4 0.98 nM 2.9e5 2.9e-4 S309-12 (N55Q) 1.2 nM 2.7e5 3.2e-4 1.3 nM 2.7e5 3.6e-4 S309-13 (W50F) 1.7 nM 2,8e5 4.6e-4 1.8 nM 3,0e5 5.3e-4 S309-14 (W105F) 14 nM Adjustment to a stable state 7.9 nM 9.8e5 7.7e-3 S309-15 (W50F/G56A/W105F) 37 nM Adjustment to a stable state Adjustment to a stable state is not possible

Binding of recombinant antibody S309 and four engineered variants to the RBD was measured by surface plasmon resonance (SPR) using the same procedure described above except using purified recombinant antibodies rather than cell culture supernatant. The results are shown in Table 10.

Table 10 Glycosylated RBD Deglycosylated RBD S309 WT or VH variant K _D K _a (1/Ms) K _d (1/s) K _D K _a (1/Ms) K _d (1/s) S309-11 (WT) 0.26 nM 9,3e4 2,4e-5 0.67 nM 3,4e5 2,3e-4 S309-12 (N55Q) 0.39 nM 8,5e4 3,3e-5 1.1 nM 3,1e5 3.2e-4 S309-13 (W50F) 0.39 nM 9,2e4 3.6e-5 1.4 nM 3,5e5 4.9e-4 S309-14 (W105F) 0.35 nM 9,6e5 3,4e-5 5.1 nM 1,5e6 7.9e-3 S309-15 (W50F/G56A/W105F) 1.6 nM 9,4e4 1.5e-4 more than 10 nM calculated Kd with adjustment to steady state S309 G56A 0.54 nM 9,3e4 5.1e-5 0.70 nM 3,4e5 2,4e-4

Example 19

Neutralization of SARS-CoV-2 infection using S309 antibodies

The neutralizing activity of S309 and the four engineered S309 variants described in Examples 17 and 18 ("S309-12" through "S309-15") was determined using a VSV-based pseudotyping luciferase reporter system (Kerafast). VSV pseudotyped particles and antibody were mixed in DMEM and incubated for 30 min at 37°C. The infection mixture was then allowed to incubate with Vero E6 cells for 1 h at 37°C, followed by the addition of DMEM with Pen-Strep and 10% FBS (the infection mixture was not removed). The cells were incubated at 37°C for 18-24 h. Luciferase was measured using an Ensight plate reader (Perkin Elmer) after addition of Bio-Glo reagent (Promega). The results are shown in Fig. 28. In Fig. 28, variants 11 - 15 correspond to S309-11 - S309-15, respectively. The calculated EC50 values based on this experiment are given in Table 11.

Table 11 Antibody EC50 (ng/ml) S309-11 (WT VH) 109 S309-12 (N55Q VH) 103 S309-13 (W50F VH) 97 S309-14 (W105F VH) 65 S309-15 (W50F/G56A/W105F VH) 53

Example 20

Antibody-dependent activation of human FcγRIIIa or FcγRIIa

Antibody-dependent activation of human FcγRIIIa or FcγRIIa was examined. ExpiCHO cells were transiently transfected with SARS-CoV-2 (BetaCoV/Wuhan-Hu-1/2019) and incubated with titrated concentrations of antibody for 10 min. ExpiCHO cells were then incubated with Jurkat cells expressing FcγRIIIa or FcγRIIa on their surface and stably transfected with the NFAT-driven luciferase gene (Promega, Cat# G9798 and G7018) at an effector-to-target ratio of 6:1 for FcγRIIIa and 5:1 for FcγRIIa. Activation of human FcγR in this bioassay results in NFAT-mediated expression of the luciferase reporter gene. Luminescence was measured after 21 h of incubation at 37 °C with 5% CO ₂ using the Bio-Glo-TM Luciferase Assay Reagent according to the manufacturer's instructions. Antibodies S303, S304, S306, S309, S315, and a combination of S309 and S315, as well as the reference antibody S230, were assayed. The results are shown in Figs. 31 and 32.

Example 21

Sequence analysis of SARS-CoV-2 S-glycoprotein

Analysis of the S-glycoprotein sequences of 2,229 SARS-CoV-2 isolates revealed that several mutations with variable frequencies occurred in the SARS-CoV-2 S ectodomain. Figure 35A shows the spike protein variants occurring with a frequency n greater than 1 as spheres displayed in the closed and open forms of the complete trimeric spike ectodomain. The RBD and other domains of the spike protein are shown as indicated. Forty mutations (out of a total of 2,229) are shown. Due to the lack of detail in the PDB structures, only residue 367 (n is 8) is highlighted in the RBD, while residues 476 (n is 7) and 483 (n is 17) are not highlighted. Figure 35B shows the amino acid distribution of the spike glycoprotein variants. Each dot is an individual variant. The locations of the A domain and RBD are shown. The variants corresponding to the 0.1% frequency threshold are as indicated.

Further analysis of S-glycoprotein sequences was performed using 11,839 SARS-CoV-2 isolates. Figure 43 shows variants supported by at least two sequences (>0.01% prevalence), visualized as the indicated spheres displayed in the closed (left) and open (right) forms of the complete trimeric spike ectodomain. Each dot is an individual variant. Figure 43 shows spike protein variants supported by at least two sequences, as indicated, as spheres displayed in the closed (left) and open (right) forms of the complete trimeric spike ectodomain. RBD and other spike protein domains are shown as indicated. A total of 171 variants (out of 11,839 spike protein sequences analyzed) are shown. Variants are flagged if their prevalence exceeds 1% (D614G only) or if they are within the RBD. The location of the N343 conservation is also indicated.

Example 22

Competition of S309 antibody with antibodies isolated from patients with SARS-CoV-2

Human monoclonal antibodies isolated from patients who recovered from SARS-CoV-2 infection were tested for overlapping RBD binding sites with the S309 antibody (VH: SEQ ID NO:105; VL: SEQ ID NO:113). Competition assays were performed using Octet (instrument: Octet Red96, ForteBio). Anti-HIS sensors (BIOSENSOR ANTI-PENTA-HIS (HIS1K)1*1ST) were used to immobilize in-house produced His-tagged SARS-CoV-2 RBD (residues 331-550 of the spike protein from BetaCoV/Wuhan-Hu-1/2019, Cat. No. MN908947) at a concentration of 3 μg/mL. The antibodies were associated for 6 min at 15 μg/mL. All proteins were diluted in kinetic buffer (KB). Competing antibodies were then associated at the same concentration for an additional 6 minutes. Two antibodies were shown to compete with S309 for binding to the RBD, but unlike S309, they did not neutralize SARS-CoV-2. Data not shown.

Example 23

Selection of SARS-CoV-2 Resistance Against Monoclonal Antibody S309-12-MLNS

To study resistance selection, SARS CoV-2 was passaged for more than one month in the presence of Vero E6 cells and fixed concentrations of the S309 N55Q MLNS GAALIE antibody (VH with SEQ ID NO:113 and VL with SEQ ID NO:168, with G236A, A330L, I332E, M428L, and N434S mutations in Fc). The experimental setup is shown in Fig. 44A. Details of infection and continuous virus culture are summarized in Fig. 44B. Cytopathogenic effect (CPE) was assessed by visual inspection of the plates. Even in the absence of CPE, virus titers were assessed by methylcellulose overlay plaque assay. The results are shown in Fig. 44C. No evidence of viral breakthrough was observed in antibody-treated wells even at the lowest antibody concentration tested. Data are representative of triplicate wells.

Example 24

Neutralization of SARS-CoV-2 infection of human lung cells Calu-3 by the antibody S309

The S309 N55Q MLNS antibody (VH SEQ ID NO:113 and VL SEQ ID NO:168, with M428L and N434S mutations in Fc) was tested for its ability to neutralize live SARS-CoV-2 infection of human lung Calu-3 cells (which are positive for the transmembrane protease TMPRSS2) and VeroE6 cells using a nanoluciferase assay. The results, including calculated IC50 values, are shown in Fig. 46.

Example 25

Neutralization of SARS-CoV-2 infection using S309 antibody

The S309 antibody was tested for its ability to neutralize live SARS-CoV-2 virus infection using nanoluciferase assay and IFA assay. Briefly, Vero E6 cells were infected with live SARS-CoV-2 luciferase virus for six hours. Data were collected using three different antibody concentrations: 1, 0.1, and 0.01 MOI. The nanoluciferase assay results are shown in Fig. 47. The IFA assay results are shown in Fig. 48A (representative wells counted in IFA) and 48B (quantitative data using citation 5). The calculated IC50 values for each MOI are shown in the boxes below the graph in Fig. 47 and 48B. Notably, no clusters of infection (or foci) were observed in this infection format.

