ANTI-SARS-COV-2 ANTIBODIES AND USE THEREOF IN THE TREATMENT OF

SARS-CoV-2 INFECTION

Introduction

The project leading to this application has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under Grant Agreement n° 101005077. This Joint Undertaking receives the support from the European Union’s Horizon 2020 research and innovation programme and EFPIA.

FIELD OF THE INVENTION

The invention provides anti-SARS-CoV-2 antibodies and use thereof in prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection.

BACKGROUND OF THE INVENTION

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) was first reported in December 2019. Since then SARS- CoV-2 has emerged as a global pandemic with an ever-increasing number of severe cases requiring specific and intensive treatments that threatens to overwhelm healthcare systems. While it remains unclear why COVID-19 patients experience a spectrum of clinical outcomes ranging from asymptomatic to severe disease and mortality, the COVID-19 pandemic is a major challenge for governments, businesses, healthcare systems and people around the globe seeking ways to safely return to work/healthcare/travel/leisure. Testing for this highly infectious and often asymptomatic disease is burdensome with limited availability; treatments and vaccines are still emerging and not completely proven. Indeed, there are now several vaccines in clinical trials that demonstrate a high level of efficacy, however there is still no data indicating the durability of this vaccine induced protection. In addition, it is likely that at-risk individuals that includes the elderly population and immunosuppressed subjects (e.g. patients undergoing cancer therapy and those that have undergone an organ transplants) will only have a partial or transient protection induced by these vaccines. Thus in the ongoing COVID-19 pandemic, there is a large unmet medical need for therapeutic interventions that can protect at-risk individuals, be of significant importance to protect individuals that are less able to mount an effective anti- SARS-CoV-2 immune response following vaccination and treat those already infected with the virus. SUMMARY OF THE INVENTION

An aspect of the present invention provides an anti-SARS-CoV-2 antibody, or an antigen binding fragment thereof, comprising a heavy chain variable region (VH) that comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) domains; and a light chain variable region (VL) that comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) domains, wherein the HCDR1 sequence is SEQ ID NO: 107, the HCDR2 sequence is selected from SEQ ID NO: 108, 181-191, and the HCDR3 sequence is selected from SEQ ID NO: 109, 192-198; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively (antibody P2G3).

A further aspect of the present invention provides an anti-SARS-CoV-2 antibody, or an antigen binding fragment thereof comprising a heavy chain variable region amino acid sequence comprising or consisting of the amino acid sequence selected from SEQ ID NO: 105 and SEQ ID NO: 163-180, and a light chain variable region amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 106.

Another aspect of the present invention provides an anti-SARS-CoV-2 antibody, or an antigen binding fragment thereof, that specifically binds an epitope on the SARS-CoV-2 Spike protein, wherein the epitope comprises at least one amino acid in the Spike protein RBD selected from Asn343, Ala344, Thr345, Arg346, Asn440, Leu441, Asp442, Ser443, Lys444, Val445, Gly446, Gly447, Asn448, Tyr449, Asn450, Tyr451 in SEQ ID NO: 199.

Another aspect of the present invention provides a pharmaceutical composition comprising one or more anti-SARS-CoV-2 antibodies, or an antigen-binding fragments thereof, of the invention and a pharmaceutically acceptable carrier.

Another aspect of the present invention provides a method for detecting a SARS-CoV-2 virus in a sample, the method comprising contacting the sample with the anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, of the invention and detecting the antibody in the sample.

Another aspect of the present invention provides the anti-SARS-CoV-2 antibody, or an antigen binding fragment thereof, of the invention for use as a pharmaceutical. Another aspect of the present invention provides the anti-SARS-CoV-2 antibody, or an antigen binding fragment thereof, of the invention for use in a method of prophylaxis, treatment, and/or attenuation of a SARS-CoV-2 virus infection in a subject, wherein the method comprises administering to the subject an effective amount of the one or more antibody, or an antigen binding fragment thereof, of the invention.

Another aspect of the present invention provides an isolated nucleic acid encoding the anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention.

Another aspect of the present invention provides a vector comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention.

Another aspect of the present invention provides a host cell comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or comprising the vector of the invention.

Another aspect of the present invention provides a method of producing the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention comprising culturing a host cell comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention under a condition suitable for expression of the nucleic acid; and recovering the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, produced by the cell.

Another aspect of the present invention provides a kit for detecting SARS-CoV-2 virus in a sample, the kit comprising the one or more anti-SARS-CoV-2 antibodies, or an antigen-binding fragment thereof, of the invention and instructions for use.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows neutralization activity associated with antibodies cell culture supernatants from immortalized B cells with B cell supernatants.

Figure 2 shows activity of anti-SARS-CoV-2 antibodies in the Spike pseudotyped lentivirus luciferase reporter neutralization assay. Curves fitting for the anti-viral neutralization effect of the newly reported antibodies are shown with solid lines while the reference antibodies tested in parallel are represented with dashed lines.

Figure 3 shows activity of anti-SARS-CoV-2 antibodies in the live virus SARS-CoV-2 cytopathic effect neutralization assay. Curves fitting for the anti-viral neutralization effect of the newly reported antibodies are shown with solid lines while the reference antibodies tested in parallel are represented with dashed lines.

Figure 4 shows the activity of anti-SARS-CoV-2 antibody Fab fragments in blocking the interaction between the ACE-2 protein and trimeric Spike proteins expressed as A) wild type and B) - H) mutant versions (B - mutation M153I; C - mutation N439K; D - mutation S459Y; E - mutation S477N; F - mutation S477R; G - mutation E-484K; H - mutation N501T) that correspond to circulating viral strains.

Figure 5 shows the activity of anti-SARS-CoV-2 antibodies in the live virus SARS-CoV-2 cytopathic effect neutralization assay using different variant of concern viruses. Curves fitting for the anti-viral neutralization effect of select newly reported antibodies are shown for the 2019-nCoV -D614G mutant (A), Alpha / B.EE7 UK variant (B), Beta / B.E351 South African variant (C) a mink variant (D), Delta / B.E617.2 variant (E), Omicron BA.l (B.EE529.1) variant (F), Omicron BA.2 variant (G) evaluated with the live virus CPE assay and Omicon BA.l (H), Omicron BA.2.12.1 (I) and Omicron BA.4 (J) evaluated in the pseudoviral neutralization assay.

Figure 6 shows the activity of individual and combinations of mAbs in the biochemical Spike- ACE2 surrogate neutralization assay performed with 2019-nCoV (A) Omicron BA.l variant (B). Omicron BA.1.1 (R346K) variant (C), and Omicron BA.2 variant (D) Spike protein. The corresponding IC50 and ICxo values for P2G3 and P5C3 are shown in (E) relative to a panel of approved anti-SARS-CoV-2 mAbs and those that are advanced in the clinic. P5C3 and P2G3 mab were also evaluated in an antibody dependent cellular phagocytosis (F) where synergy was observed with the combined mAh use (G) and in an antibody dependent cellular cytotoxicity assay (H).

Figure 7 shows the cryo-electron microscopy structure of P5C3 Fab in complex with the Spike trimer and the overlap in binding to RBD between P5C3 and ACE2. Figure 8 shows the cryo-electron microscopy structure of P5C3 Fab and P2G3 Fab in complex with the Omicron variant Spike trimer.

Figure 9 shows the evaluated the neutralizing potency of P5C3 and P2G3 in vivo in a prophylactic hamster challenge model of SARS-CoV-2 infection.

Figure 10 shows neutralizing activity of P5C3 antibodies with (A) mutations N58, M74 and N100, and (B) mutations T28, G52, S53, G54 and R70.

Figure 11 shows neutralizing activity of P2G3 antibodies with (A) mutations N54, (B) mutations N56, and (C) mutations D103 and N110.

Figure 12 shows binding activity of P5C3 LS antibody, P5C3 LS N100Q antibody, P2G3 LS antibody and P2G3 LS N54S/N56Q antibody to (A) Spike 2019nCoV, (B) Spike Alpha variant, (C) Spike Beta variant, (D) Spike Gamma variant, and (E) Spike Delta variant.

Figure 13 shows neutralization activity of P5C3 LS, P5C3 LS N100Q, P2G3 LS and P2G3 LS N54S/N56Q antibodies of (A) Spike 2019nCoV D614G, (B) Spike Beta variant and (C) Spike Delta variant pseudotyped viruses.

Figure 14 shows identification of P2G3, a human mAh with high affinity binding to trimeric Spike protein with mutations found in VOCs. Competitive binding studies between antibodies binding to the Spike RBD protein. RBC coupled beads pre-incubated with saturating concentrations of competitor antibody were used for binding studies with mAbs or ACE2. Competitors induced either strong blocking (Red boxes), partial competition (orange boxes) or non-competitive (white boxes) binding with the corresponding mAh to RBD. Red and yellow hashed lines indicate incomplete blocking of the Spike-ACE2 interaction with Alpha, Gamma and Omicron Spike variant proteins. Data presented is representative of 2-4 independent experiments with each concentration response tested in duplicate.

Figure 15 shows P2G3 demonstrates potent and broad activity in a surrogate neutralization assay and against Spike-coated pseudoviruses and live virus SARS-CoV-2 VOCs. A-B) Blocking activity of individual and combinations of anti-Spike mAbs in a biochemical Spike- ACE2 surrogate neutralization assay using A) the original 2019-nCoV or B) Omicron variant Spike trimer proteins. C) Neutralization of lentiviral particles pseudotyped with SARS-CoV-2 Spike expressing variants of concern in a 293T-ACE2 infection assay. All Spike proteins contained the D614G substitution that became dominant early in the pandemic. D) Neutralization of lentiviral particles pseudotyped with the Omicron variant Spike. Antibody cocktails representing a 1:1 mix of each mAh to give the indicated final concentration. E) Neutralization activity of P2G3 performed in a live SARS-CoV-2 infectious virus cytopathic effect assay. The indicated SARS-CoV-2 variants were used to infect Vero E6 in vitro in the absence and presence of concentration response of the indicated mAh evaluated in duplicates or triplicates. F-G) Neutralization activity of individual and mAbs combinations in F) a live virus Omicron variant or G) Delta variant cytopathic effect assay. Results shown in A - E are the average of two to three independent experiments with each concentration response tested in duplicate or triplicate. Results in F-G are representative data for two separate experiments. Mean values ± SEM are shown.

Figure 16 shows P2G3 LS confers potent in vivo efficacy in the hamster challenge model for SARS-CoV-2 infection. A) Overview of study design for the SARS-CoV-2 hamster challenge model. Animals were administered intraperitoneally 5.0, 1.0 or 0.5 mg/kg of P2G3, 5 mg/kg of REGN10933 positive control or 5 mg/kg of an IgGl isotype control and challenged two days later (Day 0) with an intranasal inoculation of SARS-CoV-2 virus (2.4 xl06 TCID50). B) Median levels of infectious virus. Only 1 out of 6 hamsters in the 0.5 mg/kg group had detectable levels of infectious virus that none the less showed a ~1.5 log reduction in infectious virus compared to the isotype mAb-treated control animals. Complete prophylactic protection in these studies was observed with P2G3 mAh plasma levels >6.2 μg/ml at the time of viral inoculation. C) Viral RNA copies/mg lung tissue in each of the study arms are shown on day 4 post-inoculation with SARS-CoV-2 virus. All P2G3 treatment groups showed a significant ~4-log reduction of genomic viral RNA levels relative to control animals at day 4 of the study. A total of 4-6 hamsters were used per P2G3 treatment arm. Non-parametric Mann-Whitney El- tests were used to evaluate the statistical difference between the treatment conditions with p<0.009 (**).

Figure 17 shows two potent anti-SARS-CoV-2 antibodies P2G3 and P5C3 bind the full-length Omicron Spike. A) Cryo-EM composite density map of the full-length Omicron Spike bound to one P5C3 and three P2G3 Fab fragments. Each Spike protomer is coloured independently as are the Fabs. B) Surface representation of the RBD in the up configuration (green) bound to both P5C3 (dark red and pink) and P2G3 (black and gray). The buried surface area formed by the Fabs are depicted on the RBD surface and coloured correspondingly. The Omicron mutations are shown in yellow as balls-and-sticks and transparent surfaces. The Fab heavy and light chains are depicted as licorice ribbons. N-linked glycans at asparagine 331 and 343 are shown as sticks. C) Zoomed-in view of the interacting region of P2G3. Specific CDR loops of the heavy and light chains are indicated. Omicron mutations in the region of the Fab are highlighted in yellow. Interacting residues of the Fab are shown as sticks. D) Detailed atom level analysis of the interactions between the Omicron RBD shown as ribbons (green) and the P2G3 Fab heavy and light chains shown as licorice (black and gray). Residues at the interface are shown as sticks with potential interactions of interest as dashed lines. Omicron mutations are shown as balls- and-sticks in yellow.

Figure 18 shows buried surface area and angle of attack of Class 3 anti-SARS-CoV-2 mAh A) Surface representation of the Omicron RBD-up coloured green. Structures of the several class 3 antibodies (Fabs) bound to RBDs were superimposed on the Omicron RBD. The buried surface area formed by the indicated Fabs are outlined on the RBD surface and coloured correspondingly (Fab-RBD structures AZD1061, PDB-7L7E; REGN10987, PDB-6XDG; S309, PDB-7BEP). The Omicron mutations are shown in yellow as balls-and-sticks and transparent surfaces. B) Angle of attack of Fabs to the RBD is defined as the line connecting the centroid of the Fab to the centroid of the surface area of the RBD that the Fabs bury. Angle of attack of P2G3 compared to other class 3 antibodies viewed from multiple angles. RBD is coloured as in panel a. C-F) P2G3, REGN10987, AZD1061 and S309/Sotrovimab Fabs in context of the full Omicron trimer were modelled by superimposing the Fabs on to the RBD of each protomer. The trimer is shown from three different perspectives to visualize the different angle of attack the Fabs have depending on the Spike protomer. C) P2G3 Fabs are able to bind all RBD-up and RBD-down conformations simultaneously. D) REGN10987 Fab bind to the green RBD-up conformation but modelled REGN10987 Fab bound to either the RBD-down of the orange or blue protomer would clash sterically with the adjacent blue RBD-down or green RBD-up protomers, respectively. Together it is predicted REGN10987 is able to bind only RBD-up. E) AZD1061 binds the RBD-up form and RBD-down form of the blue protomer butAZD1061 Fab modelled binding to the RBD-down of the orange protomer can potentially clash with the adjacent green RBD-up. E) S309/Sotrovimab is able to bind all RBDs simultaneously as shown for P2G3.

Figure 19 shows P2G3 LS and P5C3 LS show Fc-mediated functional activity in ADCC cell killing and ADCP phagocytosis of Spike coated beads in in vitro assay. Antibody dependent cellular cytotoxicity assay (ADCC) performed with CEM NKR Luc cells stably expressing cell surface 2019-nCoV Spike. P2G3 exhibits potent ADCC activity in killing Spike positive cells. ADCC experiments performed with five replicates per condition using effector cells from five different healthy donors. CEM NKR Spike cells were incubated with 300 ng/ml of the indicated human IgG a mAbs. Statistical difference evaluated by Two-way ANOVA.

Figure 20 shows P2G3 LS and P5C3 LS show Fc-mediated functional activity in ADCC cell killing and ADCP phagocytosis of Spike coated beads in in vitro assay. A) Antibody dependent cellular cytotoxicity assay performed with 2019-nCoV Spike coated fluorescent beads mixed with the indicated antibody concentration then incubated with the U937 effector cell line. B) MacSynergy plot of the P2G3 / P5C3 interactions were performed using a synergy plot stringency of 99.9% and synergy/antagonism values calculated with a Bonferroni correction. In this analysis, drug combinations in the absence of synergy or antagonism are expected to fall on the X-, Y-axis plane with a value of zero. Values falling above or below the plane represent additive/synergistic or antagonistic interactions, respectively. At the indicated concentrations of P2G3 and P5C3 mAbs, red regions of the graph above the X- / Y-axis plane correspond to synergistic interactions. In standard evaluation of synergy plot values, ranges between -25 to 25 = additivity, 25 to 50= slight synergy, 50 to 100= moderate synergy and >100 [mAb] ² % = strong synergy. Results are the average of 2-4 independent experiments with each concentration response tested in triplicates.

Figure 21 shows Cryo-EM processing of the Omicron Spike. Cryo-EM processing workflow performed in CryoSPARC v.3.3.1.