Example 26

Neutralization of live SARS-CoV-2 infection by S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies

The S309 N55Q MLNS (also referred to herein as S309 N55Q LS, containing the Fc mutations M428L/N434S) and S309 N55Q MLNS GAALIE (also referred to herein as S309 N55Q LS GAALIE, containing the Fc mutations G236A, A330L, I332E, M428L, and N434S) antibodies were assayed for their ability to neutralize live SARS-CoV-2 virus infection. Each of the S309 N55Q MLNS and S309 N55Q MLNS GAALIE comprises a VH having the amino acid sequence set forth in SEQ ID NO: 113 and a VL having the amino acid sequence set forth in SEQ ID NO: 168. The results are shown in Fig. 49. The calculated EC50 for S309 N55Q MLNS was 100.1 ng/mL. The calculated EC50 for S309 N55Q MLNS GAALIE was 78.3 ng/mL.

Example 27

Neutralization of SARS-CoV-2 pseudotyped virus by S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies

The neutralization of SARS-CoV-2 pseudotyped virus was tested using the S309 N55Q MLNS (also referred to herein as S309 N55Q LS) and S309 N55Q MLNS GAALIE (also referred to herein as S309 N55Q MLNS GAALIE) antibodies. S309 N55Q MLNS and S309 N55Q MLNS GAALIE each comprise a VH having the amino acid sequence shown in SEQ ID NO: 113 and a VL having the amino acid sequence shown in SEQ ID NO: 168. The pseudotyped virus was VSV pseudotyped with the SARS-CoV-2 spike protein. The results are shown in Fig. 50A (S309 N55Q MLNS) and Fig. 50B (S309 N55Q MLNS GAALIE). The calculated EC50 value for S309 N55Q MLNS was 24.06 ng/mL. The calculated EC50 value for S309 N55Q MLNS GAALIE was 22.09 ng/mL.

Example 28

Binding of S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to the RBD of SARS-CoV-2

The binding of S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to the RBD of SARS-CoV-2 was measured using surface plasmon resonance (SPR). Each of S309 N55Q MLNS and S309 N55Q MLNS GAALIE comprises a VH having the amino acid sequence shown in SEQ ID NO: 113 and a VL having the amino acid sequence shown in SEQ ID NO: 168. The results are shown in Fig. 51A (S309 N55Q MLNS) and Fig. 51B (S309 N55Q MLNS GAALIE).

Example 29

Binding of S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to the SARS-CoV-2 spike protein

The binding of S309 N55Q MLNS (also referred to herein as S309 N55Q LS) and S309 N55Q MLNS GAALIE (also referred to herein as S309 N55Q LS GAALIE) antibodies to SARS-CoV-2 to the SARS-CoV-2 spike protein was measured by flow cytometry. S309 N55Q MLNS and S309 N55Q MLNS GAALIE each comprise a VH having the sequence shown in SEQ ID NO: 113 and a VL having the sequence shown in SEQ ID NO: 168. The results are shown in Fig. 51A (S309 N55Q MLNS) and Fig. 51B (S309 N55Q MLNS GAALIE). Data are expressed as the percentage of cells identified as positive for antibody binding.

Example 30

Binding of S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to human Fcγ receptors

Binding of the S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to human Fcγ receptors was analyzed using SPR. Binding to FcγRIIa (both low-affinity R131 and high-affinity H131 alleles), FcγRIIIa (both low-affinity F158 and high-affinity V158 alleles), and FCγRIIb was measured. S309 N55Q MLNS and S309 N55Q MLNS GAALIE each comprise a VH having the sequence recited in SEQ ID NO: 113 and a VL having the sequence recited in SEQ ID NO: 168.

Biotin CAPture reagent (modified streptavidin) was injected into all flow wells of the CAP sensor chip mounted in a Biacore T200 (Cytiva). Biotinylated Fc receptors at a concentration of 1 μg/mL were injected through one flow cell at a flow rate of 10 μL/min for 60 s (one receptor per flow cell), with one flow cell reserved as a reference surface. Antibody at a concentration of 100 μg/mL (diluted in HBS-EP+) was injected into all flow cells for 200 s using a flow rate of 30 μL/min, and association was monitored. Dissociation was monitored for an additional 200 s after injection. Data were collected at 10 Hz. After each binding measurement, CAP Regeneration reagent was injected to prepare the surface for a new cycle. Experiments were performed at 25°C, with samples maintained at 15°C in the instrument prior to injection. The results are shown in Fig. 53 (where the MLNS mutation is designated as "LS" in the figure key).

Example 31

Binding of S309 MLNS, S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to complement component C1q

The binding of the S309 MLNS (also referred to herein as S309 LS), S309 N55Q MLNS (also referred to herein as S309 N55Q LS) and S309 N55Q MLNS GAALIE (also referred to herein as S309 N55Q LS GAALIE) antibodies to the complement component C1q was measured by biolayer interferometry (BLI) on an Octet instrument. S309 MLNS comprises a VH having the sequence set forth in SEQ ID NO: 105 and a VL having the sequence set forth in SEQ ID NO: 168. Each of S309 N55Q MLNS and S309 N55Q MLNS GAALIE comprises a VH having the sequence set forth in SEQ ID NO: 113 and a VL having the sequence set forth in SEQ ID NO: 168.

Anti-human Fab (CH1-specific) sensors were used to capture antibody at 10 μg/ml for 10 min. The IgG-loaded sensors were then exposed to kinetic buffer containing 3 μg/ml purified human C1q for 4 min, followed by a dissociation step in the same buffer for an additional 4 min. The association and dissociation profiles were measured in real time as changes in the interference pattern. The results are shown in Fig. 54.

Example 32

Activation of human Fc gamma receptors in vitro by S309 MLNS, S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies

The ability of the S309 MLNS, S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies to induce antibody-dependent activation of human Fcγ receptors was analyzed in vitro. S309 MLNS comprises a VH having the sequence presented in SEQ ID NO: 105 and a VL having the sequence presented in SEQ ID NO: 168. Each of S309 N55Q MLNS and S309 N55Q MLNS GAALIE comprises a VH having the sequence presented in SEQ ID NO: 113 and a VL having the sequence presented in SEQ ID NO: 168.

Each of S309 MLNS (also referred to herein as S309 LS), S309 N55Q MLNS (also referred to herein as S309 N55Q LS), S309 N55Q MLNS GAALIE (also referred to herein as S309 N55Q LS GAALIE), and the control antibody S309-GRLR were serially diluted 6-fold in assay buffer from 10,000 ng/mL to 0.006 ng/mL. Nine-point serial dilutions of the antibody were incubated with 12,500 (for FcγRIIIa and FcγRIIb) or 10,000 (for FcγRIIa) CoV-2 spike protein-coated CHO cells per 96-well plate in a white flat-bottomed plate for 15 minutes at room temperature. Jurkat effector cells expressing the indicated FcγRs and stably transfected with the NFAT-driven luciferase gene were thawed, diluted in assay buffer, and added to the plate at an effector-to-target cell ratio of 6:1 for FcRγIIIa and FcγRIIb or 5:1 for FcγRIIa. Control wells were included to measure antibody-independent activation (containing target and effector cells but no antibody) and background plate luminescence (wells containing assay buffer only). Plates were incubated for 18 h at 37°C with 5% _CO2 . Activation of human FcγRs in this assay results in NFAT-mediated expression of the luciferase reporter gene. Luminescence was measured using a luminometer after addition of Bio-Glo™ Luciferase Assay Reagent according to the manufacturer's instructions. The results are shown in Fig. 55.

Example 33

Effector function of S309 MLNS, S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies

Antibodies S309 MLNS (also referred to herein as S309 LS), S309 N55Q MLNS (also referred to herein as S309 N55Q LS) and S309 N55Q MLNS GAALIE (also referred to herein as S309 N55Q LS GAALIE) were assayed for their ability to stimulate NK cell-dependent antibody-dependent cell-mediated cytotoxicity (ADCC) and monocyte-mediated antibody-dependent cellular phagocytosis (ADCP) against cells expressing the CoV-2 spike protein.

S309 MLNS comprises a VH having the sequence presented in SEQ ID NO:105 and a VL having the sequence presented in SEQ ID NO:168. Each of S309 N55Q MLNS and S309 N55Q MLNS GAALIE comprises a VH having the sequence presented in SEQ ID NO:113 and a VL having the sequence presented in SEQ ID NO:168.

ADCC was measured in vitro by exposing freshly isolated human NK cells from two genotyped donors expressing homozygous low-affinity (F/F158) or high-affinity (V/V158) FcγRIIIa to antibody pre-incubated with CHO-CoV-2 spike protein cells and measuring LDH release as a readout according to the manufacturer's instructions (LDH Detection Kit, Roche) after 4 h of incubation at 37°C. Briefly, plates were centrifuged for 4 min at 400 x g and 35 μl of the supernatant was transferred to a flat 384-well plate. LDH reagent was prepared and 35 μl was added to each well. Using the kinetic protocol, the absorbance at 490 nm and 650 nm was measured once every 2 minutes for 8 minutes, and the slope of the kinetic curve was used as the result. The percentage of specific lysis was determined using the following formula: (specific release - spontaneous release) / (maximum release - spontaneous release) × 100. The results are shown in Fig. 56.

The ability of S309 MLNS, S309 N55Q MLNS, S309 N55Q MLNS GAALIE antibodies and control antibody S309-GRLR to stimulate ADCP by primary CD14+ monocytes was measured in vitro by exposing freshly isolated human PBMC (labeled with cell trace violet) to CoV-2 spike protein-expressing CHO cells (labeled with PKH67 Fluorescent Cell Linker Kit (Sigma Aldrich)) that had been pre-incubated with the antibody. Serial dilutions of mAb (5-fold serially diluted from 5,000 ng/mL to 0.32 ng/mL in RPMI-1640 (PPMI - Roswell Park Cancer Institute) + L-glutamine supplemented with 10% Hyclone FBS + 2x anti-anti (antibiotic-antimycotic)) were incubated with 10,000 CoV-2 spike protein-coated CHO cells per well of a 96-well polypropylene plate for 10 min. Primary PBMCs were fluorescently labeled with Cell Trace Violet according to the manufacturer's instructions. Target cell-antibody mixtures were then incubated with labeled PBMCs at an effector/target ratio of 16:1. ADCP activity was measured after overnight incubation by labeling the monocyte population for CD14 and measuring the percentage of Cell trace violet ⁺ PKH67 ⁺ among CD14 ⁺ monocytes by flow cytometry. The results are shown in Fig. 57.

Example 34

Effect of S309 antibody on SARS-CoV-2 spike protein-mediated cell fusion

The effect of the S309 antibody (VH: SEQ ID NO:105; VL: SEQ ID NO:168) on SARS-CoV-2 spike protein-mediated fusion was tested using cells engineered to overexpress the spike protein on the cell surface. Addition of S309 to these cell cultures inhibited cell-cell fusion. The results are shown in Fig. 58A (photomicrographs) and 58B (quantitative data).

Example 35

Effect of S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies on SARS-CoV-2 replication

The effect of S309 N55Q MLNS and S309 N55Q MLNS GAALIE antibodies on SARS-CoV-2 replication was tested in VeroE6 cells, PBMCs, and dendritic cells. Each of S309 N55Q MLNS and S309 N55Q MLNS GAALIE comprises a VH having the sequence presented in SEQ ID NO: 113 and a VL having the sequence presented in SEQ ID NO: 168.

SARS-CoV-2 virus was incubated with S309 N55Q MLNS or S309 N55Q MLNS GAALIE for one hour. The virus/antibody mixture was then added to VeroE6 cells, PBMCs, or monocyte-derived dendritic cells (MoDCs) seeded on the plate. After incubating the cells with the virus/antibody mixture for one hour at 37°C, the cells were washed and incubated for another 72 hours in fresh medium. The supernatant from the cultured cells was then analyzed for plaque-forming units (FFU). The supernatant was diluted 1:5 and added to VeroE6 cells. After one hour at 37°C, VeroE6 cells were overlaid with methylcellulose. After 24 hours of further incubation, the VeroE6 cell cultures were stained for SARS-CoV-2 nucleoprotein. The results are shown in Fig. 59. Data for the S309 N55Q MLNS antibody are shown in the top graph. Data for the S309 N55Q MLNS GAALIE antibody are shown in the bottom graph. These 72-hour replication data are representative of the 24- and 48-hour results.

Example 36

Materials and methods

Flow cytometry-based screening for binding to CoV S protein expressed on mammalian cells

ExpiCHO cells were transfected with SARS-CoV-2, SARS-CoV, and MERS-CoV S protein or an empty plasmid as a negative control. The monoclonal antibodies were then tested by flow cytometry at a concentration of 10 μg/mL for their ability to stain ExpiCHO cells expressing 2019-nCoV, SARS-CoV, MERS-CoV S protein or mock transfectant cell.

Transient expression of recombinant SARS-CoV-2 protein

The full-length S gene of SARS-CoV-2 (2019-nCoV-S) isolate BetaCoV/Wuhan-Hu-1/2019 (Cat. No. MN908947) was codon-optimized for human cell expression and cloned into the phCMV1 expression vector (Genlantis). Expi-CHO cells were transiently transfected with phCMV1-SARS-CoV-2-S, phCMV1-MERS-CoV-S (London1/2012), SARS-spike_pcDNA.3 (SARS strain), or empty phCMV1 (mock) using Expifectamine CHO Enhancer. Two days after transfection, cells were harvested, fixed or fixed and permeabilized with saponin for immunostaining with a panel of monoclonal antibodies reactive with the receptor binding domain (RBD) of SARS-CoV. Alexa647-labeled anti-human IgG Fc secondary antibody was used for detection. Antibody binding to transfected cells was analyzed by flow cytometry using a ZE5 Cell Analyzer (Biorard) and FlowJo software (TreeStar). Positive binding was determined by differential staining of CoV-S transfectants compared to mock transfectants.

Competition experiments using Octet (BLI, biolayer interferometry)

Unless otherwise stated herein, anti-His sensors (BIOSENSOR ANTI-PENTA-HIS (HIS1K)) were used to immobilize the SARS-CoV S1 subunit (Sino Biological Europe GmbH). The sensors were hydrated for 10 min with kinetic buffer (KB; 0.01% endotoxin-free BSA, 0.002 μg Tween-20, 0.005% NaN3 in PBS). Then, the SARS-CoV S1 subunit was loaded for 8 min at a concentration of 10 μg/mL in KB. Antibodies were associated for 6 min at 15 μg/ml for full-length nCoV-10 and nCov-6 mAb or 5 μg/ml for nCoV-4 Fab and in a subsequent experiment including nCoV-1 all at 10 μg/ml. Competing antibodies were then associated at the same concentration for an additional 6 min.

Competition experiments using Octet (BLI, biolayer interferometry)

For ACE2 competition experiments, ACE2-His (Bio-Techne AG) was loaded for 30 min at 5 μg/ml in KB onto anti-HIS biosensors (HIS2) (Molecular Devices-ForteBio). SARS-CoV-1 RBD-rabbitFc or SARS-CoV-2 RBD-mouseFc (Sino Biological Europe GmbH) at 1 μg/ml was associated for 15 min after pre-incubation with or without antibody (30 μg/ml, 30 min). Dissociation was monitored for 5 min.

Affinity determination using Octet (BLI, biolayer interferometry)

To determine the _KD of full-length antibodies, Protein A Biosensors (Pall ForteBio) were used to immobilize recombinant antibodies at 2.7 μg/mL for 1 min after a 10 min hydration step with kinetic buffer. Association curves were recorded for 5 min by incubating the antibody-coated sensors with different concentrations of SARS-CoV-1 RBD (Sino Biological) or SARS-CoV-2 RBD (produced in-house; BetaCoV/Wuhan-Hu-1/2019 spike protein residues 331-550, Cat# MN908947). The highest RBD concentration tested was 10 μg/mL, which was then serially diluted 1:2.5 s. Dissociation was recorded for 9 min by moving the sensors into wells containing KB. K _D values were calculated using a global fit model (Octet). Octet Red96 equipment (ForteBio) was used.

To determine the K _D of full-length antibodies versus Fab fragments, His-tagged SARS-CoV-1 or SARS-CoV-2 RBDs were loaded at 3 μg/mL in KB for 15 min onto anti-HIS biosensors (HIS2) (Molecular Devices, ForteBio). Association of full-length antibody and Fab was performed in KB at 15 μg/mL and 5 μg/mL, respectively, for 5 min. Dissociation in KB was measured for 10 min.

Binding ELISA

The reactivity of mAbs with the SARS-CoV spike protein subunit S1 (WH20 strain) was determined using an enzyme-linked immunosorbent assay (ELISA). Briefly, 96-well plates were coated with 3 μg/mL recombinant SARS-CoV spike protein subunit S1 (Sino. Biological). Wells were washed and blocked with PBS+1%BSA for 1 h at room temperature, and then incubated with serially diluted mAbs for 1 h at room temperature. Bound mAbs were detected by incubating alkaline phosphatase-conjugated goat anti-human IgG (Southern Biotechnology: 2040-04) for 1 h at room temperature and developed with 1 mg/ml p-nitrophenyl phosphate substrate in 0.1 M glycine buffer (pH 10.4) for 30 min at room temperature. Optical density (OD) was measured at 405 nm in an ELISA reader (Powerwave 340/96 spectrophotometer, BioTek).