Figure 22 shows details of Cryo-EM processing and Resolution of Cryo-EM maps A) Raw representative micrograph. B) Representative 2D class averages. C) Enlarged 2D class showing the Omicron Spike with bound Fabs. D) Direction distribution plot and FSC curves indicating a resolution of 3.04 A (FSC 0.143) of the Global (Class 9) map off the full-length Omicron Spike bound to Fabs. E) Direction distribution plot and FSC curves indicating a resolution of 3.84 A (FSC 0.143) of the P2G3-bound-RBD-down (Class 5) locally refined map F) Direction distribution plot and FSC curves indicating a resolution of 4.01 A (FSC 0.143) of the P5C3-P2G3-bound-RBD-up (Class 4) locally refined map. Global and focussed refined maps coloured by local resolution.

Figure 23 shows analysis of the P5C3 Fab-Omicron RBD interacting surfaces.

A) The buried surface area of P5C3 (pink) overlay ed on the RBD surface (green). P5C3 buries around 500 A of the surface of the Omicron RBD. Specific CDR loops of the heavy and light chains are indicated. Omicron mutations are shown as balls-and-sticks and transparent surfaces in yellow. B) Zoomed-in view of the interacting region of P2G3. Specific CDR loops of the heavy and light chains are indicated. Omicron mutations in the region of the Fab are highlighted in yellow. Interacting residues of the Fab are shown as sticks. C) Detailed atom level analysis of the interactions between the Omicron RBD shown as ribbons (green) and the P5C3 Fab heavy and light chains shown as licorice (dark red and pink). Residues at the interface are shown as sticks with potential interactions of interest as dashed lines. Omicron mutations are shown as balls-and-sticks in yellow. D) Superposition of the P5C3-Omicron-RBD interface with the P5C3-wild-type-RBD interface (PDB; 7PHG)

Figure 24 shows additional views of the P2G3 Fab-Omicron RBD interacting surfaces.

A) The buried surface area of P2G3 (gray) overlay ed on the RBD surface (green). P2G3 buries 705 A of surface of the Omicron RBD. Specific CDR loops of the heavy and light chains are indicated. Omicron mutations are shown as balls-and-sticks and transparent surfaces in yellow.

B) Stick representation of the P2G3 interface of CDRH3 and the RBD region containing residues 440-451. The mesh represents the Cryo-EM density. C) Superposition of the P2G3- RBD-up interface with the P2G3-RBD-down interface shows no significant differences and conservation of interactions. D) Stick representation of the P2G3 interface of CDRH2 residue W53 that forms a potential cation-pi interaction with RBD residue R346. The mesh represents the Cryo-EM density.

Figure 25 shows regions of the Omicron Spike and Fabs with Cryo-EM density maps.

The Cryo-EM density is rendered as a mesh. The atomic model is shown as ribbon or stick representation. Figure 26 shows buried surface area and angle of attack of Class 3 antibodies A) Surface representation of the Omicron RBD-up coloured green. Structures of the several class 3 antibodies (Fabs) bound to RBDs were superimposed on the Omicron RBD. The buried surface area formed by the indicated Fabs are outlined on the RBD surface and coloured correspondingly (Fab-RBD structures AZD1061, PDB-7L7E; REGN10987, PDB-6XDG; S309, PDB-7BEP). The Omicron mutations are shown in yellow as balls-and-sticks and transparent surfaces. B) Angle of attack of Fabs to the RBD is defined as the line connecting the centroid of the Fab to the centroid of the surface area of the RBD that the Fabs bury. Surface representation of the P2G3 Fab (grays) bound to the RBD-up in ribbons, coloured as in panel A. The pink balls are markers indicating the centroids (calculated in ChimeraX) of the P2G3 Fab and the RBD surface area buried by P2G3. A stick connects the two markers. C) Angle of attack of P2G3 compared to other class 3 antibodies viewed from multiple angles. RBD is coloured as in panel A.

Figure 27 shows P5C3 and P2G3 are incompatible with ACE2 binding.

A) Two orientations of an Omicron RBD in the up position bound to P5C3 and P2G3. The RBD is shown as a green ribbon and the two Fabs with a surface representation. ACE2 directly clashes with P5C3. P2G3 shares a minor overlapping surface. B) Two orientations of the full- length Omicron Spike with only P5C3 or P2G3 shown for clarity, (top) P5C3 directly blocks the binding of ACE2 by overlapping with its binding site, (bottom) P2G3 bound to the up-RBD minimally overlaps with the ACE2 surface (as in Panel A) however P2G3 bound to the down- RBD sterically clashes with potential ACE2 binding.

Figure 28 shows P2G3 and P5C3 demonstrates potent neutralization assay against Spike- coated pseudoviruses with R346K mutation.

Neutralization of lentiviral particles pseudotyped with SARS-CoV-2 Spike expressing variants of concern in a 293T-ACE2 infection assay. In addition to the R346K mutation, spike protein contained the D614G substitution that became dominant early in the pandemic. Results shown are representative of two independent experiments with a R346K encoding pseudovirus with each concentration response tested in triplicate. Mean values ± SEM are shown.

Figure 29 shows enhance activity in ADCP assay with Omicron Spike coated beads in an antibody dependent cellular phagocytosis assay. Antibody dependent cellular cytotoxicity assay performed with trimeric Omicron variant Spike protein that was biotinylated and bound to streptavidin coated fluorescent beads. PBS washed beads were mixed with the indicated antibody concentration and then the bead-antibody mix was incubated with the U937 effector cell line. Analysis of antibody-mediated phagocytosis of the Spike coated beads was evaluated by flow cytometry.

Figure 30 shows the identification and characterization of viral escape mutations to P2G3 and P5C3.

Figure 31 shows that P2G3 LS confers potent in vivo efficacy in the non-human primate (NHP) challenge model for Omicron BA.l SARS-CoV-2 infection.

Figure 32 shows that P2G3 LS + P5G3 LS combination exerts a strong in vivo therapeutic efficacy in the non-human primate Omicron SARS-CoV-2 challenge model.

DETAILED DESCRIPTION OF THE INVENTION

All, documents, patents, patent applications, publications, product descriptions, and protocols which are cited throughout this application are incorporated herein by reference in their entireties for all purposes. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. In addition, the materials, methods, and examples are illustrative only and are not intended to be limiting.

In the case of conflict, the present specification, including definitions, will control. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the subject matter herein belongs. As used herein, the following definitions are supplied in order to facilitate the understanding of the present invention.

The term “comprise” is generally used in the sense of include, that is to say permitting the presence of one or more features or components. Also as used in the specification and claims, the language "comprising" can include analogous embodiments described in terms of "consisting of “ and/or "consisting essentially of’. As used in the specification and claims, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise.

As used in the specification and claims, the term "and/or" used in a phrase such as "A and/or B" herein is intended to include "A and B", "A or B", "A", and "B".

As used herein, the terms "subject" and “patient” are well-recognized in the art, and, are used herein to refer to a mammal, and most preferably a human. In some embodiments, the subject is a subject in need of treatment and/or a subject being infected by a SARS-CoV-2 virus and/or a subject that should be protected from a SARS-CoV-2 virus infection. The term does not denote a particular age or sex. Thus, individuals of all ages, from newborn to adult, whether male or female, are intended to be covered.

As used herein, the term a "neutralizing antibody" means an immunoglobulin, antigen-binding fragment, or derivative thereof, that specifically binds and recognizes a SARS-CoV-2 Spike protein and/or an epitope on the RBD, an antigenic fragment thereof, or a dimer or multimer of the antigen. The "neutralizing antibody" is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms "neutralizing antibody" and "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. These antibodies can be used alone, or in combination, as prophylactic or therapeutic agents upon appropriate formulation, in association with active vaccination, as a diagnostic tool, or as a production tool as described herein. The term "antibody" is used herein in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments, so long as they exhibit the desired antigen-binding activity. Non-limiting examples of antibodies include, for example, intact immunoglobulins and variants and fragments thereof that retain binding affinity for the antigen. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (such as scFv); and multispecific antibodies formed from antibody fragments. Antibody fragments include antigen binding fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies. As used herein, an "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g. scFv); and multispecific antibodies formed from antibody fragments.

As used herein, an "effective amount" of an agent, e.g., a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. An effective amount can be provided in one or more administrations.

As used herein, a "therapeutically effective amount" is at least the minimum concentration required to effect a measurable improvement of a particular disorder (e.g., SARS-CoV-2 infection). A therapeutically effective amount herein may vary according to factors such as the disease state, age, sex, and weight of the patient, and the ability of the anti-SARS-CoV-2 antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the anti-SARS-CoV-2 antibody are outweighed by the therapeutically beneficial effects.

As used herein, a "prophylactically effective amount" refers to an amount effective, at the dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, a prophylactically effective amount may be less than a therapeutically effective amount.

As used herein, the terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. As used herein, the term "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention designed to alter the natural course of the individual or cell being treated during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease. In some embodiments, the disease is an SARS-CoV-2-associated disease. In some embodiments, the SARS-CoV-2-associated disease is SARS-CoV-2 infection. An individual is successfully "treated", for example, if one or more symptoms associated with SARS-CoV-2 infection are mitigated or eliminated.

As used herein, the term "prevention" or "prophylaxis" includes providing prophylaxis with respect to occurrence or recurrence of a disease in an individual. An individual may be predisposed to, susceptible to a SARS-CoV-2-associated disorder, or at risk of developing a SARS-CoV-2-associated disorder, but has not yet been diagnosed with the disorder. In some embodiments, a SARS-CoV-2-associated disorder is SARS-CoV-2 infection. In some embodiments, a SARS-CoV-2-associated disorder includes fever, cough, shortness of breath and myalgia or fatigue.

As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

SARS-CoV-2 is an enveloped virus, wherein the viral envelope is typically made up of three proteins that include the membrane protein (M), the envelope protein (E), and the spike protein (S). As compared to the M and E proteins that are primarily involved in virus assembly, the S protein plays a crucial role in penetrating host cells and initiating infection. One of the key biological characteristics of SARS-CoV-2 is the presence of spike proteins that allow these viruses to penetrate host cells through cell receptor proteins, such as angiotensin-converting enzyme 2 (ACE-2) receptor and cause infection. The S protein is a highly glycosylated and large type I transmembrane fusion protein that is made up of 1,160 to 1,400 amino acids, depending upon the type of virus. In addition to its role in penetrating cells, the S protein of the SARS-CoV-2 virus is a major inducer of neutralizing antibodies. Coronavirus S (spike) protein is initially synthesized as a precursor protein. Individual precursor S polypeptides form a homotrimer and undergo glycosylation within the Golgi apparatus as well as processing to remove the signal peptide, and cleavage by a cellular protease to generate separate SI and S2 polypeptide chains, which remain associated as S1/S2 protomers within the homotrimer and is therefore a trimer of heterodimers. The SI subunit is distal to the virus membrane and contains the receptor-binding domain (RBD) that mediates virus attachment to its host (cell) receptor. The S2 subunit contains fusion protein machinery, such as the fusion peptide, two heptad-repeat sequences (HR1 and HR2) and a central helix typical of fusion glycoproteins, a transmembrane domain, and the cytosolic tail domain. A structural conformation adopted by the ectodomain of the coronavirus S protein following processing into a mature coronavirus S protein in the secretory system, and prior to triggering of the fusogenic event that leads to transition of coronavirus S to the postfusion conformation.

The present disclosure provides a panel of antibodies that bind the SARS-CoV-2 RBD and/or Spike protein or fragment thereof and have potent neutralizing activity against the SARS-CoV- 2 virus. These antibodies could form the basis of a monotherapy or combination (cocktail) therapy consisting of two or more antibodies for use in prophylactic protection of individuals from SARS-CoV-2 infection and/or therapeutic agents that could ameliorate the clinical outcome of individuals already infected with the SARS-CoV-2 virus.

An aspect of the present invention provides an anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, comprising a heavy chain variable region (VH) that comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) domains; and a light chain variable region (VL) that comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) domains, wherein the HCDR1 sequence is SEQ ID NO: 107, the HCDR2 sequence is selected from SEQ ID NO: 108, 181-191, and the HCDR3 sequence is selected from SEQ ID NO: 109, 192-198; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively (antibody P2G3). According to an embodiment, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein the heavy chain variable region HCDR1, HCDR2, and HCDR3 sequences are as set forth in SEQ ID NO: 107, SEQ ID NO: 108, and SEQ ID NO: 109, respectively; and the light chain variable region LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 110, SEQ ID NO: 111, and SEQ ID NO: 112, respectively.

According to a further embodiment, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 107-109, 181- 198, and wherein the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 110-112.

According to another embodiment, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein the heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 107-109, and wherein the light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 110-112.

A further aspect of the present disclosure provides an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof comprising a heavy chain variable region amino acid sequence comprising or consisting of the amino acid sequence selected from SEQ ID NO: 105 and SEQ ID NO: 163-180, and a light chain variable region amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 106.

According to an embodiment, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof comprising a heavy chain variable region amino acid sequence comprising or consisting of the amino acid sequence SEQ ID NO: 105, and a light chain variable region amino acid sequence comprising or consisting of the amino acid sequence of SEQ ID NO: 106. According to a further embodiment, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof comprising a human heavy chain variable (VH) region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NO: 105 and SEQ ID NO: 163-180, and a human light chain variable (VL) region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 106.

According to another embodiment, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof comprising a human heavy chain variable (VH) region comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence SEQ ID NO: 105, and a human light chain variable (VL) region that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 106.

In another aspect, the present disclosure provides an anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, comprising a heavy chain variable region (VH) that comprises a heavy chain CDR1 (HCDR1), a heavy chain CDR2 (HCDR2), and a heavy chain CDR3 (HCDR3) domains; and a light chain variable region (VL) that comprises a light chain CDR1 (LCDR1), a light chain CDR2 (LCDR2), and a light chain CDR3 (LCDR3) domains, wherein: a) the HCDR1 sequence is selected from SEQ ID NO: 27, 135-138, the HCDR2 sequence is selected from SEQ ID NO: 28, 139-150, and the HCDR3 sequence is selected from SEQ ID NO: 29, 157-162; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively (antibody P5C3); b) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 51, SEQ ID NO: 52, and SEQ ID NO: 53, respectively (antibody P1G17); c) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 24, SEQ ID NO: 25, and SEQ ID NO: 26, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 54, SEQ ID NO: 55, and SEQ ID NO: 56, respectively (antibody P7K18); d) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 30, SEQ ID NO: 31, and SEQ ID NO: 32, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 60, SEQ ID NO: 61, and SEQ ID NO: 62, respectively (antibody P2B11); e) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 63, SEQ ID NO: 64, and SEQ ID NO: 65, respectively (antibody P1H23); f) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 36, SEQ ID NO: 37, and SEQ ID NO: 38, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 66, SEQ ID NO: 67, and SEQ ID NO: 68, respectively (antibody P6E16); g) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 39, SEQ ID NO: 40, and SEQ ID NO: 41, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively (antibody PI 06); h) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 42, SEQ ID NO: 43, and SEQ ID NO: 44, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 72, SEQ ID NO: 73, and SEQ ID NO: 74, respectively (antibody P1M12); i) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 45, SEQ ID NO: 46, and SEQ ID NO: 47, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 75, SEQ ID NO: 76, and SEQ ID NO: 77, respectively (antibody P1L7); j) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 48, SEQ ID NO: 49, and SEQ ID NO: 50, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 78, SEQ ID NO: 79, and SEQ ID NO: 80, respectively (antibody P1L4); k) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 87, SEQ ID NO: 88, and SEQ ID NO: 89, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98, respectively (antibody MS31); l) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively (antibody MS35); m) the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 93, SEQ ID NO: 94, and SEQ ID NO: 95, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 102, SEQ ID NO: 103, and SEQ ID NO: 104, respectively (antibody MS42).

In an embodiment, the present disclosure provides an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 27, SEQ ID NO: 28, and SEQ ID NO: 29, respectively; the LCDR1, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 57, SEQ ID NO: 58, and SEQ ID NO: 59, respectively (antibody P5C3).

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the heavy chain variable (VH) region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 1 - 10, 81 - 83 and 113-134, and wherein the light chain variable (VL) region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NOs: 11-20 and 84-86. In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises or consists of an amino acid sequence selected from SEQ ID NOs: 1 - 10, 81 - 83, and 113-134 and wherein the VL region comprises or consists of an amino acid sequence selected from SEQ ID NOs: 11-20 and 84-86.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of one of SEQ ID NO: 3 and 113-134 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH region comprises or consists of the amino acid sequence of one of SEQ ID NO: 3 and 113-134 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 3 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 133 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 134 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 13. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P5C3. In some embodiments, the present disclosure provides an anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein the human heavy chain variable (VH) region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO: 133 and SEQ ID NO: 134, and the human light chain variable (VL) region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 13.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 1 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 1 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 11. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P1G17.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 2 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 12. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 2 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 12. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P7K18.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 4 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 16. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 4 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 16. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P2B11.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 5 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 14. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 5 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 14. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P1H23.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 6 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 15. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 6 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 15. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P6E16.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 7 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 17. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 7 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 17. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as PI 06.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 8 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 18. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 8 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 18. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P1M12.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 9 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 19. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 9 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 19. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P1L7.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 10 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 20. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 10 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 20. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as P1L4. In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 81 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 84. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 84. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as MS31.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 82 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 85. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 85. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as MS35.

In some embodiments, the present disclosure provides an anti-SARS-CoV-2 antibody, or antigen binding fragment thereof, wherein the VH region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 83 and wherein the VL region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ ID NO: 86. In some embodiments, the VH region comprises or consists of the amino acid sequence of SEQ ID NO: 83 and the VL region comprises or consists of the amino acid sequence of SEQ ID NO: 86. In some embodiments, such an antibody, or antigen binding fragment thereof, is referred to herein as MS42.

In some embodiments, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein: a. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 3 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 13; b. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 133 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 13; c. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 134 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 13; d. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 1 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 11; e. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 2 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 12; f. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 4 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 16; g. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 5 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 14; h. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 6 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 15; i. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 7 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 17; j. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 8 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 18; k. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 9 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 19; l. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 10 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 20; m. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 81 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 84; n. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 82 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 85; or o. the heavy chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 83 and the light chain variable region amino acid sequence comprises or consists of the amino acid sequence of SEQ ID NO: 86.

In some embodiments, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein a. the human heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 3, and the human light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; b. the human heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

133, and the human light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13; c. the human heavy chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO:

134, and the human light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13.

In some embodiments, the present disclosure provides the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, wherein a. the human heavy chain variable region amino acid sequence comprises or consists of SEQ ID NO: 3, and the human light chain variable region amino acid sequence comprises or consists of SEQ ID NO: 13; b. the human heavy chain variable region amino acid sequence comprises or consists of SEQ ID NO: 133, and the human light chain variable region amino acid sequence comprises or consists of SEQ ID NO: 13; c. the human heavy chain variable region amino acid sequence comprises or consists of SEQ ID NO: 134, and the human light chain variable region amino acid sequence comprises or consists of SEQ ID NO: 13. SEQ ID No. 106: (P2G3)

DIQLTQSPSFLSASVGDRVTVTCRASQGISSYVAWYQQKAGKAPTLLIYTASTLQSG VP SRF SGSGSGTEFTLTIS SLQPEDFAT YYCQQLHS YP VTFGQGTRLDIER

Table 1 : Heavy chain CDR sequences for anti-SARS-CoV-2 antibodies

Table 2 : Light chain CDR sequences for anti-SARS-CoV-2 antibodies

In one embodiment, the anti-SARS-CoV-2 antibody of the invention is an isolated monoclonal antibody.

In another embodiment, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention exhibits neutralization of SARS-CoV-2 Spike pseudotyped lentivirus and/or the SARS-CoV-2 live virus at a concentration less than 10 μg/ml.

In another embodiment, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention is derived from a human antibody, human IgG, human IgGl, human IgG2, human IgG2a, human IgG2b, human IgG3, human IgG4, human IgM, human IgA, human IgAl, human IgA2, human IgD, human IgE, canine antibody, canine IgGA, canine IgGB, canine IgGC, canine IgGD, chicken antibody, chicken IgA, chicken IgD, chicken IgE, chicken IgG, chicken IgM, chicken IgY, goat antibody, goat IgG, mouse antibody, mouse IgG, pig antibody, and rat antibody.

In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention is selected from a human antibody, a canine antibody, a chicken antibody, a goat antibody, a mouse antibody, a pig antibody, a rat antibody, a shark antibody, a camelid antibody.

In some other embodiments of the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention : the antibody is a human antibody selected from a human IgG (including human IgGl, human IgG2, human IgG2a, human IgG2b, human IgG3, and human IgG4), a human IgM, a human IgA (including human IgAl and human IgA2), a human IgD, and a human IgE, the antibody is a canine antibody selected from a canine IgGA, a canine IgGB, a canine IgGC, a canine IgGD, the antibody is a chicken antibody selected from a chicken IgA, a chicken IgD, a chicken IgE, a chicken IgG, a chicken IgM, and a chicken IgY, the antibody is a goat antibody including a goat IgG, the antibody is a mouse antibody including a mouse IgG.

In some other embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention is a mono-specific antibody, a bispecific antibody, a trimeric antibody, a multi-specific antibody, or a multivalent antibody.

In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention is a humanized antibody, a caninized antibody, a chimeric antibody (including a canine-human chimeric antibody, a canine-mouse chimeric antibody, and an antibody comprising a canine Fc), or a CDR-grafted antibody.

In some embodiments of the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention, the antigen binding fragment is selected from the group consisting of an Fab, an Fab2, an Fab’ single chain antibody, an Fv, a single chain variable fragment (scFv), and a nanobody . Another aspect of the present disclosure provides a derivative of the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention, wherein the derivative is selected from the group consisting of an Fab, Fab2, Fab’ single chain antibody, Fv, single chain, mono-specific antibody, bispecific antibody, trimeric antibody, multi-specific antibody, multivalent antibody, chimeric antibody, canine-human chimeric antibody, canine-mouse chimeric antibody, antibody comprising a canine Fc, humanized antibody, human antibody, caninized antibody, CDR-grafted antibody, shark antibody, nanobody, and canelid antibody.

In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention, or the derivative of the invention, further comprising a detectable label fixably attached thereto, wherein the detectable label is selected from the group consisting of fluorescein, DyLight, Cy3, Cy5, FITC, HiLyte Fluor 555, HiLyte Fluor 647, 5-carboxy-2,7- dichlorofluorescein, 5 -carboxy fluorescein, 5-FAM, hydroxy tryptamine, 5 -hydroxy tryptamine (5-HAT), 6-carboxyfluorescein (6-FAM), FITC, 6-carboxy-l,4-dichloro-2’,7’- di chi oroHl uorescei n (TET), 6-carboxy- 1 ,4-di chi oro-2 ’ ,4 ’ , 5 ’ , 7 ’ -tetra^chl orofl uorescei n (HEX), 6-carboxy -4’, S’-dichloro^’^’-dimethoxy-fluorescein (6-JOE), an Alexa fluor, Alexa fluor 350, Alexa fluor 405, Alexa fluor 430, Alexa fluor 488, Alexa fluor 500, Alexa fluor 514, Alexa fluor 532, Alexa fluor 546, Alexa fluor 555, Alexa fluor 568, Alexa fluor 594, Alexa fluor 610, Alexa fluor 633, Alexa fluor 635, Alexa fluor 647, Alexa fluor 660, Alexa fluor 680, Alexa fluor 700, Alexa fluor 750, a BODIPY fluorophores, BODIPY 492/515, BODIPY 493/503, BODIPY 500/510, BODIPY 505/515, BODIPY 530/550, BODIPY 542/563, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650-X, BODIPY 650/665-X, BODIPY 665/676, FL, FL ATP, Fl-Ceramide, R6G SE, TMR, TMR-X conjugate, TMR-X, SE, TR, TR ATP, TR-X SE, a rhodamine, rhodamine 110, rhodamine 123, rhodamine B, rhodamine B 200, rhodamine BB, rhodamine BG, rhodamine B extra, 5-carboxytetramethylrhodamine (5-TAMRA), 5 GLD, 6-carboxyrhodamine 6G, Lissamine, Lissamine Rhodamine B, Phallicidine, Phalloidine, rhodamine red, Rhod-2, 6- carboxy-X-rhodamine (ROX), carboxy-X-rhodamine (5-ROX), Sulphorhodamine B can C, Sulphorhodamine G Extra, 6-carboxytetram ethyl ^“ rhodamine (TAMRA), tetramethylrhodamine (TRITC), rhodamine WT, Texas Red, and Texas Red-X.

In another aspect, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention comprises a heavy chain variable region (VH) sequence and/or a light chain variable region (VL) sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 81, 82, 83, 105, 113 to 134, 163 to 179 or 180 and/or SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 84, 85, 86 or 106. In certain embodiments, a VH sequence and/or VL sequence having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (such as conservative substitutions), insertions, or deletions relative to the reference sequence, but a anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising that sequence retains the ability to bind to SARS-CoV-2 virus (via for example RBD, Spike protein or fragment thereof). In certain embodiments, a total of 1 to 10 amino acids, such as 1, 2, or 3 amino acids have been substituted, inserted and/or deleted in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 81, 82, 83, 105, 113 to 134, 163 to 179 or 180 and/or in SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 84, 85, 86 or 106. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (for example in the FRs). Optionally, the anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprises the VH sequence and/or VL sequences SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 81, 82, 83, 105, 113 to 134, 163 to 179 or 180 and/or SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 84, 85, 86 or 106, including post-translational modifications of that sequence.

In another aspect, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention comprises a heavy chain variable region that comprises CDR1, CDR2, and CDR3 domains sequences and/or a light chain variable region that comprises CDR1, CDR2, and CDR3 domains sequences having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of one or more SEQ ID NOs: 21 to 80, 87 to 104, 107 to 112, 135 to 150, 157 to 162, and 181 to 198. In certain embodiments, the CDR domains sequences having at least 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity contains substitutions (such as conservative substitutions), insertions, or deletions relative to the reference sequence, but a anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising that sequence retains the ability to bind to SARS-CoV-2 virus (via for example RBD, Spike protein or fragment thereof). In certain embodiments, a total of 1 to 10 amino acids, such as 1, 2 or 3 amino acids, have been substituted, inserted and/or deleted in one or more SEQ ID NOs: 21 to 80, 87 to 104, 107 to 112, 135 to 150, 157 to 162, and 181 to 198. In certain embodiments, substitutions, insertions, or deletions occur in regions outside the HVRs (for example in the FRs). Optionally, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprises the CDR domains sequences SEQ ID NOs: 21 to 80, 87 to 104, 107 to 112, 135 to 150, 157 to 162, and 181 to 198, including post-translational modifications of that sequence.

In certain embodiments, an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention has a dissociation constant (Kd) of <0.1 nM between the P2G3 antibody and either the Omicron BA.1 or Omicron BA.2 Spike trimer recombinant protein. The estimated Kon rate for P2G3 is 5 x 10 ⁵ s ^"1 IYT'and Koff rate of <5 x 10 ^"5 s ^"1 .

In certain embodiments, an anti-SARS-CoV-2 antibody of the invention is an antibody fragment. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, Fv, and scFv fragments. Diabodies are antibody fragments with two antigen-binding sites that may be bivalent or bispecific. See, for example, EP 404,097; WO 1993/01161; Hudson et al., Nat. Med. 9: 129-134 (2003); and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are also described in Hudson et al., Nat. Med. 9: 129-134 (2003). Single-domain antibodies are antibody fragments comprising all or a portion of the heavy chain variable domain or all or a portion of the light chain variable domain of an antibody. In certain embodiments, a single-domain antibody is a human single-domain antibody. Antibody fragments can be made by various techniques, including but not limited to proteolytic digestion of an intact antibody as well as production by recombinant host cells (e.g. E. cob or phage).

In certain embodiments, an anti-SARS-CoV-2 antibody of the invention is a chimeric antibody. In one example, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse, rat, hamster, rabbit, or non-human primate, such as a monkey) and a human constant region. In a further example, a chimeric antibody is a "class switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof. In certain embodiments, a chimeric antibody is a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity to humans, while retaining the specificity and affinity of the parental non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which HVRs, such as CDRs, (or portions thereof) are derived from a non-human antibody, and FRs (or portions thereof) are derived from human antibody sequences. A humanized antibody optionally will also comprise at least a portion of a human constant region. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (such as the antibody from which the HVR residues are derived), for example to restore or improve antibody specificity or affinity.

In certain embodiments, an anti-SARS-CoV-2 antibody provided herein is a human antibody. Human antibodies can be produced using various techniques known in the art. Human antibodies may be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigenic challenge. Such animals typically contain all or a portion of the human immunoglobulin loci, which replace the endogenous immunoglobulin loci, or which are present extrachromosomally or integrated randomly into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci have generally been inactivated. Human variable regions from intact antibodies generated by such animals may be further modified, e.g., by combining with a different human constant region. Human antibodies can also be made by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described. Human antibodies generated via human B-cell hybridoma technology are also know in the art. Human antibodies may also be generated by isolating Fv clone variable domain sequences selected from human- derived phage display libraries. Such variable domain sequences may then be combined with a desired human constant domain.

In certain embodiments, an anti-SARS-CoV-2 antibody of the invention is a multispecific antibody, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites. In certain embodiments, bispecific antibodies may bind to two different epitopes of SARS-CoV-2 virus, such as the amino acid loops on the RBD that form the major contact sites with the ACE-2 receptor and a second epitope that may be non-overlapping with the first on the RBD, SI domain or within any regions of the Spike trimer. It is conceivable that in binding to two different epitopes on the Spike trimer simultaneously, the resulting bispecific will have an enhanced binding affinity, enhanced neutralization activity and/or greater potential in neutralizing viruses that encode variant amino acid residues within the Spike protein. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments. Techniques for making multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain - light chain pairs having different specificities (known in the art), and "knob-in-hole" engineering (also known in the art, see for example U.S. Patent No. 5,731,168). Multispecific antibodies may also be made by engineering electrostatic steering effects for making antibody Fc-heterodimeric molecules (see for example WO 2009/089004A1); cross-linking two or more antibodies or fragments (see for example US Patent No. 4,676,980); using leucine zippers to produce bispecific antibodies; using "diabody" technology for making bispecific antibody fragments; and using single-chain Fv (sFv) dimers; and preparing trispecific antibodies. Engineered antibodies with three or more functional antigen binding sites, including "Octopus antibodies," are also included herein (see for example US 2006/0025576A1). The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention also includes a "Dual Acting FAb" or "DAF" comprising an antigen binding site that binds to Spike protein as well as another, different antigen. In this regard, a DAF could be generated using an anti-SARS-CoV-2 antibody described herein combined with an ACE-2 binding antibody fragment that would be capable of blocking the interaction between the viral Spike and ACE-2 receptor used by the virus to enter and infect host target cells.

In some embodiments, the anti-SARS-CoV-2 antibodies described herein demonstrate advantageous properties over other anti-SARS-CoV2 antibodies described in the art. In some embodiments, the anti-SARS-CoV-2 antibodies described herein demonstrate improved affinity for a SARS-CoV-2 virus compared to antibodies described in the art (See e.g. , Example 3). In some embodiments, the anti-SARS-CoV-2 antibodies described herein demonstrate improved neutralization of SARS-CoV-2 (See e.g., Examples 4 and 8). In some embodiments, the anti-SARS-CoV-2 antibodies described herein demonstrate improved disruption of the interaction between the SARS-CoV-2 virus and the ACE-2 receptor (See e.g, Example 9).