Neutralization Analysis

Unless otherwise stated, murine leukemia virus (MLV) pseudotyped with SARS-CoV-2 spike protein (SARS-CoV-2pp) or SARS-CoV-1 spike protein (SARS-CoV-1pp) was used. DBT cells stably transfected with ACE2 (DBT-ACE2) were used as target cells. SARS-CoV-2pp or SARS-CoV-1pp were activated with TPCK trypsin at a concentration of 10 μg/mL. Activated SARS-CoV-2pp or SARS-CoV-1pp was added to a serial dilution of antibodies (starting with a final concentration of 50 μg/mL per antibody, 3-fold dilution). DBT-ACE2 cells were added to the antibody-virus mixtures and incubated for 48 h. Luminescence was measured after aspiration of cell culture supernatant and addition of stable GLO substrate (Promega).

Unless otherwise stated, pseudotyped particle neutralization assays use the VSV-based luciferase reporter pseudotyping system (Kerafast). VSV pseudotyped particles and antibody were mixed in DMEM and incubated for 30 min at 37°C. The infection mixture was then incubated with Vero E6 cells for 1 h at 37°C, followed by the addition of DMEM with Pen-Strep and 10% FBS (the infection mixture is not removed). Cells were incubated at 37°C for 18–24 h. Luciferase was measured using an Ensight plate reader (Perkin Elmer) after addition of Bio-Glo reagent (Promega).

SPR of the single-cycle kinetic approach

SPR experiments were performed using a Biacore T200 instrument using a single-cycle kinetic approach. S309 IgG was captured on the surface and injected with increasing concentrations of purified glycosylated or deglycosylated SARS-CoV-2 RBD. Association and dissociation kinetics were monitored and fitted to a binding model to determine affinity.

Expression of recombinant antibodies

Recombinant antibodies were expressed in ExpiCHO cells transiently cotransfected with plasmids expressing the heavy and light chains as described previously. (Stettler et al. (2016) Specificity, cross-reactivity, and function of antibodies elicited by Zika virus infection. Science, 353(6301), 823–826) Monoclonal antibodies S303, S304, S306, S309, S310, and S315 were expressed as rIgG-MLNS antibodies. The MLNS mutation provides a longer half-life in vivo. (Zalevsky et al. (2010) Enhanced antibody half-life improves in vivo activity. Nature Biotechnology, 28(2), 157–159)

Sequence alignment

The SARS-CoV-2 genomic sequences were downloaded from GISAID on March 29, 2020, using the filters “full (>29,000 bp)” and “low coverage exclusion”. Bat and pangolin sequences were removed to yield human-only sequences. The spike protein ORF was localized by performing a genome alignment of the reference protein (YP_009724390.1) with GeneWise2. Incomplete matches and ORFs containing insertion/deletion were retained and included in subsequent analysis. Nucleotide sequences were translated in silico using seqkit. Sequences with more than 10% undefined amino acids (due to N base recognition) were removed. Multiple sequence alignment was performed using MAFFT. Variants were identified by comparing aligned sequences (n is 2229) with the reference sequence using the R/Bioconductor Package Biostrings. A similar strategy was used to extract and translate spike protein sequences from SARS-CoV genomes obtained from ViPR (search criteria: SARS-related coronavirus, full-length genomes, human host deposited before December 2019 to exclude SARS-CoV-2, n is 53). The raw SARS-CoV genome sequences contained all major published strains such as Urbani, Tor2, TW1, P2, Frankfurt1, and others. Pangolin sequences shown in Tsan-Yuk Lam et al. were obtained from GISAID. Bat sequences from three sarbecovirus clades as shown in Lu et al. (Lancet 2020) were obtained from Genbank. Civet and raccoon dog sequences were similarly obtained from Genbank.

Example 37

ACE2-Independent Mechanism of SARS-CoV2 Neutralization by S309 Antibody

In the following experiments, the S309 antibody (VH SEQ ID NO: 105, VL SEQ ID NO: 168) was expressed as a recombinant IgG1 with the Fc mutations M428L and N434S. The effect of ACE2 overexpression on the neutralization of infection by the S309 antibody was investigated. Vero E6 or Vero E6-TMPRSS2 cells were infected with SARS-CoV-2 (isolate USA-WA1/2020) at an MOI of 0.01 in the presence of S309 (10 μg/ml). The cells were fixed 24 h after infection, and the viral nucleocapsid protein was immunostained and quantified. Nucleocapsid staining was virtually absent in antibody-treated cells. S309 had an IC50 (ng/ml) in Vero E6 cells of 65 and in Vero E6-TMPRSS2 cells of 91 (data not shown).

A panel of 7 cell lines (HeLa, 293T (wt), Vero E6, Huh7, 293T ACE2, MRC 5-ACE2-TMPRSS2, A549-ACE2-TMPRSS2 clone 5, A549-ACE2-TMPRSS2 clone 10) were infected with SARS-CoV-2-Nluc or VSV pseudotyped SARS-CoV-2 spike protein in the presence of S309. Luciferase signal was quantified 24 h post infection. The maximum S309 neutralization values were as shown in Table 12.

Table 12. Maximum neutralization values using S309 Cell type Virus/pseudotype SARS-CoV-2-Nluc Pseudotyped VSV Vero E6 more than 99% more than 99% Vero E6-TMPRSS2 more than 99% 96% Huh7 98% 78% 293T ACE2 26% 34% MRC5-ACE2-TMPRSS2 87% 45% A549-ACE2-TMPRSS2 clone 5 89% 65% A549-ACE2-TMPRSS2 clone 10 81% 42%

Binding of purified, fluorescently labeled SARS-CoV-2 spike protein to these cell lines was quantified by flow cytometry. HeLa and 239T WT cells had the lowest MFI values, followed by Huh7 and VeroE6 cells. 293T ACE2 (maximum neutralization), MRC 5-ACE2-TMPRSS2 (third highest neutralization value), clone 5 A549-ACE2-TMPRSS2 (fourth highest neutralization value), and clone 10 A549-ACE2-TMPRSS2 (second highest neutralization value) had higher MFI values. A correlation analysis was performed between the maximum neutralization potential of binding to the S309 additive; the Spearman S309 correlation values were: r is -0.94 for both viral models. p is 0.017.

To further characterize SARS-CoV-2-susceptible cell lines, the seven cell lines described above were incubated with purified, fluorescently labeled SARS-CoV-2 spike protein or RBD protein, and protein binding was quantified by flow cytometry. In descending order of MFI, the cell lines were: A549-ACE2-TMPRSS2 clone 10; 293T ACE2; MRC 5-ACE2-TMPRSS2; A549-ACE2-TMPRSS2 clone 5; Vero E6; Huh7; 293T (wt); and HeLa.

The selected lectins and published receptor candidates were screened using HEK293T cells infected with SARS-CoV-2 pseudotyped VSV viruses. ACE2, DC-SIGN, L-SIGN and SIGLEC-1 gave the highest signals. ACE2 provided a signal of approximately 10 ⁵ relative luminescent units (RLU), while DC-SIGN, SIGLEC-1 and L-SIGN had signals of approximately 10 ⁴ RLU. All other lectins/candidates tested gave signals of approximately 10 ² -10 ³ RLU.

HEK 293T, HeLa, and MRC5 cells were transiently transduced to overexpress DC-SIGN, L-SIGN, SIGLEC1, or ACE2 and infected with SARS-CoV-2-pseudotiform VSVs. Uninfected cells and non-transduced cells were included as controls. In HEK293T cells, ACE2, DC-SIGN, SIGLEC-1, and L-SIGN significantly enhanced infection. In HeLa and MRC5 cells, only ACE2 enhanced infection.

Stable HEK293T cell lines overexpressing DC-SIGN, L-SIGN, SIGLEC-1, or ACE2 were infected with authentic SARS-CoV-2 (MOI 0.1), fixed, and immunostained 24 h later for SARS-CoV-2 nucleoprotein. Wild-type cells (infected and uninfected) were used as controls. Enhanced staining was observed in cells overexpressing DC-SIGN, L-SIGN, or SIGLEC-1, and staining was significantly increased in cells overexpressing ACE2.

Stable cell lines were infected with SARS-CoV-2-Nluc and luciferase levels were quantified after 24 h. In order of increasing RLU: uninfected (around 10 ² -10 ³ RLU); parental 293T (around 10 ⁴ RLU); DC-SIGN (around 10 ⁵ RLU); L-SIGN (around 10 ⁵ RLU); SIGLEC-1 (around 10 ⁵ -10 ⁶ RLU); ACE2 (more than 10 ⁷ RLU).

Stable cell lines were incubated with varying concentrations of anti-SIGLEC1 mAb (clone 7-239) and infected with SARS-CoV-2-Nluc. Infection as a percentage of untreated cells remained close to exceeding 100% in 293T cells expressing DC-SIGN, L-SIGN, or ACE2, but dropped below 50% (0.2 μg/ml anti-SIGLEC) to close to 0 (1 μg/ml or 5 μg/ml anti-SIGLEC) in 293T cells expressing SIGLEC-1.

Single-cell expression levels of individual potential SARS-CoV-2 coreceptor candidates were determined in different lung cell types obtained from the Human Lung Cell Atlas (nature.com/articles/s41586-020-2922-4). DC-SIGN, L-SIGN, and SIGLEC-1 are expressed in different cell types in the lung at levels similar to or even higher than ACE2.

Binding of antibodies targeting DC-/L-SIGN, DC-SIGN, SIGLEC1, or ACE2 to HEK293T cells stably overexpressing the respective binding receptor was analyzed by flow cytometry and immunofluorescence. HEK 293T cells overexpressing the respective binding receptors were infected with VSV pseudotyped with wild-type spike protein, or with the SARS-COV-2 B1.1.7 mutant spike protein. Luminescence was analyzed the day after infection. Infection was enhanced in cells expressing the binding receptors. Infection with VSV pseudotyped with either spike protein was similar for each test group. Cells expressing ACE2 gave the highest luminescence signal.

Vero E6 cells, in vitro differentiated moDCs, or PBMCs were infected with SARS-CoV-2 at an MOI of 0.01. At 24 h post-infection, cells were fixed, immunostained for viral nucleocapsid protein, and infected cells were quantified. Only VeroE6 cells showed infection (approximately 7% of cells). Supernatant from infected cells was collected at 24, 48, and 72 h, and infectious virus titer was quantified using the FFU assay on Vero E6 cells.

The major cell types with detectable SARS-CoV-2 genome in bronchoalveolar lavage fluid (BALF) and sputum of patients with severe COVID-19 were assessed. A t-SNE plot was constructed, and the number of each SARS-CoV-2+ cell type was determined (total n is 3085 cells from 8 subjects in Ren et al. Cell 2021). Cell types included T cells, NK cells, plasma cells, neutrophils, macrophages, ciliated cells, squamous cells, and secretory cells. The expression of ACE2, DC-SIGN, L-SIGN, SIGLEC-1, and their combinations were assessed for each cell type.

The amounts of ACE2, DC-SIGN (CD209), L-SIGN (CLEC4M), SIGLEC1 transcripts were correlated with the amount of SARS-CoV-2 RNA in macrophages and secretory cells. The correlation was based on counts (before log transformation) from Ren et al. (Cell 2021).

Representative data demonstrating receptor expression in stable HEK293T cell lines are shown in Fig. 60. Cell lines were generated to overexpress DC-SIGN, L-SIGN, or ACE2 by transducing HEK293T cells with a lentivirus encoding the transgene, and immunofluorescence assays were performed to assess transgene expression.

Representative data demonstrating the ability of pseudotyped VSV expressing the SARS-CoV-2 S protein with a luciferase reporter to infect HEK293T cells (using a luminescence assay) are shown in Fig. 61; expression of DC-SIGN or L-SIGN increased pseudotyped virus infection levels by more than 10-fold compared to infection of HEK293T WT cells, and expression of ACE2 increased pseudotyped virus infection levels by more than 100-fold compared to infection of HEK293T WT cells.

The neutralizing activity of the candidate mAb S309 against pseudotyped VSV was assessed in engineered HEK293T cells. Data are shown in Fig. 62; S309 completely neutralized infection by DC-SIGN and L-SIGN and, to a lesser extent, ACE2.

The ability of live SARS-CoV-2 with a luciferase reporter to infect HEK293T cells was examined using a luminescence assay. Data are shown in Fig. 63; expression of DC-SIGN or L-SIGN increased live virus infection levels by more than 3-fold compared to infection of HEK293T WT cells, and expression of ACE2 increased live virus infection levels by more than 100-fold compared to infection of HEK293T WT cells.

The neutralizing activity of mAb S309 against pseudotyped VSV was assessed in engineered HEK293T cells. Data are shown in Fig. 64; S309 completely neutralized infection by DC-SIGN and L-SIGN and, to a lesser extent, neutralized infection via ACE2.

Experiments were conducted to examine whether the S309 antibody could neutralize SARS-CoV-2 entry through SIGLEC-1. Briefly, stable HEK293T cell lines were generated as described above to overexpress DC-SIGN/L-SIGN, DC-SIGN, SIGLEC-1, or ACE2. Expression data are shown in Fig. 65. As shown in Fig. 66; expression of DC-SIGN, L-SIGN, or SIGLEC increased live virus infection levels by more than 10-fold compared to infection of HEK293T WT cells, and expression of ACE2 increased pseudotyped virus infection levels by more than 100-fold compared to infection of HEK293T WT cells. As shown in Fig. 67, S309 completely neutralized infection through DC-SIGN, L-SIGN, and SIGLEC-1.

Expression of DC-SIGN (CD209) and other cell surface receptor proteins, including SIGLEC-1 and other SIGLECs, was determined on various cell types. Data are summarized in Fig. 68A and 68B.

Further experiments were conducted to investigate the function(s) of DC-SIGN, L-SIGN, and SIGLEC-1 in SARS-CoV-2 infection. In one set of experiments, HEK293T cells stably expressing DC-SIGN, L-SIGN, SIGLEC-1, or ACE2 were infected with live SARS-CoV-2 Nluc at three different multiplicities of infection (MOIs): 0.01, 0.1, and 1). Infection was determined using relative luminescent units and compared to infection in HEK293T cells (parental). The data are shown in Fig. 69. At the lowest MOI tested, an increase in infection was observed in cells expressing DC-SIGN, L-SIGN, or SIGLEC. At the highest MOI tested, infection was not further enhanced compared to parental DC-SIGN, L-SIGN, or SIGLEC expression. These data show that 293T parent cells are susceptible to SARS-CoV-2 infection and L-SIGN, DC-SIGN and SIGLEC-1 enhance infection levels but do not function as primary infection receptors.

In another set of experiments, 293T cells, HeLa cells, and MRC5 cells were transiently transduced with lentivirus encoding DC-SIGN, L-SIGN, SIGLEC-1, or ACE2 and infected with pseudotyped VSV three days after transduction. The data are shown in Fig. 70. While 293T cells showed a low level of susceptibility (compared to uninfected and untransduced cells), HeLa and MRC5 cells were completely resistant to the virus. The low level of infection in 293T cells could be increased by expression of L-SIGN, DC-SIGN, or SIGLEC-1, consistent with a role for these proteins as well as tethering factors. HeLa and MRC5 cells remained resistant to infection even after expression of L-SIGN, DC-SIGN, or SIGLEC-1 and became susceptible only after expression of ACE2. These data indicate that L-SIGN, DC-SIGN, and SIGLEC-1 are not the primary receptors for SARS-CoV-2.

Example 38

In vivo efficacy of S309 antibody

The efficacy of S309 was investigated in Syrian hamsters, an animal model that represents the most relevant SARS-CoV-2 infection model to date that does not require in vivo ACE2 overexpression to sustain productive infection and disease. Prophylactic administration of S309 induced dose-dependent protection against SARS-CoV-2 infection and tissue injury in hamsters, as demonstrated by viral RNA levels, viral load, and lung histopathology (Figures 73A-7C). These data demonstrate that poor and incomplete neutralization of S309 entry in vitro using ACE2-overexpressing cells did not compromise the in vivo efficacy of RBM-deficient mAbs.

S309 carrying the N297A mutation has a reduced ability to trigger effector functions due to reduced interaction with Fcγ receptors. This was further confirmed by reduced binding of the S309-N297A variant to hamster monocytes in the spleen. In vivo potency measured with the N297A mAb is similar to or just slightly inferior to wt S309, indicating that the neutralizing capacity of the mAb outweighs its ability to exert effector function under these conditions. The serum concentration of S309 required to reduce viral RNA in the lung by 90% was 9 μg/mL, Fig. 73D.

Example 39

Antibody activity against SARS-CoV-2 variants

A number of SARS-CoV-2 variants have emerged, with an increase in variant infections reported in late 2020. The receptor binding motif (RBM) appears to be particularly variable with respect to mutation. Notable emerging variants have been observed in Scotland, the UK, South Africa, California, Columbus, and in mink in Denmark, with some mutations reported to confer antibody escape or serum neutralization. Experiments were conducted to assess the ability of S309 antibodies to neutralize variants. S309 N55Q MLNS (VH: SEQ ID NO:113; VL: SEQ ID NO:168; with Fc mutations M428L and N434S) was tested against SARS-CoV-2, with a panel of the 20 most common SARS-CoV-2 RBD variant mutations as determined by sequence reads. Antibodies REGN10933 and REGN10987 (Hansen et al., Science 369(6506):1010–1014; eabd0827-0810 (2020) and PDB 6XDG (rcsb.org/structure/6XDG)) were included for comparison. The results are summarized in Table 13.