In some embodiments, the anti-SARS-CoV-2 antibodies, such as P2G3 antibody, described herein demonstrate improved neutralization of a SARS-CoV-2 virus compared to anti-SARS- CoV2 antibodies known in the art. In some embodiments, the anti-SARS-CoV-2 antibodies, or an antigen-binding fragments thereof, such as P2G3 antibody, exhibit an in vitro neutralization IC50 of a SARS-CoV-2 virus at a concentration less than 10 pg/mL. In some embodiments, the anti-SARS-CoV-2 antibodies, or an antigen-binding fragment thereof, such as P2G3 antibody, exhibit an in vitro neutralization IC50 of a SARS-CoV-2 virus of less than 25 ng/mL, less than 20 ng/mL, less than 15 ng/mL, less than 10 ng/mL, or less than 5 ng/mL. In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention, such as P2G3 antibody, exhibits an in vitro neutralization IC50 of a SARS- CoV-2 virus of between 4 ng/mL and 21 ng/mL, between 4 ng/mL and 14 ng/mL, between 4 ng/mL and 11 ng/mL, between 4 ng/mL and 8 ng/mL, between 5 ng/mL and 15 ng/mL, between 5 ng/mL and 10 ng/mL, between 5 ng/mL and 8 ng/mL, or between 5 ng/mL and 7 ng/mL. In some embodiments, anti-SARS-CoV-2 antibodies, or an antigen-binding fragment thereof, described herein, such as P2G3 antibody, exhibit an in vitro neutralization IC50 of a SARS- CoV-2 virus of about 4 ng/mL, 5 ng/mL, 6 ng/mL, 7 ng/mL, 8 ng/mL, 9 ng/mL, 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, or 22 ng/mL.

In some embodiments, the anti-SARS-CoV-2 antibodies, such as P2G3 antibody, described herein demonstrate improved affinity for a SARS-CoV-2 virus compared to antibodies described in the art. In some embodiments, the anti-SARS-CoV-2 antibodies, or an antigenbinding fragments thereof, such as P2G3 antibody, described herein exhibit an in vitro affinity IC80 for the SARS-CoV-2 spike protein of between 10 and 40 ng/mL. In some embodiments, the IC80 is between 10 ng/mL and 35 ng/mL, between 10 ng/mL and 28 ng/mL, between 10 ng/mL and 24 ng/mL, between 10 ng/mL and 21 ng/mL, between 10 ng/mL and 19 ng/mL, between 16 ng/mL and 36 ng/mL, between 16 ng/mL and 25 ng/mL, between 16 ng/mL and 20 ng/mL, or between 16 ng/mL and 19 ng/mL. In some embodiments, the anti-SARS-CoV-2 antibodies, or an antigen-binding fragments thereof, such as P2G3 antibody, described herein exhibit an in vivo affinity IC80 for the SARS-CoV-2 spike protein of less than 35 ng/mL, less than 28 ng/mL, less than 24 ng/mL, less than 21 ng/mL, less than 19 ng/mL, or less than 17 ng/mL. In some embodiments, the in vivo IC80 is about 10 ng/mL, 11 ng/mL, 12 ng/mL, 13 ng/mL, 14 ng/mL, 15 ng/mL, 16 ng/mL, 17 ng/mL, 18 ng/mL, 19 ng/mL, 20 ng/mL, 21 ng/mL, 22 ng/mL, 23 ng/mL, 24 ng/mL, 25 ng/mL, 26 ng/mL, 27 ng/mL, 28 ng/mL, 29 ng/mL, 30 ng/mL, 31 ng/mL, 32 ng/mL, 33 ng/mL, 34 ng/mL, 35 ng/mL, 36 ng/mL, 37 ng/mL, 38 ng/mL, 39 ng/mL, or 40 ng/mL.

In some embodiments, the neutralization capability and/or affinity of an anti-SARS-CoV2 antibody described herein is determined by binding to a coronavirus spike protein. In some embodiments, the spike protein is displayed as part of a lentivirus pseudotyped with the SARS- CoV2 spike protein. In some embodiments, the spike protein is part of a live SARS-CoV-2 virus. In some embodiments, the live SARS-CoV-2 virus is selected from wild type SARS- CoV-2 or a variant of SARS-CoV-2 selected from Alpha/B.1.1.7, Beta/B.1.351, Mink variant 16, Gamma/P.1, Delta/Bl.617.2, Omicron BA.l/B.1.1.529, Omicron BA.2/B.1.1.529.2, CAL.C20, C.37, and B.1.621; preferably the live SARS-CoV-2 virus is selected from wild type SARS-CoV-2 or a variant of SARS-CoV-2 selected from Alpha/B.1.1.7, Beta/B.1.351, Mink variant 16, Gamma/P.1, Delta/Bl.617.2, Omicron BA.l/B.1.1.529, Omicron BA.2/B.1.1.529.2.

A neutralizing antibody may be one that exhibits the ability to neutralize, or inhibit, infection of cells by the SARS-CoV-2 virus. In general, a neutralization assay typically measures the loss of infectivity of the virus through reaction of the virus with specific antibodies. Typically, a loss of infectivity is caused by interference by the bound antibody with any of the virus replication steps including but not limited to binding to target cells, entry, and/or viral release. The presence of un-neutralized virus is detected after a predetermined amount of time, for example one, two, three, four, five, six, seven, eight, nine, 10, 12 or 14 days, by measuring the infection of target cells using any of the systems available to the person skilled on the art (for example a luciferase-based system or a cytopathic effect infection assay).

A non-limiting example of a neutralization assay may include combining a given amount of a virus or a SARS-CoV-2 Spike pseudotyped virus (see below) and different concentrations of the test or control (typically positive and negative controls assayed separately) antibody or antibodies are mixed under appropriate conditions (for example one (1) hour at room temperature) and then inoculated into an appropriate target cell culture (for example Vero cells or 293T ACE-2 stable cell line). For instance, the neutralizing antibody-producing cells (for example B cells producing antibodies) may be assayed for the production of SARS-CoV-2 Spike or RBD antibodies by seeding such cells in separate plates as single cell micro-cultures on human feeder cells in the presence of Epstein-Barr Virus (EBV) (which also stimulate polyclonally memory B cells), a cocktail of growth factors (for example TLR9 agonist CpG- 2006, IL-2 (1000 IU/ml), IL-6 (10 ng/ml), IL-21 (10 ng/ml), and anti-B cell receptor (BCR) goat antibodies (which trigger BCRs). After an appropriate time (e.g., 14 days), supernatants of such cultures may tested in a primary binding assay (e.g. Luminex assay using Spike trimer coupled beads) and a cell based neutralization assays to monitor B cell clones that produce antibodies capable of preventing viruses or pseudoviruses from productively infecting a target cell. The pseudoviruses may be incubated with B cell culture supernatants for an appropriate time and temperature (for example one (1) h at 37% (5% CO2)) before the addition of host cells (for example 3000 293T ACE-2 stable cells). Incubation for an appropriate time (for example 72 hours) may then follow, after which the supernatant may be removed and Steadylite reagent (Perkin Elmer) added (for example 15 mΐ). Luciferase activity may then be determined (for example five minutes later) on a Synergy microplate luminometer (BioTek). Decreased luciferase activity relative to a negative control typically indicates virus neutralization. Neutralization assays such as these, suitable for analyzing the neutralizing antibodies, or antigen-binding fragments thereof the neutralizing antibody, or an antigenbinding fragment thereof (binding agents) of this disclosure, are known in the art (see, e.g., Crawford et al Viruses. 2020 May 6; 12(5):513. and Nie et al, Nat Protoc. 2020 Nov;15(ll):3699-3715). In some embodiments, neutralization may be determined as a measure of the concentration (for example μg/ml) of monoclonal antibody capable of neutralizing any of about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of viral infection (as may be measured by percent neutralization and/or by determining an “IC ₅₀ ” and/or “ICxo” value).

In some embodiments, an antibody, or an antigen-binding fragment thereof may be considered neutralizing if it is able to neutralize 50% of viral infection at a concentration of, for instance, about any of 10 ^"5 , 10 ^"4 , 10 ^"3 , 10 ^"2 , 10 ^'1 , 10°, 10 ¹ , 10 ² , or 10 ³ μg/ml (e.g., an IC ₅₀ value as shown in Figures 2 and 3). In some embodiments, the ability of a neutralizing antibody to neutralize viral infection may be expressed as a percent neutralization (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% (e.g., as in Figures 2 and 3)). And in some embodiments, as in the Examples herein, the ability of a neutralizing antibody to neutralize viral infection may be expressed as, and, in preferred embodiments, the IC ₅₀ and/or ICso value is below 25 μg/ml, and is even more preferably below about any of 15, 10, 5, 2, 1, 0.5, 0.25, 0.1, 0.05, or 0.01 μg/ml (see, e.g., Figures 2 and 3). Other measures of neutralization may also be suitable as may be determined by those of ordinary skill in the art.

Another aspect of the present invention provides an anti- SARS-CoV-2 antibody, or an antigenbinding fragment thereof, such as P2G3 antibody, wherein the antibody or antigen-binding fragment thereof specifically binds to an epitope in the SARS-CoV-2 Spike protein, wherein the epitope comprises at least one amino acid in the Spike protein RBD selected from Asn343, Ala344, Thr345, Arg346, Asn440, Leu441, Asp442, Ser443, Lys444, Val445, Gly446, Gly447, Asn448, Tyr449, Asn450, Tyr451 in SEQ ID NO: 199. In some embodiments, the epitope comprises each of Asn343, Ala344, Thr345, Arg346, Asn440, Leu441, Asp442, Ser443, Lys444, Val445, Gly446, Gly447, Asn448, Tyr449, Asn450, Tyr451 in SEQ ID NO: 199. In some embodiments, the epitope comprises amino acids 343-346 and/or 44045 lof SEQ ID NO: 199.

In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, such as P2G3 antibody, neutralizes SARS-CoV-2 in an in vitro and/or in vivo SARS-CoV-2 neutralization assay and/or specifically binds to an epitope in the SARS-CoV-2 Spike protein that comprises at least one amino acid in the Spike protein selected from Asn343, Ala344, Thr345, Arg346, Asn440, Leu441, Asp442, Ser443, Lys444, Val445, Gly446, Gly447, Asn448, Tyr449, Asn450, Tyr451 in SEQ ID NO: 199. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, such as P2G3 antibody, neutralizes SARS-CoV- 2 in an in vitro and/or in vivo SARS-CoV-2 neutralization assay and/or specifically binds to an epitope in the SARS-CoV-2 Spike protein that comprises each of Asn343, Ala344, Thr345, Arg346, Asn440, Leu441, Asp442, Ser443, Lys444, Val445, Gly446, Gly447, Asn448, Tyr449, Asn450, Tyr451 in SEQ ID NO: 199.

In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, such as P2G3 antibody, has a greater affinity for a SARS-CoV-2 spike protein compared to previously described anti-SARS-CoV2 antibodies and specifically binds to an epitope in the SARS-CoV-2 Spike protein that comprises at least one amino acid in the Spike protein selected from Asn343, Ala344, Thr345, Arg346, Asn440, Leu441, Asp442, Ser443, Lys444, Val445, Gly446, Gly447, Asn448, Tyr449, Asn450, Tyr451 in SEQ ID NO: 199. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof, such as P2G3 antibody, has a greater affinity for a SARS-CoV-2 spike protein compared to previously described anti- SARS-CoV2 antibodies and specifically binds to an epitope in the SARS-CoV-2 Spike protein that comprises each of Asn343, Ala344, Thr345, Arg346, Asn440, Leu441, Asp442, Ser443, Lys444, Val445, Gly446, Gly447, Asn448, Tyr449, Asn450, Tyr451 in SEQ ID NO: 199.

In certain embodiments, amino acid sequence variants of the anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of the antibody. Amino acid sequence variants of an antibody may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the antibody. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, such as antigen-binding.

In certain embodiments, anti-SARS-CoV-2 antibody variants, or antigen binding fragments thereof variants, having one or more amino acid substitutions are provided herein. Sites of interest for substitutional mutagenesis include theHVRs and FRs. More substantial changes are provided in Table A under the heading of "exemplary substitutions" and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into an antibody of interest and the products screened for a desired activity, for example, retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

Table A Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, lie;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class. One type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (such as a humanized or human antibody). Generally, the resulting variant(s) selected for further study will have modifications (such as improvements) in certain biological properties (for example increased affinity, reduced immunogenicity) relative to the parent antibody and/or will have substantially retained certain biological properties of the parent antibody. An exemplary substitutional variant is an affinity matured antibody, which may be conveniently generated, for example, using phage display-based affinity maturation techniques such as those described herein. Briefly, one or more HVR residues are mutated and the variant antibodies displayed on phage and screened for a particular biological activity (such as binding affinity). Alterations (such as substitutions) may be made in HVRs, for example, to improve antibody affinity. Such alterations may be made in HVR "hotspots," i.e., residues encoded by codons that undergo mutation at high frequency during the somatic maturation process (see for example Chowdhury, Methods Mol. Biol. 207: 179-196 (2008)), and/or SDRs (a-CDRs), with the resulting variant VH or VL being tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries has been described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178: 1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001).) In some embodiments of affinity maturation, diversity is introduced into the variable genes chosen for maturation by any of a variety of methods (such as, error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. Another method to introduce diversity involves HVR-directed approaches, in which several HVR residues (for example, 4-6 residues at a time) are randomized. HVR residues involved in antigen binding may be specifically identified, for example, using alanine scanning mutagenesis or modelling. CDR-H3 and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions may occur within one or more HVRs and/or CDRs so long as such alterations do not substantially reduce the ability of the antibody to bind antigen. For example, conservative alterations (such as conservative substitutions as provided herein) that do not substantially reduce binding affinity may be made in HVRs and/or CDRs. Such alterations may be outside of HVR "hotspots" or SDRs. In certain embodiments of the variant VH, VL and CDR sequences provided above, each HVR and/or CDR either is unaltered, or contains no more than one, two or three amino acid substitutions.

A useful method for identification of residues or regions of an antibody that may be targeted for mutagenesis is called "alanine scanning mutagenesis" as described by Cunningham and Wells (1989) Science, 244: 1081-1085. In this method, a residue or group of target residues (for example, charged residues such as Arg, Asp, His, Lys, and Glu) are identified and replaced by a neutral or negatively charged amino acid (such as alanine or polyalanine) to determine whether the interaction of the antibody with antigen is affected. Further substitutions may be introduced at the amino acid locations demonstrating functional sensitivity to the initial substitutions. Alternatively, or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact residues and neighboring residues may be targeted or eliminated as candidates for substitution. Variants may be screened to determine whether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g. for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

In certain embodiments, an anti-SARS-CoV-2 antibody, or an antigen binding fragment thereof, of the invention is altered to increase or decrease the extent to which the antibody is glycosylated. Addition or deletion of glycosylation sites to an antibody may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites is created or removed. Where the antibody comprises an Fc region, the carbohydrate attached thereto may be altered. Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region. The oligosaccharide may include various carbohydrates, such as, mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention may be made in order to create antibody variants with certain improved properties.

In one embodiment, antibody variants are provided having a carbohydrate structure that lacks fucose attached (directly or indirectly) to an Fc region. For example, the amount of fucose in such antibody may be from 1% to 80%, from 1% to 65%, from 5% to 65% or from 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297, relative to the sum of all glycostructures attached to Asn 297 (for example, complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as described in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc region (Eu numbering of Fc region residues); however, Asn297 may also be located about + 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells.

Antibodies variants are further provided with bisected oligosaccharides, for example, in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and/or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003/011878, US Patent No. 6,602,684 and US 2005/0123546. Antibody variants with at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997/30087, WO 1998/58964 and WO 1999/22764. In certain embodiments, one or more amino acid modifications may be introduced into the Fc region of an anti-SARS-CoV-2 antibody of the invention, thereby generating an Fc region variant. The Fc region variant may comprise a human Fc region sequence (such as a human IgGl, IgG2, IgG3 or IgG4 Fc region) comprising an amino acid modification (such as a substitution) at one or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the antibody in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. Alternatively, non-radioactive assays methods may be employed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, for example, in an animal model. Clq binding assays may also be carried out to confirm that the antibody is unable to bind Clq and hence lacks CDC activity. To assess complement activation, a CDC assay may be performed. FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art.

In certain embodiments, it may be desirable to create cysteine engineered antibodies, for example "thioMAbs," in which one or more residues of an antibody are substituted with cysteine residues. In particular embodiments, the substituted residues occur at accessible sites of the antibody. By substituting those residues with cysteine, reactive thiol groups are thereby positioned at accessible sites of the antibody and may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to create an immunoconjugate.

In certain embodiments, an anti-SARS-CoV-2 antibody of the invention may be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available. The moieties suitable for derivatization of the antibody include but are not limited to water soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone, poly-1, 3- dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymer, polyaminoacids (either homopolymers or random copolymers), and dextran or poly(n-vinyl pyrrolidone)polyethylene glycol, propropylene glycol homopolymers, proly propylene oxide/ethylene oxide copolymers, polyoxy ethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may be of any molecular weight, and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer are attached, they can be the same or different molecules. In general, the number and/or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative will be used in a therapy under defined conditions, etc.