Y indicates less than a three-fold reduction in neutralization of live virus or pseudotyped virus;

N means more than three-fold reduction in neutralization of live virus or pseudotyped virus;

P represents neutralization by the antibody is predicted because the variant amino acid is outside the epitope;

? means unknown.

Table 13. Summary of neutralization results using antibodies against SARS CoV-2 coronavirus variants Mutation variant S309 N55QMLNS REGN10933 REGN10987 N501Y (British, South African and Brazilian mutant) Y Y Y S477N Y Y Y N439K (Scottish mutant) Y Y N L452R (California mutant) Y P P E484K (South African and Brazilian mutant) Y N Y Y453F (Mink Mutant) Y N N (4x reduction) A520S Y Y Y K417N (South African mutant) Y (K417N/V) N Y (K417N/E/V) S494P Y N P S477R P ? P V367F Y Y Y P384L Y P P A522S Y P P A522V Y P P V382L Y P P N501T Y P P P330S Y P P T478I Y ? P S477I Y ? P P479S Y P P

Y indicates less than a three-fold reduction in neutralization of live virus or pseudotyped virus;

N means more than three-fold reduction in neutralization of live virus or pseudotyped virus;

P represents neutralization by the antibody is predicted because the variant amino acid is outside the epitope;

? means unknown.

The total number of sequenced SARS-CoV-2 mutants known to escape antibodies (as of January 29, 2021) was: S309 N55Q MLNS = 29; REGN10987 = 10425; REGN10933 = 3621.

Binding of S309 antibodies to the SARS-CoV-2 RBD variant was assessed by BLI. S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) with wild-type Fc and S309 N55Q (VH: SEQ ID NO:113; VL: SEQ ID NO:168) carrying the MLNS or MLNS + GAALIE Fc mutations were evaluated. REGN10987 and REGN10933 were included as a comparison. Briefly, antibodies were diluted in kinetic buffer at 3 μg/mL and loaded onto Protein A sensors for 75 seconds. After a short equilibration step in kinetic buffer, the loaded sensors were transferred to wells containing RBD variants at 5 μg/mL in kinetic buffer and association was recorded for 3 minutes. The complex was dissociated in kinetic buffer for 3 min. Data are shown in Fig. 71A-71B; "WT" = Wuhan-Hu-1 with D614G; "Triple mutant" in the bottom row = Wuhan-Hu-1 with D614G and the addition of the South African variant B.1.351 with the RBD mutations K417N, E484K and N501Y. Other mutations present in the South African variant B.1.351 were not present in the "SA" RBD tested.

Neutralization by S309 antibodies against SARS-CoV-2 variants was assessed using MLV pseudotyped virus and Vero-E6 target cells expressing TMPRSS2. S309 (VH: SEQ ID NO:105; VL: SEQ ID NO:168) with wild-type Fc and S309 N55Q (VH: SEQ ID NO:113; VL: SEQ ID NO:168) carrying the MLNS or MLNS + GAALIE Fc mutations were evaluated. REGN10987, REGN10933, and the combination of REGN10987 + REGN10933 were also evaluated. Data are shown in Fig. 72. “WT” indicates Wuhan-Hu-1; “UK” indicates SARS-CoV-2 variant B.1.1.7; and “SA” indicates variant B.1.351.

The various embodiments described herein may be combined to produce additional embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications referenced in this specification and/or listed on the application data sheet, including U.S. Patent Application Ser. No. 62/981,984, filed February 26, 2020, U.S. Patent Application Ser. No. 62/982,661, filed February 27, 2020, U.S. Patent Application Ser. No. 62/987,298, filed March 9, 2020, U.S. Patent Application Ser. No. 62/989,522, filed March 13, 2020, U.S. Patent Application Ser. No. 62/990,369, filed March 16, 2020, U.S. Patent Application Ser. No. 62/992,082, filed March 19, 2020, U.S. Patent Application Ser. No. 62/993,500, filed March 18, 2020, U.S. Patent Application Ser. No. 62/995,500, filed March 18, 2020, U.S. Patent Application Ser. No. 62/996,500, filed March 18, 2020, U.S. Patent Application Ser. No. 62/998,500, filed March 18, 2020, U.S. Patent Application Ser. No. 62/9 ... 62/994,235, filed March 24, 2020, U.S. Patent Application 63/001,204, filed March 27, 2020, U.S. Patent Application 63/003,214, filed March 31, 2020, U.S. Patent Application 63/005,206, filed April 3, 2020, U.S. Patent Application 63/010,589, filed April 15, 2020, U.S. Patent Application 63/011,971, filed April 17, 2020, U.S. Patent Application 63/014,024, filed April 22, 2020, U.S. Patent Application 63/023,788, filed May 12, 2020, U.S. Patent Application 63/025,133, filed May 14, 2020, U.S. Patent Application 63/039,813, filed June 16, 2020, U.S. Patent Application 63/043,653, filed June 24, 2020, U.S. Patent Application 63/050,331, filed July 10, 2020, and U.S. Patent Application 63/052,810, filed July 16, 2020, are herein incorporated by reference in their entireties. Aspects of the embodiments may be modified if necessary to utilize the concepts of various patents, applications, and publications to provide still further embodiments.

These and other changes may be made to the present embodiment in light of the above detailed description. In general, the terms used in the following claims should not be construed as limiting the claims to the specific embodiments disclosed in the description and claims, but should be construed as including all possible embodiments together with the full scope of equivalents to which such claims extend. Accordingly, the claims are not limited by the description.

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SEQUENCE LIST

<110> Intervet International BV

<120> HVT Polyvalent Vector Vaccine

<130> 24912

<160> 6

<170> Patentln version 3.5

<210> 1

<211> 5498

<212> DNA

<213> Artificial sequence

<220>

<223> Combined Single Expression Cassette

<220>

<221> promoter

<222> (1} .. (1391)

<223> murine cytomegalovirus immediate early gene 1 promoter-enhancer

<220>

<221> gene

<222> (1423) .. (2781}

<223> IВDV strain F 52/70, VP2 gene

<220>

<221> terminator

<222> (2812) .. (3021}

<223> SV40 polyA signal + terminator

<220>

<221> promoter <222> (3160) .. (3520}

<223> human cytomegalovirus immediate-early gene 1 core promoter

<220>

<221> gene

<222> (3545) .. (5206)

<223> NDV Clone 30 F-gene

<220>

<221> terminator

<222> (5218) .. (5498)

<223> hCMV IE gene terminator

<400> 1

aаstssgсss gttttatgaс tagааасsaat agtttttaat gсsaaatgсa сtgaaаааааааааааааааааааааааааааааааааааааааааTER 60