In another embodiment, conjugates of an antibody and nonproteinaceous moiety that may be selectively heated by exposure to radiation are provided. In one embodiment, the nonproteinaceous moiety is a carbon nanotube. The radiation may be of any wavelength, and includes, but is not limited to, wavelengths that do not harm ordinary cells, but which heat the nonproteinaceous moiety to a temperature at which cells proximal to the antibody- nonproteinaceous moiety are killed.

As an additional distinction of the anti-SARS-CoV-2 antibodies of the invention, mutations in the antibody Fc domain were engineered to extend the in vivo half-life of these candidates. These mutations include LS (M428L/N434S), YTE (M252Y/S254T/T256E), DF215 (T307Q/Q311 V/A378V) and DF228 (T256D/H286D/T307R/Q311V/A378V) substitutions. These modified Fc antibodies are expected to extend the half-life of the antibodies by >4-fold compared to wild type IgGl antibodies which could allow for the prophylactic protect of an individual for up to 4 to 6 months with one antibody dose.

Antibody drugs with the extended in vivo half-life mutations discussed above would allow for circulating levels of antibody to remain high for up to 4 to 6 months with administration of only one therapeutic antibody dose. Given the potency of the discovered antibodies, this single dose is expected to provide an extended prophylactic protection to subjects at risk of infection. The extended half-life mutations investigated with the most potent anti-SARS-CoV-2 antibodies disclosed herein also represent a significant advantage compared to antibodies in the clinic. The mutations under investigation include LS (M428L/N434S), YTE (M252Y/S254T/T256E), DF215 (T307Q/Q311V/A378V) and DF228 (T256D/H286D/N286D/T307R/A378V) substitutions that can improve the pharmacokinetic properties of the anti-SARS-CoV-2 antibodies (extended half-life, higher Cmax, higher AUC and reduced clearance) and potentially improve some of the overall antibody stability properties. Apart from the PK considerations, the LS, DF215 and DF228 substitutions can increase the antibody dependent cytotoxicity (ADCC) and complement dependent cytotoxicity (CDC) functional activities of an antibody such that they have a greater capacity to kill cells infected with the SARS-CoV-2 virus. This increased activity may translate into an additional clinical advantage for the anti-SARS-CoV-2 antibodies of the invention.

Any method known to those of ordinary skill in the art may be used to generate the anti-SARS- CoV-2 antibodies, or antigen-binding fragments thereof, of the invention having specificity for (for example binding to) SARS-CoV-2 virus. For instance, to generate and isolate monoclonal antibodies from an animal such as a mouse may be administered (for example immunized) with one or more SARS-CoV-2 proteins. Animals exhibiting serum reactivity to SARS-CoV-2 expressed on virus infected cells (as determined by, for instance, flow cytometry and / or microscopy) may then be selected for generation of anti- SARS-CoV-2 hybridoma cell lines. This may be repeated for multiple rounds. Screening may also include, for instance, affinity binding and / or functional characterization to identify the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof (binding agent) as being specific for SARS-CoV-2. In some embodiments, such as in the Examples herein, subjects (such as humans) may be screened for the expression of antibodies against SARS-CoV-2. In some embodiments, plasma samples of subjects (such as humans) infected by SARS-CoV-2 may be screened to identify subjects expressing anti-SARS-CoV-2 antibodies, and in particular, anti-SARS-CoV-2 antibodies against the virus. Anti-SARS-CoV-2 antibody -producing cells of such subjects may then be isolated, followed by the isolation and characterization of the antibodies produced thereby (as in the Examples herein). An anti-SARS-CoV-2 antibody may be one that exhibits the ability to neutralize, or inhibit, infection of cells by the SARS-CoV-2 virus. In general, a neutralization assay typically measures the loss of infectivity of the virus through reaction of the virus with specific antibodies. Typically, a loss of infectivity is caused by interference by the bound antibody with any of the virus replication steps including but not limited to binding to target cells, entry, and/or viral release. The presence of un-neutralized virus is detected after a predetermined amount of time, for example one, two, three, four, five, six, seven, eight, nine, 10, 12 or 14 days, by measuring the infection of target cells using any of the systems available to the person skilled on the art (for example a luciferase-based system or a cytopathic effect infection assay). A non-limiting example of a neutralization assay may include combining a given amount of a virus or a SARS-CoV-2 Spike pseudotyped virus (see below) and different concentrations of the test or control (typically positive and negative controls assayed separately) antibody or antibodies are mixed under appropriate conditions (for example one (1) hour at room temperature) and then inoculated into an appropriate target cell culture (for example Vero cells or 293T ACE-2 stable cell line). For instance, the anti-SARS-CoV-2 antibody-producing cells (for example B cells producing antibodies) may be assayed for the production of SARS-CoV-2 Spike or RBD antibodies by seeding such cells in separate plates as single cell micro-cultures on human feeder cells in the presence of Epstein-Barr Virus (EBV) (which also stimulate polyclonally memory B cells), a cocktail of growth factors (for example TLR9 agonist CpG-2006, IL-2 (1000 IU/ml), IL-6 (10 ng/ml), IL-21 (10 ng/ml), and anti-B cell receptor (BCR) goat antibodies (which trigger BCRs). After an appropriate time (e.g., 14 days), supernatants of such cultures may be tested in a primary binding assay (e.g. Luminex assay using Spike trimer coupled beads) and a cell based neutralization assays to monitor B cell clones that produce antibodies capable of preventing viruses or pseudoviruses from productively infecting a target cell. The pseudoviruses may be incubated with B cell culture supernatants for an appropriate time and temperature (for example one (1) h at 37% (5% CO2)) before the addition of host cells (for example 3000 293T ACE-2 stable cells). Incubation for an appropriate time (for example 72 hours) may then follow, after which the supernatant may be removed and Steadylite reagent (Perkin Elmer) added (for example 15 mΐ). Luciferase activity may then be determined (for example five minutes later) on a Synergy microplate luminometer (BioTek). Decreased luciferase activity relative to a negative control typically indicates virus neutralization. Neutralization assays such as these, suitable for analyzing the neutralizing antibodies, or antigen-binding fragments thereof the neutralizing antibody, or an antigen-binding fragment thereof (binding agents) of this disclosure, are known in the art (see, e.g., Crawford et al Viruses. 2020 May 6;12(5):513. and Nie et al, Nat Protoc. 2020 Nov;15(l 1):3699-3715). In some embodiments, neutralization may be determined as a measure of the concentration (for example μg/ml) of monoclonal antibody capable of neutralizing any of about 50%, 60%, 70%, 80%, 90%, 95%, or 99% of viral infection (as may be measured by percent neutralization and/or by determining an “IC50” and/or “ICxo” value). In some embodiments, a neutralizing antibody, or an antigen-binding fragment thereof (binding agent) may be considered neutralizing if it is able to neutralize 50% of viral infection at a concentration of, for instance, about any of 10 ^"5 , 10 ^"4 , 10 ^"3 , 10 ^"2 , 10 ^'1 , 10°, 10 ¹ , 10 ² , or 10 ³ μg/ml (e.g., an IC50 value as shown in Figures 2 and 3). In some embodiments, the ability of a neutralizing antibody to neutralize viral infection may be expressed as a percent neutralization (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 99% (e.g., as in Figures 2 and 3)). And in some embodiments, as in the Examples herein, the ability of a neutralizing antibody to neutralize viral infection may be expressed as, and, in preferred embodiments, the IC50 and/or ICxo value is below 25 μg/ml, and is even more preferably below about any of 15, 10, 5, 2, 1, 0.5, 0.25, 0.1, 0.05, or 0.01 μg/ml (see, e.g., Figures 2 and 3). Other measures of neutralization may also be suitable as may be determined by those of ordinary skill in the art.

The invention also provides methods of producing the anti-SARS-CoV-2 antibodies, or the antigen-binding fragments thereof, of the invention using recombinant techniques. For example, polypeptides can be prepared using isolated nucleic acids encoding such antibodies or fragments thereof, vectors and host-cells comprising such nucleic acids.

An aspect of the present invention provides an isolated nucleic acid encoding the anti-SARS- CoV-2 antibody, or an antigen-binding fragment thereof, of the invention. Another aspect of the present invention provides a vector comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention. In an embodiment, the vector of the invention is an expression vector.

Another aspect of the present invention provides a host cell comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or comprising the vector of the invention. In an embodiment, the host cell of the invention is prokaryotic or eukaryotic.

Antibodies may be produced using recombinant methods and compositions, such as described in U.S. Patent No. 4,816,567. In one embodiment, isolated nucleic acid encoding an anti-SARS- CoV-2 antibody of the invention is provided. Such nucleic acid may encode an amino acid sequence comprising the VL and/or an amino acid sequence comprising the VH of the antibody (e.g., the light and/or heavy chains of the antibody). In some embodiments, the isolated nucleic acid encodes a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 1- 10, 81-83 and 105. In some embodiments, the isolated nucleic acid encodes a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 11-20, 84-86 and 106.

For recombinant production of anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention, nucleic acids encoding the desired antibodies or antibody fragments of the invention, are isolated and inserted into a replicable vector for further cloning (amplification of the DNA) or for expression. In a further embodiment, one or more vectors (such as expression vectors) comprising such nucleic acid are provided. In some embodiments, a vector comprises a nucleic acid encoding a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 81-83 and 105. In some embodiments, a vector comprises a nucleic acid encoding a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 11-20, 84-86 and 106. DNA encoding the polyclonal or monoclonal antibodies is readily isolated (for example, with oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of the antibody) and sequenced using conventional procedures. Many cloning and/or expression vectors are commercially available. Vector components generally include, but are not limited to, one or more of the following, a signal sequence, an origin of replication, one or more marker genes, a multiple cloning site containing recognition sequences for numerous restriction endonucleases, an enhancer element, a promoter, and a transcription termination sequence.

The anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof of the invention may be produced recombinantly not only directly, but also as a fusion protein, where the antibody is fused to a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. The heterologous signal sequence selected preferably is one that is recognized and processed (i.e., cleaved by a signal peptidase) by eukaryotic host-cells. For prokaryotic host- cells that do not recognize and process native mammalian signal sequences, the eukaryotic (i.e., mammalian) signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group consisting of leader sequences from alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II genes. For yeast secretion the native signal sequence may be substituted by, for example, the yeast invertase leader, factor leader (including Saccharomyces and Kluyveromyces -factor leaders), or acid phosphatase leader, the C. albicans glucoamylase leader, or the signal described in WO 90/13646. In mammalian cell expression, mammalian signal sequences as well as viral secretory leaders, for example, the herpes simplex virus gD signal, are available. The DNA for such precursor region is ligated in reading frame to the DNA encoding the antibodies or fragments thereof.

Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host-cells. Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2m plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, vesicular stomatitis virus ("VSV") or bovine papilloma virus ("BPV") are useful for cloning vectors in mammalian cells. Generally, the origin of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used only because it contains the early promoter).

Expression and cloning vectors may also contain a selection gene, known as a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of a host-cell. Those cells that are successfully transformed with a heterologous gene produce a protein conferring drug resistance and thus survive the selection regimen. Examples of such dominant selection strategies use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody- or antibody fragment-encoding nucleic acids, such as dihydrofolate reductase ("DHFR"), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like. For example, cells transformed with the DHFR selection gene are first identified by culturing all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR. An exemplary host-cell strain for use with wild-type DHFR is the Chinese hamster ovary ("CHO") cell line lacking DHFR activity (such as ATCC CRL-9096).

Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the antibody- or antibody fragment-encoding nucleic acids, such as dihydrofolate reductase ("DHFR"), glutamine synthetase (GS), thymidine kinase, metallothionein-I and -II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, and the like.

Alternatively, cells transformed with the GS (glutamine synthetase) gene are identified by culturing the transformants in a culture medium containing L-methionine sulfoximine (Msx), an inhibitor of GS. Under these conditions, the GS gene is amplified along with any other cotransformed nucleic acid. The GS selection/amplification system may be used in combination with the DHFR selection/amplification system described above.

Alternatively, host-cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encoding anti-CD83 agonist antibodies or fragments thereof, wild-type DHFR protein, and another selectable marker such as aminoglycoside 3 '-phosphotransferase ("APH") can be selected by cell growth in medium containing a selection agent for the appropriate selectable marker, such as an aminoglycosidic antibiotic, such as kanamycin, neomycin, or G418.

A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid YRp7. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow medium containing tryptophan (such as ATCC No. 44076 or PEP4-1). The presence of the trpl lesion in the yeast host-cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Similarly, Lew2-deficient yeast strains (such as ATCC 20,622 or 38,626) can be complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 pm circular plasmid pKDl can be used for transformation of Kluyveromyces yeasts. Alternatively, an expression system for large-scale production of recombinant calf chymosin was reported for K. lactis. Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of Kluyveromyces have also been disclosed.

Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the nucleic acid encoding the anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof of the invention. Promoters suitable for use with prokaryotic hosts include the phoA promoter, lactamase and lactose promoter systems, alkaline phosphatase promoter, a tryptophan promoter system, and hybrid promoters such as the tac promoter, although other known bacterial promoters are also suitable. Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the antibodies and antibody fragments.

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT -rich region located approximately 25 to 30 bases upstream from the site where transcription is initiated. Another sequence found 70 to 80 bases upstream from the start of transcription of many genes is a CNCAAT region where N may be any nucleotide. At the 3' end of most eukaryotic genes is an AATAAA sequence that may be the signal for addition of the polyA tail to the 3' end of the coding sequence. All of these sequences may be inserted into eukaryotic expression vectors.

Examples of suitable promoter sequences for use with yeast hosts include the promoters for 3- phosphogly cerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase, 3 -phosphogly cerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Inducible promoters in yeast have the additional advantage of permitting transcription controlled by growth conditions. Exemplary inducible promoters include the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657. Yeast enhancers also are advantageously used with yeast promoters. Transcription of nucleic acids encoding antibodies or fragments thereof from vectors in mammalian host-cells can be controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and most preferably Simian Virus 40 (SV40), by heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and by heat-shock gene promoters, provided such promoters are compatible with the desired host-cell systems.

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of the human cytomegalovirus is conveniently obtained as a Hindlll E restriction fragment. A system for expressing DNA in mammalian hosts using the bovine papilloma virus as a vector is disclosed in U.S. Patent No. 4,419,446. A modification of this system is described in U.S. Patent No. 4,601,978. Alternatively, the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

Transcription of a DNA encoding the antibodies or fragments thereof by higher eukaryotes is often increased by inserting an enhancer sequence into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a-fetoprotein, and insulin). Typically, however, one of ordinary skill in the art will use an enhancer from a eukaryotic virus. Examples include the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the antibody-or antibody-fragment encoding sequences, but is preferably located at a site 5' of the promoter.

Expression vectors used in eukaryotic host-cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding antibodies or fragments thereof. One useful transcription termination component is the bovine growth hormone polyadenylation region. Suitable host cells for cloning or expressing nucleic acid encoding the anti-SARS-CoV-2 antibodies or the antigen-binding fragments thereof of the invention in the vectors described include prokaryotic or eukaryotic cells described herein. For example, antibodies may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of antibody fragments and polypeptides in bacteria, see for example U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. After expression, the antibody may be isolated from the bacterial cell paste in a soluble fraction and can be further purified.

In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for antibody-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized," resulting in the production of an antibody with a partially or fully human glycosylation pattern.

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

Plant cell cultures can also be utilized as hosts. See for example US Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technology for producing antibodies in transgenic plants).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7); human embryonic kidney line (293 or 293 cells; baby hamster kidney cells (BHK); mouse Sertoli cells (TM4 cells); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human liver cells (Hep G2); mouse mammary tumor (MMT 060562); TRI cells; MRC 5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR- CHO cells; and myeloma cell lines such as Y0, NSO and Sp2/0.

In some embodiments, host-cells are transformed with the above-described expression or cloning vectors for anti-SARS-CoV-2 antibody or antigen-binding fragment production are cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host-cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host-cell selected for expression, and will be apparent to the person skilled in the art.