ctaatttgca aagccaaacg ccccctatgt gagtaatacg gggacttttt acccaatttc 120

ccaagcggaa agccccctaa tacactcata tggcatatga atcagcacgg tcatgcactc 180

taatggcggc ccatagggac tttccacata gggggcgttc accatttccc agcatagggg 240

tggtgactca atggccttta. cccaagtaca ttgggtcaat gggaggtaag ccaatgggtt 300

tttcccatta ctggcaagca cactgagtca aatgggactt tccactgggt tttgcccaag 360

tacattgggt caatgggagg tgagccaatg ggaaaaaccc attgctgcca agtacactga 420

ctcaataggg actttccaat gggtttttcc attgttggca agcatataag gtcaatgtgg 480

gtgagtcaat agggactttc cattgtattc tgcccagtac ataaggtcaa tagggggtga 540

atcaacagga aagtcccatt ggagccaagt acactgcgtc aatagggact ttccattggg 600

ttttgcccag tacataaggt caatagggga tgagtcaatg ggaaaaaccc attggagcca 660

agtacactga ctcaataggg actttccatt gggttttgcc cagtacataa ggtcaatagg 720

gggtgagtca acaggaaagt cccattggag ccaagtacat tgagtcaata gggactttcc 780

aatgggtttt gcccagtaca taaggtcaat gggaggtaag ccaatgggtt tttcccatta 840

ctggcacgta tactgagtca ttagggactt tccaatgggt tttgcccagt acataaggtc 900

aataggggtg aatcaacagg aaagtcccat tggagccaag tacactgagt caatagggac 960

tttсattgg gttttgсса gtaсaaaagg tсaatagggg gtgagtсaat gggtttttсс 1020

cattattggc acgtacataa ggtcaatagg ggtgagtcat tgggtttttc cagccaattt 1080

aattaaaacg ccatgtactt tcccaccatt gacgtcaatg ggctattgaa actaatgcaa 1140

cgtgaccttt aaacggtact ttcccatagc tgattaatgg gaaagtaccg ttctcgagcc 1200

aatacacgtc aatgggaagt gaaagggcag ccaaaacgta acaccgcccc ggttttcccc 1260

tggaaattcc atattggcac gcattctatt ggctgagctg cgttctacgt gggtataaga 1320

ggcgcgacca gcgtcggtac cgtcgcagtc ttcggtctga ccaccgtaga acgcagagct 1380

cctcgctgca ggcggccgct ctagaactcg tcgatcgcag cgatgacaaa cctgcaagat 1440

caaacccaac agattgttcc gttcatacgg agccttctga tgccaacaac cggaccggcg 1500

tccattccgg acgacaccct ggagaagcac actctcaggt cagagacctc gacctacaat 1560

ttgactgtgg gggacacagg gtcagggcta attgtctttt tccctggatt ccctggctca 1620

attgtgggtg ctcactacac actgcaaagc aatgggaact acaagttcga tcagatgctc 1680

ctgactgccc agaacctacc ggccagctac aactactgca gactagtgag tcggagtctc 1740

acagtgaggt caagcacact ccctggtggc gtttatgcac taaacggcac cataaacgcc 1800

gtgaccttcc aaggaagcct gagtgaactg acagatgtta gctacaatgg gttgatgtct 1860

gcaacagcca acatcaacga caaaattggg aatgtcctgg taggggaagg ggtcactgtc 1920

ctcagcctac ccacatcata tgatcttggg tatgtgaggc ttggtgaccc cattcccgct 1980

atagggcttg acccaaaaat ggtagctaca tgcgacagca gtgacaggcc cagagtctac 2040

accataactg cagccgatga ttaccaattc tcatcacagt accaaccagg tggggtaaca 2100

atcacactgt tctcagccaa cattgatgct atcacaagcc tcagcattgg gggagagctc 2160

gtgtttcaaa caagcgtcca aggccttgta ctgggcgcca ccatctacct tataggcttt 2220

gatgggactg cggtaatcac cagagctgta gccgcagata atgggctgac ggccggcacc 2280

gacaatctta tgccattcaa tcttgtcatt ccaaccaatg agataaccca gccaatcaca 2340

tccatcaaac tggagatagt gacctccaaa agtggtggtc aggcaaggga tcagatgtca 2400

tggtcggcaa gtgggagcct agcagtgacg atccatggtg gcaactatcc aggggccctc 2460

cgtcccgtca cactagtagc ctacgaaaga gtggcaacag gatccgtcgt tacggtcgct 2520

ggggtgagta acttcgagct gattccaaat cctgaactag caaagaacct ggttacagaa 2580

tacggccgat ttgacccagg agccatgaac tacacaaaat tgatactgag tgagagggac 2640

cgtcttggca tcaagaccgt ctggccaaca agggagtaca ctgattttcg tgagtacttc 2700

atggaggtgg ccgacctcaa ctctcccctg aagattgcag gagcatttgg cttcaaagac 2760

ataatccggg ctataaggag gtaagcttga tctagagcgg ccgcggggat ccagacatga 2820

taagatacat tgatgagttt ggacaaacca caactagaat gcagtgaaaa aaatgcttta 2880

tttgtgaaat ttgtgatgct attgctttat ttgtaaccat tataagctgc aataaacaag 2940

ttaacaacaa caattgcatt cattttatgt ttcaggttca gggggaggtg tgggaggttt 3000

ttteggateс tctagagtcg acaattattt catttaataa catatagccc aaagacctct 3060

atgaacattt agtttcccgt atactcaacg gcgcgtgtac acacaagggc gaattccaca 3120

gtggatatca agcttaatta agtaccgagc tcgaattggc gcgccaggtc aattccctgg 3180

cattatgccc agtacatgac cttatgggac tttcctactt ggcagtacat etaegtatta 3240

gtcatcgcta ttaccatggt gatgcggttt tggcagtaca tcaatgggcg tggatagegg 3300

tttgactcac ggggatttcc aagtctccac cccattgacg tcaatgggag tttgttttgg 3360

caccaaaatc aaegggaett tccaaaatgt cgtaacaact ccgccccatt gacgcaaatg 3420

ggeggtagge gtgtacggtg ggaggtctat ataagcagag ctcgtttagt gaaccgtcag 3480

atcgcctgga. gacgccatcc acgctgtttt gacctccata gaagacaccg ggcgcgccgg 3540

atccatgggc cccagacctt ctaccaagaa cccagtacct atgatgctga ctgtccgagt 3600

cgcgctggta ctgagttgca tctgtccggc aaactccatt gatggcaggc ctcttgcggc 3660

tgcaggaatt gtggttacag gagacaaagc cgtcaacata tacacctcat cccagacagg 3720

atcaatcata gttaagctcc tcccgaatct gcccaaggat aaggaggcat gtgegaaage 3780

ccccttggat gcatacaaca ggacattgac cactttgctc accccccttg gtgactctat 3840

ccgtaggata caagagtctg tgactacatc tggagggggg agacaggggc gccttatagg 3900

cgccattatt ggcggtgtgg ctcttggggt tgcaactgcc gcacaaataa cagcggccgc 3960

agctctgata caagccaaac aaaatgctgc caacatcctc cgacttaaag agagcattgc 4020

cgcaaccaat gaggctgtgc atgaggtcac tgacggatta tcgcaactag cagtggcagt 4080

tgggaagatg cagcagtttg ttaatgacca atttaataaa acagctcagg aattagactg 4140

catcaaaatt gcacagcaag ttggtgtaga gctcaacctg tacctaaccg aattgactac 4200

agtattcgga ccacaaatca cttcacctgc tttaaacaag ctgactattc aggcacttta 4260

caatctagct ggtggaaata. tggattactt attgactaag ttaggtgtag ggaacaatca 4320

actcagctca ttaatcggta gcggcttaat caccggtaac cctattctat acgactcaca 4380

gactcaactc ttgggtatac aggtaactct accttcagtc gggaacctaa ataatatgeg 4440

tgccacctac ttggaaacct tatccgtaag cacaaccagg ggatttgcct cggcacttgt 4500

cccaaaagtg gtgacacagg tcggttctgt gatagaagaa cttgacacct catactgtat 4560

agaaactgac ttagatttat attgtacaag aatagtaacg ttccctatgt cccctggtat 4620

ttattcctgc ttgageggea ataegtegge ctgtatgtac tcaaagaccg aaggcgcact 4680

tactacacca tacatgacta. tcaaaggttc agtcatcgcc aactgcaaga tgacaacatg 4740

tagatgtgta aaccccccgg gtatcatatc gcaaaactat ggagaagccg tgtctctaat 4800

agataaacaa teatgeaatg ttttatcctt aggegggata actttaaggc tcagtgggga 4860

attcgatgta acttatcaga agaatatctc aatacaagat tctcaagtaa taataacagg 4920

caatcttgat atctcaactg agcttgggaa tgtcaacaac tcgatcagta atgctttgaa 4980

taagttagag gaaagcaaca gaaaactaga caaagtcaat gtcaaactga ctagcacatc 5040

tgctctcatt acstatateg ttttgactat catatctctt gtttttggta tacttagccc 5100

gattctagca tgctacctaa tgtacaagca aaaggcgcaa caaaagacct tattatggct 5160

tgggaataat actctagatc agatgagagc cactacaaaa atgtgaggat ctctcgagga 5220

attctagate ccacgtcact attgtatact ctatattata ctctatgtta tactctgtaa 5280

tcctactcaa taaacgtgtc acgcctgtga aaccgtacta agtctcccgt gtcttcttat 5340

caccatcagg tgacatcctc gcccaggctg tcaatcatgc cggtatcgat tccagtagca 5400

ccggccccac gctgacaacc cactcttgca gcgttagcag cgcccctctt aacaagccga 5460

cccccaccag cgtcgcggtt actaacactc ctctcccc 5498

<210> 2

<211> 2459

<212> DNA

<213> Artificial sequential

<220>

<223> Expression Cassette 3

<220>

<221> promoter

<222> (20) .. (701)

<223> PRV gB gene promoter, enlarged

<220>

<221> gene

<222> (713) . . (2395)

<223> AIV HA H9 gene, synthetic, codon-optimized

<220>

<221> terminator

<222> (2404) .. (2458)

<223> FHV1 Us9 gene terminator

<400> 2

agcttaatta aggcgcgcca gatctcgctg ctgcacacgt acgtggcggt ggccgccggg 60

ttccgcgcac ggcgcgcgtt ctgcgaggcc gccgcgcgcg cgggcaccgt cgtggacgag 120

cgcgagacgg getgettega cgcgcacagc ttcatgaagg ccacggtgca gcgccacccc 180

gtggacgccg cgctcctccc ggcgctcacg cacaagttct tcgagctcgt caacgggccg 240

ctcttcgcgc acgacacgca. cgccttcgcc cagtccccca acacggcgct ctactttgcg 300

gtggagaacg tgggcctcct gccgcacctg aaggaggagc tggcgcgctt catggtggcc 360

cgcgattggt gcgtcagtga gttccgcggc ttctaccgct tccagacggc cggcgtaacc 420

gccacccagc ggcaggcctg gcgatatatc cgcgagctgg tgctggcggt tgcagtcttc 480

aggtccgtct tccactgcgg ggacgtcgag gtcctccgcg eggategсtt cgccggacgc 540

gacgggctgt acctgaccta cgaggcgtct tgccccgctg gtggcggtct ttggcgcggg 600

sssgcgggc atcggcccgg gcaccacggc ggtgctggcc tcggacgtct ttggcctgct 660

cccacaccacg ctgctgctgc gcggggcgcc gtcgcgctag agatctaaag ccatggagac 720

catctccctg atgactatcc tgctcgtcgt gaccacctcc aacgccgaca agatttgcat 780

cggtcaccag tccaccaact ctaccgagac cgtggacacc ctgaccgaga ctaacgtccc 840

tgtcacccac gccaaggagc tgctgcacac tgagcacaac ggaatgctct gcgctaccaa 900

cctcggtcac ccactcatcc tggacacctg cactatcgag ggtctcatct acggcaaccc 960

stcttgсgaс сtgсtсstgg gaggtсgtga gtggtсstaс atсgtсgagс gtссttсgс 1020

tgtgaacggc acttgctacc ccggtaacgt ggagaacctc gaggagctcc gtactctctt 1080

ctcctcttcc tccagctacc agcgtatcca aatcttcccc gacacgatct ggaacgtgac 1140

ctacaccggt acctccaagt cctgcagcga ctctttctac cgcaacatgc gctggctgac 1200

ccagaagaac ggactgtacc ctgtgcagga cgctcagtac accaacaacc gcggaaagga 1260

catcctgttc gtctggggta. tccaccaccc tcccactgac accgctcaga ccaacctgta 1320

cactcgcacc gacactacca ctagcgtcac taccgagaac ctggaccgca ctttcaagcc 1380

actgatcggt cctcgtcctc tggtgaacgg tctgatcggc aggatcaact actactggag 1440

cgtgctcaag cccggacaga ctctgagggt gcgtagcaac ggcaacctga tcgccccttg 1500

gttcggtcac gtgctgtctg gagagtctca cggacgtatc ctgaagaccg acctgaactc 1560

cggtaactgc gtggtccagt gccagactga gaagggtggc ctcaactcca ctctgccctt 1620

ccacaacatc tccaagtacg ccttcggcac ctgccccaag tacatcggtg tgaagtccct 1680

gaagctcgct atcggactgc gtaacgtccc tgctaggtcc agcaggggtc tgttcggcgc 1740

tatcgctggt ttcatcgagg gtggatggcc tggtctggtg gctggttggt acggcttcca 1800

gcactctaac gaccagggtg tcggaatggc cgctgaccgt gactctactc agaaggccgt 1860

cgacaagatc acctccaagg tgaacaacat cgtcgacaag atgaacaagc agtacgagat 1920

catcgaccac gagttctccg aggtggagac tcgcctcaac atgatcaaca acaagatcga 1980

cgaccagatc caggacgtct gggcttacaa cgctgagctc ctggtgctcc tcgagaacca 2040

gaagactctg gacgagcacg acgccaacgt caacaacctc tacaacaagg tgaagcgtgc 2100

cctgggctcc aacgctatgg aggacggcaa gggttgcttc gagctctacc acaagtgcga 2160

cgaccagtgc atggagacca tcaggaacgg gacctacaac cgccgtaagt acaaggagga 2220

gagcaggctg gagcgtcaga agatcgaggg cgtcaagctg gagtctgagg gcacctacaa 2280

gatcctgact atctactcca ccgtcgcctc tagcctcgtg ctcgctatgg gcttcgctgc 2340

cttcctgttc tgggccatgt ccaacggttc ctgccgttgc aacatctgca tctaattaat 2400

taacaataaa catagcatac gttatgacat ggtctaccgc gtcttatatg gggacgaca 2459

<210> 3

<211> 20

<212> DNA

<213> Herpesvirus of turkeys

<220>

<221> primer

<222> (1) . . (19)

<223> guide RNA for UL40-41 insertion

<400> 3

acctaaagta cacgtgaatc

20

<210> 4

<211> 20

<212> DNA

<213> Herpesvirus of turkeys

<220>

<221> primer

<222> (1)..(20)

<223> guide RNA for UL44-45 insertion

<400> 4

асatсgggас gtасatсatg

20

<210> 5

<211> 20

<212> DNA

<213> Herpesvirus of turkeys

<220>

<221> primer

<222> (1) . . (20)

<223> guide RNA for UL47-48 insertion

<4О0> 5

tggcggttас aatttсасасg

20

<210> 6

<211> 20

<212> DNA

<213> Herpesvirus of turkeys

<220>

<221> primer

<222> (1) . . (20)

<223> guide RNA for UL54-LORF4 insertion

<400> 6

ttagatttcc ggacagcctg

20

<---

Claims (30)

1. A recombinant turkey herpes virus (rHVT) for producing a vaccine for preventing or reducing MDV, IBDV, NDV and/or AIV infection or associated disease signs in poultry, expressing the infectious bursal disease virus (IBDV) viral protein 2 (VP2) gene and the Newcastle disease virus (NDV) fusion protein (F) gene from first and second expression cassettes which are inserted into a unique small (Us) region of the genome of said rHVT, characterized in that the first and second expression cassettes are both inserted into the Us2 gene, and in that said rHVT also expresses the avian influenza virus (AIV) hemagglutinin (HA) gene from a third expression cassette which is inserted into a unique long (UL) region of the genome of said rHVT either between the UL40 and UL41 genes or between the UL44 and UL45 genes. 2. rHVT according to claim 1, characterized in that the IBDV VP2 gene is expressed from a first expression cassette containing, in the direction from 5' to 3' and in the following order: a. mouse cytomegalovirus immediate early gene 1 (mCMV-IE1) promoter, b. IBDV VP2 gene, and c. transcription terminator, and wherein the promoter and terminator of said expression cassette are operably linked to the VP2 gene. 3. rHVT according to claim 1 or 2, characterized in that the NDV F gene is expressed from a second expression cassette comprising, in the 5' to 3' direction and in the following order: a. human cytomegalovirus immediate early gene 1 (hCMV-IE1) promoter, b. NDV F protein gene, and c. transcription terminator, and where the promoter and terminator of said expression cassette are operably linked to the F gene. 4. rHVT according to paragraphs 1-3, characterized in that the first and second expression cassettes are combined into one expression cassette. 5. rHVT according to claim 4, characterized in that the combined single expression cassette is integrated into the Us2 gene. 6. The rHVT of any one of claims 1 to 5, wherein the AIV HA gene is expressed from a third expression cassette comprising, in the 5' to 3' direction and in the following order: a. promoter of the glycoprotein B (gB) gene from mammalian herpes virus, b. the gene for the AIV HA protein, and c. transcription terminator, and where the promoter and terminator of said expression cassette are operably linked to the HA gene. 7. rHVT according to any one of claims 1-6, characterized in that the AIV HA protein gene encodes the HA protein of a serotype selected from H5, H7 and H9. 8. An avian host cell for producing a vaccine for preventing or reducing MDV, IBDV, NDV and/or AIV infection or disease signs associated therewith in poultry, comprising the rHVT of any one of claims 1-7. 9. A vaccine for preventing or reducing MDV, IBDV, NDV and/or AIV infection or disease signs associated therewith in poultry, comprising an immunologically effective amount of the rHVT of any one of claims 1-7 and/or the host cell of claim 8, and a pharmaceutically acceptable carrier. 10. The vaccine according to item 9, containing at least one additional immunoactive component. 11. A method for producing a vaccine for poultry according to paragraph 9 or 10, comprising the steps of: a. infecting host cells in vitro with rHVT according to any one of claims 1-9, b. collection of infected host cells, and c. mixing the collected infected host cells with a pharmaceutically acceptable carrier. 12. Use of rHVT according to any one of claims 1-7, a host cell according to claim 8, or any combination thereof for producing a vaccine for poultry. 13. Use of a vaccine according to claim 9 or 10 for the prevention or reduction of MDV, IBDV NDV and/or AIV infection or associated disease manifestations. 14. A method for preventing or reducing infection with MDV, IBDV, NDV and/or AIV or associated disease signs comprising administering the vaccine of any one of claims 9 or 10 to poultry. 15. A method of vaccinating poultry to prevent or reduce infection with MDV, IBDV NDV and/or AIV or disease signs associated therewith, comprising the step of inoculating said poultry with the vaccine of claim 9 or 10.