In some embodiments, a host cell comprising one or more nucleic acid encoding an anti-SARS- CoV-2 antibody or an antigen-binding fragment thereof of the invention is provided. In one such embodiment, a host cell comprises (for example, has been transformed with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid that encodes an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid that encodes an amino acid sequence comprising the VH of the antibody. In one embodiment, the host cell is eukaryotic, for example a Chinese Hamster Ovary (CHO) cell or lymphoid cell (such as Y0, NSO, Sp20 cell). In some embodiments, a host cell comprises a nucleic acid encoding a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 1-10, 81-83 and 105. In other embodiments, a host cell comprises a nucleic acid encoding a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 11-20, 84-86 and 106. In one embodiment, a method of making a anti-SARS-CoV-2 antibody of the invention is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the antibody, as provided above, under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium). In some embodiments, the host cell is a 293T cell. In some embodiments, an anti-SARS-CoV-2 antibody of the invention is produced by a method comprising culturing a host cell comprising one or more nucleic acid encoding an antibody described herein, under a condition suitable for expression of the one or more nucleic acid, and recovering the antibody produced by the cell. In a further embodiment, the one or more nucleic acid encodes a VH amino acid sequence selected from the group consisting of SEQ ID NOs: 1- 10, 81-83 and 105. In another further embodiment, the one or more nucleic acid encodes a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 11-20, 84-86 and 106. In some embodiments, the anti-SARS-CoV-2 antibody of the invention produced by a method comprising culturing a host cell comprising one or more nucleic acid encoding an antibody described herein has a lysine residue removed from the C-terminus. In some embodiments, the host cell is a 293T cell.

When using recombinant techniques, anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof of the invention can be produced intracellularly, in the periplasmic space, or secreted directly into the medium. If the antibodies are produced intracellularly, as a first step, the particulate debris from either host-cells or lysed fragments is removed, for example, by centrifugation or ultrafiltration. A procedure for isolating antibodies which are secreted to the periplasmic space of E. coli is known in the art. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonylfhioride (PMSF) over about 30 minutes. Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

The antibody or fragment thereof compositions prepared from such cells can be purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies or antibody fragments that are based on human 1, 2, or 4 heavy chains. Protein G is recommended for all mouse isotypes and for human 3 heavy chain antibodies or antibody fragments. The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrene-divinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibodies or antibody fragments comprise a CH3 domain, the Bakerbond ABX™resin is useful for purification. Other techniques for protein purification, such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, heparin, SEPHAROSE™, or anion or cation exchange resins (such as a polyaspartic acid column), as well as chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody or antibody fragment to be recovered.

Following any preliminary purification step or steps, the mixture comprising the antibody or antibody fragment of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5, preferably performed at low salt concentrations (e.g., from about 0-0.25 M salt).

In general, various methodologies for preparing antibodies for use in research, testing, and clinical applications are well-established in the art, consistent with the above-described methodologies and/or as deemed appropriate by one skilled in the art for a particular antibody of interest.

Thus, an aspect of the present invention provides a method of producing the anti-SARS-CoV- 2 antibody, or an antigen-binding fragment thereof, of the invention comprising culturing a host cell comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention under a condition suitable for expression of the nucleic acid; and recovering the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, produced by the cell. In an embodiment, the method of producing the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention further comprises purifying the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof.

Another aspect of the present invention provides a method for detecting SARS-CoV-2 virus in a cell or on a cell, the method comprising contacting a test biological sample with one or more anti-SARS-CoV-2 antibodies, or an antigen-binding fragment thereof, of the invention or one or more derivatives of the invention and detecting the one or more anti-SARS-CoV-2 antibodies, or an antigen-binding fragment thereof, bound to the biological sample or components thereof.

In one embodiment, the method for detecting SARS-CoV-2 virus in a cell or on a cell further comprises comparing the amount of binding to the test biological sample or components thereof to the amount of binding to a control biological sample or components thereof, wherein increased binding to the test biological sample or components thereof relative to the control biological sample or components thereof indicates the presence of a cell expressing SARS- CoV-2 in the test biological sample.

In another embodiment of the method for detecting SARS-CoV-2 virus in a cell or on a cell, the biological sample is selected from the group comprising blood, serum, a cell and tissue, such as liver tissue from a liver biopsy.

Another aspect of the present invention provides a method for detecting a SARS-CoV-2 virus in a sample, the method comprising contacting the sample with the anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, of the invention and detecting the antibody in the sample.

In an embodiment of the method for detecting a SARS-CoV-2 virus in a sample, the method further comprises comparing the amount of the antibody detected in the sample to the amount of the antibody detected in a control sample, wherein increased detection of the antibody in the sample relative to the control sample indicates the presence of the SARS-CoV-2 virus in the test biological sample.

In another embodiment of the method for detecting a SARS-CoV-2 virus in a sample, the SARS-CoV-2 virus is selected from a wild type SARS-CoV-2 virus or a variant selected from Alpha/B.1.1.7, Beta/B.1.351, Mink variant 16, Gamma/P.1, Delta/Bl.617.2, Omicron BA.l/B.1.1.529, Omicron BA.2/B.1.1.529.2, CAL.C20, C.37, and B.1.621.

In another embodiment of the method for detecting a SARS-CoV-2 virus in a sample, the sample is selected from the group comprising blood, serum, nasopharyngeal and/or nasal swabs, anal swabs, bronchoalveolar lavage, cerebrospinal fluid, nasal-throat swab, throat swab, sputum, a cell, and tissue. The term "detecting" as used herein encompasses quantitative or qualitative detection.

In other embodiments of the invention, any of the anti-SARS-CoV-2 antibodies, or the antigenbinding fragments thereof, of the invention is useful for detecting the presence of SARS-CoV- 2 virus and/or Spike protein or fragment thereof in a biological sample.

In another embodiment of the invention, the anti-SARS-CoV-2 antibodies, or the antigenbinding fragments thereof, of the invention for use in a method of diagnosis or detection is provided. In a further aspect, a method of detecting the presence of SARS-CoV-2 in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-SARS-CoV-2 antibody, or the antigen-binding fragment thereof, of the invention under conditions permissive for binding of the anti-SARS-CoV-2 antibody, or the antigen-binding fragment thereof, of the invention to SARS-CoV-2, and detecting whether a complex is formed between the anti-SARS-CoV-2 antibody, or the antigen-binding fragment thereof, of the invention and SARS-CoV-2. Such method may be an in vitro or in vivo method.

In a further aspect, a method of detecting the presence of RBD and/or Spike protein or fragment thereof in a biological sample is provided. In certain embodiments, the method comprises contacting the biological sample with an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention under conditions permissive for binding of the anti-SARS- CoV-2 antibody, or the antigen-binding fragment thereof to RBD and/or Spike protein or fragment thereof, and detecting whether a complex is formed between the anti-SARS-CoV-2 antibody or the antigen-binding fragment thereof and RBD and/or Spike protein or fragment thereof. Such method may be an in vitro or in vivo method.

In one embodiment, the anti-SARS-CoV-2 antibodies, or the antigen-binding fragments thereof, of the invention are used to select subjects eligible for therapy with the anti-SARS- CoV-2 antibodies, or the antigen-binding fragments thereof, of the invention, such as where SARS-CoV-2 or RBD, or Spike protein or fragment thereof is a biomarker for selection of patients.

In yet a further aspect, there is provided a diagnostic test apparatus and method for determining or detecting the presence of SARS-CoV-2 in a sample. The apparatus may comprise, as a reagent, one or more anti-SARS-CoV-2 antibodies, or the antigen-binding fragments thereof, of the invention. The antibody/ies may, for example, be immobilized on a solid support (for example, on a microtiter assay plate, or on a particulate support) and serve to "capture" SARS- CoV-2 from a sample (such as a blood or serum sample or other clinical specimen - such as a liver biopsy). The captured virus may then be detected by, for example, adding a further, labeled, reagent which binds to the captured virus. Conveniently, the assay may take the form of an ELISA, especially a sandwich-type ELISA, but any other assay format could in principle be adopted (such as radioimmunoassay, Western blot) including immunochromatographic or dipstick-type assays.

For diagnostic purposes, the anti-SARS-CoV-2 antibodies, or the antigen-binding fragments thereof, of the invention may either be labeled or unlabeled. Unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies). Alternatively, the antibodies can be directly labeled. A wide variety of labels may be employed - such as radionuclides, fluors, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc. Labels include, but are not limited to, labels or moieties that are detected directly (such as fluorescent, chromophoric, electron-dense, chemiluminescent, and radioactive labels), as well as moieties, such as enzymes or ligands, that are detected indirectly, e.g., through an enzymatic reaction or molecular interaction. Exemplary labels include, but are not limited to, the radioisotopes 32 P, 14 C, 125 I, 3 H, and 131 I, fluorophores such as rare earth chelates or fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, luceriferases, such as firefly luciferase and bacterial luciferase, luciferin, 2,3- dihydrophthalazinediones, horseradish peroxidase (HRP), alkaline phosphatase, b- galactosidase, glucoamylase, lysozyme, saccharide oxidases, such as glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase, heterocyclic oxidases such as uricase and xanthine oxidase, coupled with an enzyme that employs hydrogen peroxide to oxidize a dye precursor such as HRP, lactoperoxidase, or microperoxidase, biotin/avidin, spin labels, bacteriophage labels, stable free radicals, and the like.

In some embodiments, the test biological sample is compared to a control biological sample. In some embodiments, the control biological sample is from an individual known not to be infected with the SARS-CoV-2 virus. In some embodiments, the control biological sample is from an individual known to be infected with SARS-CoV-2. In some embodiments, any of the methods of treatment and/or attenuation of a SARS-CoV-2 virus infection described in the present disclosure are based on the determination or detection of SARS-CoV-2 in a sample by any of the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof of the invention. As used herein, "based upon" includes (1) assessing, determining, or measuring the subject's characteristics as described herein (and preferably selecting a subject suitable for receiving treatment); and (2) administering the treatment s) as described herein.

In some embodiments, a method is provided for identifying an individual suitable or not suitable (unsuitable) for treatment with the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof of the invention. In a further embodiment, an individual suitable for treatment is administered an anti-SARS-CoV-2 antibody or an antigen-binding fragment thereof of the invention.

In some embodiments, a method is providing for selecting or not selecting an individual for treatment with the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof of the invention, the method comprising: a) assessing the viral load and/or viral titer in a biological sample from the individual, and b) selecting the individual for treatment with an anti-SARS- CoV-2 antibody or an antigen-binding fragment thereof of the invention if the viral load is at least 5 IU/mL. In some embodiments, the viral load is at least 5xl0 ² copies per ml, 10 ³ copies per ml, 10 ⁴ copies per ml, 10 ⁵ copies per ml, 10 ⁶ copies per ml, 10 ⁷ copies per ml, or > 10 ⁷ copies per ml inclusive, including any values in between these numbers.

In a further aspect of the invention, there is provided an assay method for identifying an agent that improves or enhances the efficacy of the neutralizing activity of the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof of the invention. Provided herein is an assay method for identifying an agent that improves or enhances the efficacy of the neutralizing activity of the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof of the invention against SARS-CoV-2 virus, comprising the steps of: (a) contacting said anti-SARS- CoV-2 antibody or antigen-binding fragment thereof with an agent to be tested; and (b) determining whether the agent improves or enhances the efficacy of the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof in neutralizing the infectivity of SARS-CoV-2 virus. In some embodiments, the ability of the agent to improve or enhance the efficacy of the neutralizing activity of the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of the invention against SARS-CoV-2 virus is compared to a control. In some embodiments, the control is the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof of the invention in the absence of the agent. In some embodiments, the control is humanized antibody or fragment thereof with a placebo, e.g., water, saline, sugar water, etc. As used herein, the term "agent" may be a single entity or it may be a combination of entities. The agent may be an organic compound or other chemical. The agent may be a compound, which is obtainable from or produced by any suitable source, whether natural or artificial. The agent may be an amino acid molecule, a polypeptide, or a chemical derivative thereof, or a combination thereof. The agent may even be a polynucleotide molecule - which may be a sense or an anti-sense molecule. In some embodiments, the agent is an antibody. In some embodiments, the agent is a cytokine (such as interferon- a). In some embodiments, the agent is a direct acting antiviral agent. In further embodiments, the direct acting antiviral agent is viral protease inhibitor or a viral polymerase inhibitor. In some embodiments, the agent is an indirect acting viral agent. The agent may be designed or obtained from a library of compounds, which may comprise peptides, as well as other compounds, such as small organic molecules. By way of example, the agent may be a natural substance, a biological macromolecule, or an extract made from biological materials such as bacteria, fungi, or animal (particularly mammalian) cells or tissues, an organic or an inorganic molecule, a synthetic agent, a semi- synthetic agent, a structural or functional mimetic, a peptide, a peptidomimetics, a derivatized agent, a peptide cleaved from a whole protein, or a peptides synthesized synthetically (such as, by way of example, either using a peptide synthesizer or by recombinant techniques or combinations thereof, a recombinant agent, an antibody, a natural or a non-natural agent, a fusion protein or equivalent thereof and mutants, derivatives or combinations thereof. Typically, the agent will be an organic compound. Typically, the organic compounds will comprise two or more hydrocarbyl groups. Here, the term "hydrocarbyl group" means a group comprising at least C and H and may optionally comprise one or more other suitable substituents. Examples of such substituents may include halo-, alkoxy-, nitro-, an alkyl group, a cyclic group etc. In addition to the possibility of the substituents being a cyclic group, a combination of substituents may form a cyclic group. If the hydrocarbyl group comprises more than one C then those carbons need not necessarily be linked to each other. For example, at least two of the carbons may be linked via a suitable element or group. Thus, the hydrocarbyl group may contain hetero atoms. Suitable hetero atoms will be apparent to those skilled in the art and include, for instance, sulphur, nitrogen and oxygen. For some applications, preferably the agent comprises at least one cyclic group. The cyclic group may be a polycyclic group, such as a non-fused polycyclic group. For some applications, the agent comprises at least the one of said cyclic groups linked to another hydrocarbyl group. The agent may contain halo groups. Here, "halo" means fluoro, chloro, bromo or iodo. The agent may contain one or more of alkyl, alkoxy, alkenyl, alkylene and alkenylene groups - which may be unbranched- or branched-chain.

Another aspect of the present invention provides a kit for detecting SARS-CoV-2 virus in a cell or on a cell, the kit comprising the one or more anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, of the invention or the derivative of the invention and instructions for use. In some embodiments of the kit of the invention, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, the derivative of the invention is in lyophilized form.

Another aspect of the present invention provides a kit for detecting SARS-CoV-2 virus in a sample, the kit comprising the one or more anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention and instructions for use. In some embodiments of the kit of the invention, the anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, the derivative of the invention is in lyophilized form.

In some embodiments, the kit containing the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention useful for the detection of SARS-CoV-2 virus in a cell or on a cell, the treatment, prevention and/or diagnosis of the disorders described above is provided.

In some embodiments of the kit of the invention, the sample is selected from the group comprising blood, serum, nasopharyngeal and/or nasal swabs, anal swabs, bronchoalveolar lavage, cerebrospinal fluid, nasal-throat swab, throat swab, sputum, a cell, and tissue.

In some embodiments, the kit of the invention comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention. The label or package insert indicates that the composition is used for diagnosing and/or treating the condition of choice. Moreover, the kit may comprise (a) a first container with a composition contained therein, wherein the composition comprises an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention; and (b) a second container with a composition contained therein, wherein the composition comprises a further cytotoxic or otherwise therapeutic agent. The article of manufacture or kit in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture or kit may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In some embodiments, the kit of the invention is a diagnostic kit, for example, research, detection and/or diagnostic kit. Such kits typically contain the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention. Suitably, the antibody is labeled, or a secondary labeling reagent is included in the kit. Preferably, the kit is labeled with instructions for performing the intended application, for example, for performing an in vivo imaging assay.

Another aspect of the present invention provides a pharmaceutical composition comprising one or more the anti-SARS-CoV-2 antibodies, or an antigen-binding fragment thereof, of the invention or one or more derivatives of the invention, and a pharmaceutically acceptable carrier.

In an embodiment, the pharmaceutical composition of the invention comprises a first and a second anti-SARS-CoV-2 antibody, wherein the first anti-SARS-CoV-2 antibody is the P2G3 antibody of the invention and one or more second anti-SARS-CoV-2 antibody is selected from the group consisting of P5C3, P6E16, P1H23, MS42, P1M12, P2B11, P7K18, P1L7, P1G17, MS31 and MS35. Preferably the pharmaceutical composition of the invention comprises the anti-SARS-CoV-2 antibody P2G3 of the invention and the anti-SARS-CoV-2 antibody P5C3 of the invention. In another embodiment, the pharmaceutical composition of the invention comprises the anti- SARS-CoV-2 antibody P2G3 of the invention and the anti-SARS-CoV-2 antibody P6E16 of the invention.

In another embodiment, the pharmaceutical composition of the invention comprises the anti- SARS-CoV-2 antibody P2G3 of the invention and the anti-SARS-CoV-2 antibody P1H23 of the invention.

In another embodiment, the pharmaceutical composition of the invention comprises the anti- SARS-CoV-2 antibody P2G3 of the invention and the anti-SARS-CoV-2 antibody PMS42 of the invention.

In another embodiment, the pharmaceutical composition of the invention comprises the anti- SARS-CoV-2 antibody P2G3 of the invention and the anti-SARS-CoV-2 antibody P1M12 of the invention.

Pharmaceutical compositions and formulations of one or more anti-SARS-CoV-2 antibodies as described herein are prepared by mixing such one or more antibodies having the desired degree of purity with one or more optional pharmaceutically acceptable carriers {Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (such as Zn-protein complexes); and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include insterstitial drug dispersion agents such as soluble neutral -active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Buffers are used to control the pH in a range which optimizes the therapeutic effectiveness, especially if stability is pH dependent. Buffers are preferably present at concentrations ranging from about 50 mM to about 250 mM. Suitable buffering agents for use with the present invention include both organic and inorganic acids and salts thereof, such as citrate, phosphate, succinate, tartrate, fumarate, gluconate, oxalate, lactate, acetate. Additionally, buffers may comprise histidine and trimethylamine salts such as Tris.

Preservatives are added to retard microbial growth, and are typically present in a range from 0.2% - 1.0% (w/v). Suitable preservatives for use with the present invention include octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium halides (such as chloride, bromide, iodide), benzethonium chloride; thimerosal, phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol, 3-pentanol, and m-cresol.

Tonicity agents, sometimes known as "stabilizers" are present to adjust or maintain the tonicity of liquid in a composition. When used with large, charged biomolecules such as proteins and antibodies, they are often termed "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby lessening the potential for inter- and intra-molecular interactions. Tonicity agents can be present in any amount between 0.1% to 25% by weight, or more preferably between 1% to 5% by weight, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.

Non-ionic surfactants or detergents (also known as "wetting agents") are present to help solubilize the therapeutic agent as well as to protect the therapeutic protein against agitation- induced aggregation, which also permits the formulation to be exposed to shear surface stress without causing denaturation of the active therapeutic protein or antibody. Non-ionic surfactants are present in a range of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07 mg/ml to about 0.2 mg/ml.

Suitable non-ionic surfactants include polysorbates (20, 40, 60, 65, 80, etc.), polyoxamers (184, 188, etc.), PLURONIC® polyols, TRITON®, polyoxyethylene sorbitan monoethers (TWEEN®-20, TWEEN®-80, etc.), lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. Anionic detergents that can be used include sodium lauryl sulfate, dioctyle sodium sulfo succinate and dioctyl sodium sulfonate. Cationic detergents include benzalkonium chloride or benzethonium chloride.

The choice of pharmaceutical carrier, excipient or dilutent may be selected with regard to the intended route of administration and standard pharmaceutical practice.

Pharmaceutical compositions may comprise as - or in addition to - the carrier, excipient or dilutent any suitable binder(s), lubricant(s), suspending agent(s), coating agent(s) or solubilizing agent(s).

There may be different composition/formulation requirements dependent on the different delivery systems. By way of example, pharmaceutical compositions useful in the present invention may be formulated to be administered using a mini-pump or by a mucosal route, for example, as a nasal spray or aerosol for inhalation or ingestible solution, or parenterally in which the composition is formulated by an injectable form, for delivery, by, for example, an intravenous, intramuscular or subcutaneous route. Alternatively, the formulation may be designed to be administered by a number of routes.

In some embodiments, an anti-SARS-CoV-2 antibody formulation is a lyophilized anti-SARS- CoV-2 antibody formulation. In another embodiment, an anti-SARS-CoV-2 antibody formulation is an aqueous anti-SARS-CoV-2 antibody formulation. Exemplary lyophilized antibody formulations are described in US Patent No. 6,267,958. Aqueous antibody formulations include those described in US Patent No. 6,171,586 and W02006/044908, the latter formulations including a histidine-acetate buffer. The formulation herein may also contain more than one active ingredients, such as antiviral agents, as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. In some embodiments, an active ingredient is an antiviral agent. In other embodiments, the antiviral agent is selected from the group comprising Remdesivir, anti-inflammatory drugs, such as tocilizumab and sarilumab, and antibodies that bind to other SARS-CoV-2 proteins required by SARS-CoV-2 to infect the cell. For example, Remdesivir may be used which is a broad-spectrum antiviral medication that acts as a ribonucleotide analogue inhibitor of viral RNA polymerase. Once additional antivirals against SARS-CoV-2 are identified, it may be desirable to further provide an antiviral agent that target additional steps in the viral replication cycle or an antibody. The combination of the anti-SARS-CoV-2 antibodies described in this invention may also be used in combination with anti-inflammatory drugs, including tocilizumab and sarilumab, that have been reported to help prevent COVID-19 related deaths. Antibodies that bind to other SARS-CoV-2 proteins required by SARS-CoV-2 to infect the cell are also contemplated. In any embodiments herein, an antiviral agent as described herein can be used in a formulation with an anti-SARS-CoV-2 antibody of the invention. Such as antiviral agents described herein are suitably present in combination in amounts that are effective for the purpose intended.

Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Stability of the proteins and antibodies described herein may be enhanced through the use of non-toxic "water-soluble polyvalent metal salts". Examples include Ca2+, Mg2+, Zn2+, Fe2+, Fe3+, Cu2+, Sn2+, Sn4+, A12+ and A13+. Exemplary anions that can form water soluble salts with the above polyvalent metal cations include those formed from inorganic acids and/or organic acids. Such water-soluble salts are soluble in water (at 20°C) to at least about 20 mg/ml, alternatively at least about 100 mg/ml, alternatively at least about 200 mg/ml.

Suitable inorganic acids that can be used to form the "water soluble polyvalent metal salts" include hydrochloric, acetic, sulfuric, nitric, thiocyanic and phosphoric acid. Suitable organic acids that can be used include aliphatic carboxylic acid and aromatic acids. Aliphatic acids within this definition may be defined as saturated or unsaturated C2-9 carboxylic acids (such as aliphatic mono-, di- and tri -carboxylic acids). For example, exemplary monocarboxylic acids within this definition include the saturated C2-9 monocarboxylic acids acetic, proprionic, butyric, valeric, caproic, enanthic, caprylic pelargonic and capryonic, and the unsaturated C2- 9 monocarboxylic acids acrylic, propriolic methacrylic, crotonic and isocro tonic acids. Exemplary dicarboxylic acids include the saturated C2-9 dicarboxylic acids malonic, succinic, glutaric, adipic and pimelic, while unsaturated C2-9 dicarboxylic acids include maleic, fumaric, citraconic and mesaconic acids. Exemplary tricarboxylic acids include the saturated C2-9 tricarboxylic acids tricarballylic and 1,2,3-butanetricarboxylic acid. Additionally, the carboxylic acids of this definition may also contain one or two hydroxyl groups to form hydroxy carboxylic acids. Exemplary hydroxy carboxylic acids include glycolic, lactic, glyceric, tartronic, malic, tartaric and citric acid. Aromatic acids within this definition include benzoic and salicylic acid.

Commonly employed water soluble polyvalent metal salts which may be used to help stabilize the encapsulated polypeptides of this invention include, for example: (1) the inorganic acid metal salts of halides (such as zinc chloride, calcium chloride), sulfates, nitrates, phosphates and thiocyanates; (2) the aliphatic carboxylic acid metal salts (e.g., calcium acetate, zinc acetate, calcium proprionate, zinc glycolate, calcium lactate, zinc lactate and zinc tartrate); and (3) the aromatic carboxylic acid metal salts of benzoates (e.g., zinc benzoate) and salicylates.

Pharmaceutical formulations of anti-SARS-CoV-2 antibodies of the invention can be designed to immediately release an anti-SARS-CoV-2 antibody ("immediate-release" formulations), to gradually release the anti-SARS-CoV-2 antibodies over an extended period of time ("sustained- release," "controlled-release," or "extended-release" formulations), or with alternative release profiles. The additional materials used to prepare a pharmaceutical formulation can vary depending on the therapeutic form of the formulation (for example whether the system is designed for immediate-release or sustained-, controlled-, or extended-release). In certain variations, a sustained-release formulation can further comprise an immediate-release component to quickly deliver a priming dose following drug delivery, as well as a sustained- release component. Thus, sustained-release formulations can be combined with immediate- release formulations to provide a rapid "burst" of drug into the system as well as a longer, gradual release. For example, a core sustained-release formulation may be coated with a highly soluble layer incorporating the drug. Alternatively, a sustained-release formulation and an immediate-release formulation may be included as alternate layers in a tablet or as separate granule types in a capsule. Other combinations of different types of drug formulations can be used to achieve the desired therapeutic plasma profile.

Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antagonist, which matrices are in the form of shaped articles, such as films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2 -hydroxy ethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3-hydroxybutyric acid.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, for example by filtration through sterile filtration membranes.

The pharmaceutical compositions may be used in any of the methods described herein.

The pharmaceutical composition may be used among those subjects (such as humans) susceptible to infection with SARS-CoV-2 i.e. to prevent or reduce/decrease the onset of SARS- CoV-2 infection.

The pharmaceutical composition may be used among those subjects (such as humans) already infected with SARS-CoV-2 i.e. to treat SARS-CoV-2 infection. Such treatment may facilitate clearance of the virus from those subjects who are acutely infected.

Another aspect of the present invention provides the anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, of the invention or the derivative of the invention for use as a pharmaceutical.

Another aspect of the present invention provides a method of prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection in a subject, comprising administering to the subject an effective amount of the one or more anti-SARS-CoV-2 antibody, or an antigen- binding fragment thereof, of the invention or one or more derivative of the invention. In some embodiments of the method, the subject has been diagnosed with the SARS-CoV-2 infection or the subject has to be protected from SARS-CoV-2 virus infection. In some embodiments of the method, the subject does not have a SARS-CoV-2 infection. In some embodiments of the method, treating and/or attenuating the SARS-CoV-2 virus infection comprises reducing viral load.

In some other embodiments of the invention, the method of prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection in a subject further comprises administering an antiviral agent.

In some embodiments, the antiviral agent is selected from the group consisting of a viral protease inhibitor, a viral polymerase inhibitor, an NS5A inhibitor, an interferon, a second anti- SARS-Cov-2 antibody, and a combination thereof. In some embodiments, the antiviral agent is selected from the group comprising Remdesivir, anti-inflammatory drugs, such as tocilizumab and sarilumab, and antibodies that bind to other SARS-CoV-2 proteins required by SARS-CoV- 2 to infect the cell. In some embodiments, the antiviral agent is an antibody as described herein. In some embodiments, the antiviral agent is Remdesivir. In other embodiments, the antiviral agent is anti-inflammatory drug, preferably tocilizumab and/or sarilumab.

In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention is administered in combination with, sequential to, concurrently with, consecutively with, rotationally with, or intermittently with an antiviral agent (such as a viral RNA polymeraseinhibitor) or anti-inflammatory drug (such as an anti-IL-6 antibody). In some embodiments, the administration of the combination of an anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or a derivative of the invention and an antiviral agent and/or anti-inflammatory agent ameliorates one or more symptom of SARS-CoV-2, reduces and/or suppresses viral titer and/or viral load, and/or prevents SARS-CoV-2, and/or achieves a sustained virologic response more than treatment with the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the antiviral agent alone. In some embodiments, the anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention and the antiviral agent and/or anti-inflammatory agent are provided in separate dosage forms. In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen- binding fragment thereof, of the invention or the derivative of the invention and the antiviral agent are provided in the same dosage form.

Thus, in a further aspect the invention provides a method of prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection, comprising the use of the one or more anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention. Suitably, an effective amount of the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention is administered to the subject. In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention is administered in a therapeutic effective amount to effect beneficial clinical results, including, but not limited to neutralizing SARS- CoV-2 and/or ameliorating one or more symptoms of SARS-CoV-2 infections or aspects of SARS-CoV-2 infection. In some embodiments, the anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention is administered in a therapeutic effective amount to reduce viral titer and/or viral load of SARS-CoV-2. In some embodiments, the anti-SARS- CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention is administered in a therapeutic effective amount to achieve a sustained virologic response. As used herein, the term "sustained virologic response" refers to the absence of detectable viremia during certain period of time, such as twelve weeks, after stopping anti-SARS-CoV-2 treatment.

There is also provided the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention for use in the method of prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection in a subject, wherein the method comprises administering to the subject an effective amount of the one or more anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention.

There is also provided the use of the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention or the pharmaceutical composition of the invention in the manufacture of a composition for the prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection in a subject. In some embodiments, the prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection in a subject comprises administering to the subject an effective amount of the one or more anti-SARS-CoV- 2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention.

The anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention or a pharmaceutical composition comprising same are useful in reducing, eliminating, or inhibiting SARS-CoV-2 infection and can be used for treating any pathological condition that is characterized, at least in part, by SARS-CoV-2 infection. The anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention can be used for treating a SARS-CoV-2 infection. The anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention can also be used in prophylaxis and/or methods for preventing a SARS-CoV-2 infection. For example the anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention is administered prophylactically.

Overall, the inventors have developed some of the most potent anti-SARS-CoV-2 antibodies against the SARS-CoV-2 virus with several of the identified antibodies binding distinct, nonoverlapping epitopes on the SARS-CoV-2 RBD. As such, monotherapy or combination therapy of anti-SARS-CoV-2 antibodies could be used in both prophylactic and therapeutic treatments to combat SARS-CoV-2 viral infection. Thus in an embodiment of the method of prophylaxis, treatment and/or attenuation of a SARS-CoV-2 virus infection of the invention, a combination of one, two or more anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention can be administered to the subject.

In one aspect, the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, provided in the present invention are used as a monotherapy. In one aspect, the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, provided in the present invention are used in combination therapy. In one embodiment, the anti-SARS-CoV-2 antibodies or antigen-binding fragments thereof, provided in the present invention can be used against SARS-CoV-2 virus as a monotherapy, or in combinations thereof. For example, preferred combinations are combinations of

- the anti-SARS-CoV-2 antibody P2G3 of the invention is administered in combination with one or more anti-SARS-CoV-2 antibodies of the invention selected from the group consisting of P5C3, P6E16, P1H23, MS42, P1M12, P2B11, P7K18, P1L7, P1G17, MS31 and MS35.

- the preferred anti-SARS-CoV-2 antibody combinations are P2G3 antibody with P5C3 antibody, P2G3 antibody with P6E16 antibody, P2G3 antibody with P1H23 antibody, P2G3 antibody with MS42 antibody or P2G3 antibody with P1M12 antibody.

In some embodiments of the method of prophylaxis, treatment, and/or attenuation of a SARS- CoV-2 virus infection in a subject, the anti-SARS-CoV-2 antibody P2G3 of the invention is administered in combination with one or more anti-SARS-CoV-2 antibodies of the invention selected from P5C3, P6E16, P1H23, MS42, P1M12, P2B11, P7K18, P1L7, P1G17, MS31 and MS35; preferably the anti-SARS-CoV-2 antibody P2G3 of the invention is administered in combination with the anti-SARS-CoV-2 antibody P5C3 of the invention.

In an embodiment of the method of prophylaxis, treatment, and/or attenuation of a SARS-CoV- 2 virus infection in a subject, the P2G3 antibody of the invention and the one or more additional anti-SARS-CoV-2 antibodies are administered as part of the same composition.

In an embodiment of the method of prophylaxis, treatment, and/or attenuation of a SARS-CoV- 2 virus infection in a subject, the P2G3 antibody of the invention and the one or more additional anti-SARS-CoV-2 antibodies are administered as separate compositions.

In an embodiment of the method of prophylaxis, treatment, and/or attenuation of a SARS-CoV- 2 virus infection in a subject, the P2G3 antibody of the invention and the one or more additional anti-SARS-CoV-2 antibodies are administered sequentially or simultaneously.

In the combination therapy (combined administration) of the invention, the anti-SARS-CoV-2 antibodies of the invention are co-administered simultaneously, for example in a combined unit dose (e.g., providing simultaneous delivery). In the combination therapy (combined administration) of the invention, the anti-SARS-CoV-2 antibodies of the invention can also be co-administered separately or sequentially at a specified time interval, such as, but not limited to, an interval of minutes, hours, days, weeks or months. In some embodiments, the anti-SARS- CoV-2 antibodies of the invention for the combination therapy may be administered essentially simultaneously, for example two unit dosages administered at the same time, or a combined unit dosage of the two or more antibodies. In other embodiments, the anti-SARS-CoV-2 antibodies of the invention for combination therapy may be delivered in separate unit dosages. The anti-SARS-CoV-2 antibodies of the invention for the combination therapy may be administered in any order, or as one or more preparations that includes two or more antibodies. In a preferred embodiment, at least one administration of one antibody may be made within minutes, one, two, three, or four hours, or even within one or two days of the other antibody. In some embodiments, combination therapy of the invention provides neutralizing the SARS- CoV-2 virus through binding of anti-SARS-CoV-2 antibodies to different epitopes which has the potential effect of greater neutralization potency, reduced chance of developing viruses with mutations that confer resistance and greater breadth in neutralizing viruses with polymorphism in the general population.

In some embodiments, the methods of attenuation of a SARS-CoV-2 virus infection in a subject, such as reduction of incidence of, reduction duration of, reduction or lessen severity of, typically refers to attenuation of one or more symptoms of SARS-CoV-2 infection. Typically, the symptoms of SARS-CoV-2 include fever, cough, shortness of breath and myalgia or fatigue.

In some embodiments, the methods of the invention suppress or reduce viral titer. "Viral titer" is known in the art and indicates the amount of virus in a given biological sample.

In some embodiments, the methods of the invention suppress or reduce viremia. "Viremia" is known in the art as the presence of virus in nasopharyngeal and/or nasal swabs or other collected biological samples that could include anal swabs, bronchoalveolar lavage, cerebrospinal fluid, nasal-throat swab, throat swab or sputum testing.

In some embodiments, the methods of the invention suppress or reduce viral load. "Viral load" refers to the amount of SARS-CoV-2 virus in a person's nasopharyngeal swabs or other relevant samples. The results of a SARS-CoV-2 viral load test are usually expressed as RNA copies/mL. A subject with a SARS-CoV-2 viral load of >1 million copies/mL or more is considered to have a high viral load. Amount of virus (such as viral titer or viral load) are indicated by various measurements, including, but not limited to amount of viral nucleic acid, the presence of viral particles, replicating units (RU), plaque forming units (PFU). Amount of virus such as high viral load, low viral load or undetectable viral load can be defined according to a clinical acceptable parameter established by the person skilled in the art. In some embodiments, an undetectable viral load is defined by the limit of the assay for detecting SARS-CoV-2. Generally, for fluid samples such as blood and urine, amount of virus is determined per unit fluid, such as milliliters. For solid samples, such as tissue samples, amount of virus is determined per weight unit, such as grams. Methods for determining amount of virus are known in the art and are also described herein. In some embodiments, the methods described herein result in a sustained virologic response for at least 12 weeks after stopping the treatment.

The term "SARS-CoV-2-associated diseases" or "SARS-CoV-2-associated disorders" or “COVID-19 patients” as used herein, refers to an infection with SARS-CoV-2 or a disease or disorder that is associated with SARS-CoV-2 infection such as respiratory distress. This disease can lead to one or more of the following symptoms that include fever, dry cough, tiredness, aches and pains sore throat, diarrhea, conjunctivitis, headache, loss of taste or smell, a rash on skin, or discoloration of fingers or toes. More serious symptoms include difficulty breathing or shortness of breath chest pain or pressure, and loss of speech or movement. Patients that experience acute respiratory distress syndrome due to COVID-19 will warrant intubation and mechanical ventilation. In severe cases, progression of the disease can lead to long-term health issues or death. Accordingly, in some embodiments, an anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention prevents development of a SARS-CoV-2- associated disease.

The anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention can also be used in methods for preventing a SARS-CoV-2 infection, i.e. in prophylaxis. In some embodiments, the anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention are useful in methods of preventing an acute SARS-CoV-2 infection. In some embodiments, the anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention can be used in methods for preventing a SARS-CoV-2 infection in a subject susceptible to infection with SARS-CoV-2. In some embodiments, the anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivative of the invention and/or the pharmaceutical composition of the invention can also be used in methods for preventing a SARS-CoV-2 infection in a subject exposed to or potentially exposed to SARS- CoV-2. "Exposure" to SARS-CoV-2 denotes an encounter or potential encounter with SARS- CoV-2 which could result in a SARS-CoV-2 infection. Generally, an exposed subject is a subject that has been exposed to SARS-CoV-2 by a route by which SARS-CoV-2 can be transmitted. In some embodiments, the subject has been exposed to or potentially exposed to a subject which may or may not be infected with SARS-CoV-2 (i.e., SARS-CoV-2 infection status of the subject is unknown). SARS-CoV-2 is often transmitted by air and contact.

In a further aspect, the invention provides for the use of an anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, of the invention or the derivative of the invention in the manufacture or preparation of a medicament. In one embodiment, the medicament is for treatment of SARS-CoV-2 infection. In a further embodiment, a medicament comprising an anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, of the invention or the derivative of the invention for use in a method of treating SARS-CoV-2 infection comprises administering to an individual having a SARS-CoV-2 infection an effective amount of the medicament comprising an anti-SARS-CoV-2 antibody, or antigen-binding fragments thereof, of the invention or the derivative of the invention. In one such embodiment, the method further comprises administering to the individual an effective amount of at least one additional antiviral agent, such as agent described herein. In some embodiments, the invention provides for the use of an anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, of the invention or the derivative of the invention in combination with an antiviral agent described herein in the manufacture or preparation of a medicament.

In some embodiments of any of the methods described herein, the subject is a human.

The antibody /ies may be administered, for example, in the form of immune serum or may more preferably be a purified recombinant or monoclonal antibody. Methods of producing sera or monoclonal antibodies with the desired specificity are routine and well-known to those skilled in the art. The anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivatives of the invention can be administered to a subject in accord with known methods and any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, for example by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time-points, bolus administration, and pulse infusion are contemplated herein., such as by intravenous administration, for example as a bolus or by continuous infusion over a period of time, by subcutaneous, intramuscular, intraperitoneal, intracerobrospinal, intra-articular, intrasynovial, intrathecal, or inhalation routes, generally by intravenous or subcutaneous administration.

Suitably, a passive immunization regime may conveniently comprise administration of the anti- SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention and/or administration of antibody in combination with other antiviral agents. The active or passive immunization methods of the invention should allow for the protection or treatment of individuals against infection with viruses of SARS-CoV-2 type.

Indeed, given that P5C3, P2G3 and P6E16 are some of the most potent anti-SARS-CoV-2 antibodies identified to date, they are ideal candidate to be used in passive immunization for the prophylactically protection of uninfected individuals at risk of infection with the SARS- CoV-2 virus. This invention also describes the development of additional anti-SARS-CoV-2 antibodies, including MS35 and PI 06, that bind to non-overlapping epitopes on the viral Spike protein with P5C3 and could be used in combination with herein identified most potent antibodies to have a greater antiviral potency and breadth in neutralizing viruses with mutations. Beyond prophylactic protection, the antibodies described herein can provide therapeutic benefit to: 1) individuals recently infected through contact with a SARS-CoV-2 positive individual, 2) COVID-19 patients that mount a weak humoral immune response and 3) COVID-19 patients in general with deteriorating health due to uncontrolled viral infection.

The anti-SARS-CoV-2 antibodies, or antigen-binding fragments thereof, of the invention or the derivatives of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The antibody need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of antibody present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 to 99% of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of an anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, of the invention or a derivative of the invention (when used alone or in combination with one or more other additional antiviral agents) will depend on the type of disease to be treated, the type of antibody, the severity and course of the disease, whether the antibody is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody, and the discretion of the attending physician. The antibody is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 pg/kg to 15 mg/kg (for example 0.1 mg/kg- lOmg/kg) of antibody can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 pg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the antibody would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermitently, such as every week or every three weeks (for example such that the patient receives from about two to about twenty, or for example about two or about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered.

Although there are now several vaccines in clinical trials that demonstrate a high level of efficacy, there is still no data indicating the durability of this vaccine induced protection. In addition, it is likely that at-risk individuals that includes the elderly population and immunosuppressed subjects (such as patients undergoing cancer therapy and those that have undergone an organ transplants) will only have a partial or transient protection induced by these vaccines. As such, the anti-SARS-CoV-2 antibodies of the invention may be of significant importance to protect individuals that are less able to mount an effective anti-SARS-CoV-2 immune response following vaccination.

In one aspect, the invention provides methods for inhibiting, treating or preventing SARS-CoV- 2 virus infection in a subject comprising administering to the subject an effective amount of an anti-SARS-CoV-2 antibody (neutralizing antibody) described herein. In some embodiments, an effective amount of an anti-SARS-CoV-2 antibody is administered to a subject for inhibiting, treating or preventing SARS-CoV-2 cellular entry in a subject. In some embodiments, an effective amount of an anti-SARS-CoV-2 antibody is administered to an individual for inhibiting, treating or preventing SARS-CoV-2 spread in a subject. In some embodiments, an effective amount of an anti-SARS-CoV-2 antibody is administered to a subject for inhibiting, treating or preventing a SARS-CoV-2-associated disease in the individual.

The identified clones are among the most potent anti-SARS-CoV-2 (neutralizing) antibodies discovered against the SARS-CoV-2 virus. The P2G3 antibody has IC50 values of about 5 ng/ml that is 6- to 9-fold more potent than the clinical antibodies advanced by Regeneron. Several of potent antibodies disclosed herein also bind to non-overlapping epitopes on the viral Spike protein. This provides an antibody combination therapy that would: 1) have a more pronounced neutralizing activity of the virus, 2) neutralize a broader array of circulating viruses with mutations and 3) help to suppress the development of resistant virus that may emerge in an antibody monotherapy.

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications without departing from the spirit or essential characteristics thereof. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features. The present disclosure is therefore to be considered as in all aspects illustrated and not restrictive, the scope of the invention being indicated by the appended claims, and all changes which come within the meaning and range of equivalency are intended to be embraced therein.

The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, exemplary of methods of practising the present invention and are not intended to limit the application and the scope of the invention.

EXAMPLES

Example 1

Selection of SARS-CoV-2 infected donors, isolation and selection of anti-SARS-CoV-2 antibodies.

In order to isolate potent neutralizing antibodies against the SARS-CoV-2 virus, serum samples from COVID-19 patients were screened for the presence of high titers of antibodies able to bind to the SARS-CoV-2 Spike protein and with neutralizing activity in the SARS-CoV-2 Spike pseudoviral neutralization assay (assay methods described below). In addition to COVID-19 patients, serum samples from donors that were naturally infected with the SARS-CoV-2 virus and subsequently vaccinated >4 months later with a SARS-CoV-2 mRNA vaccine or donors that were vaccinated then became infected with the SARS-COV-2 virus, were also evaluated. This analysis resulted in the identification of twelve (12) patients with elevated serum levels of neutralizing antibodies.

In the isolation of antigen specific B cells, freshly isolated blood mononuclear cells from the selected COVID-19 patients were incubated with biotinylated Spike trimer protein, biotinylated RBD protein and a cocktail of fluorescently labeled antibodies for flow cytometry. Spike trimer proteins used in these studies corresponded to either the original 2019-nCoV first identified in Wuhan China, the Beta variant from South Africa (B.1.351) or Delta variant from India (B.1.617.2). Biotinylated proteins were stained with Streptavidin-PE and SARS-CoV-2 antigen specific B memory cells were detected and sorted separately according to Spike/RBD expression (i.e. PE fluorescence), IgG (i.e. IgD and IgM negative cells), CD19 and CD27 expression. Individual cells were seeded in separate plates as single cell micro-cultures on human feeder cells (i.e. CD40L expressing 3T3 cells) in the presence of Epstein-Barr Virus (EBV) (which also stimulate polyclonally memory B cells) and a cocktail composed TLR9 agonist CpG-2006, IL-2 (1000 IU/ml), IL-6 (10 ng/ml), IL-21 (10 ng/ml), and anti-BCR goat antibodies (BCR triggering). Supernatants from the day 14 immortalized B cell cultures were then tested for binding to the Spike trimer protein in the Luminex bead based assay. The different recombinant proteins used in binding assays were produced by transient transfection of either CHO or 293 T mammalian cell lines and enriched to >90% purity from the cell surface supernatants as described in Fenwick et al (J Virol. 2020 Nov 3:JVI.01828-20. doi: 10.1128/JVI.01828-20). In performing the antibody binding assay, Spike coupled beads are diluted 1/100 in PBS with 50 mΐ added to each well of a Bio-Plex Pro 96-well Flat Bottom Plates (Bio-Rad). Following bead washing with PBS on a magnetic plate washer (MAG2x program), 50 mΐ of individual antibodies at different dilution concentrations in PBS were added to the plate wells. Plates were sealed with adhesive film, protected from light and agitated at 500 rpm for 30 minutes on a plate shaker. Beads were then washed on the magnetic plate washer and anti-human IgG-PE secondary antibody (ThermoFisher) was added at a 1/100 dilution with 50m1 per well. Plates were agitated for 45 minutes, and then washed on the magnetic plate washer. Beads resuspended in 80 mΐ of reading buffer were agitated 5 minutes at 700 rpm on the plate shaker then read directly on a Luminex FLEXMAP 3D plate reader (ThermoFisher). Wells with supernatant contained antibody with elevated binding properties to Spike were further profiled for binding to the SARS-CoV-2 SI protein and RBD domain, and for neutralizing activity at different dilutions in the SARS-CoV-2 Spike pseudotyped lentivirus neutralization assay. Antibodies in the individual well supernatants were binned into groups based on their binding to Spike, SI and RBD (Figure 1; Dark grey circles), to Spike and SI alone (grey squares), RBD with lower binding to SI or Spike (triangles) or binding only to Spike trimer (white diamonds). Neutralization activity determined in a 96-well plate assay were antibody supernatant dilutions were mixed with the SARS-CoV-2 Spike pseudotyped lentivirus for 1 hour at 37° C (5% C02) before the addition to 293T ACE-2 cells. These were incubated for a further 72 h, after which cells were lysed and treated with the ONE-Step™ Luciferase Assay System (LabForce) and luciferase activity detected by reading the plates on a Synergy microplate luminometer (BioTek). The percent neutralization value of each antibody supernatant samples performed at two serum dilutions was then plotted as with each of the binned antibody binding classes (Figure 1).

B cells that produced antibody supernatants with the strongest neutralizing activity and those that had distinct binding properties for the Spike, SI and RBD proteins were collected with heavy and light chain antibody sequences cloned. Cloning was accomplished by standard molecular biology methods were cellular RNA was extracted using the NucleoSpin RNA XS kit (Life System Designs), reverse transcription with SMARTScribe™ Reverse Transcriptase kit (Takeda Bio Europe), PCR amplification with Platinum™ Taq DNA Polymerase High Fidelity (Life Technologies Europe) and cloning of DNA inserts corresponding to the heavy and light chain variable regions into a TA cloning vector. The resulting nucleotide sequences and corresponding amino acid sequences of the variable regions and the complementarity determining regions (CDRs) ascertained are listed in Table 1 and 2. These sequences correspond to the neutralizing antibodies termed P5C3, P6E16, P1H23, P1M12, P106, P2B11, P7K18, P1L7, P1L4, MS31, MS35, MS42, P2G3 and P1G17 that are IgGl-type fully human monoclonal antibody.

Example 2

Production of anti-SARS-CoV-2 antibodies

The heavy chain and kappa or lambda light chain sequences identified from the antigen specific B cells producing neutralizing antibody were cloned by standard molecular biology into IgG mammalian expression vectors (e.g. pFUSE expression vectors). Plasmids encoding the anti- SARS-CoV-2 antibodies with CDRS listed in Tables 1 and 2 were co-transfected into the CHO Express mammalian cell line. After incubation of transiently transfected cells for 7 days, the full-length IgGl -based antibodies were purified from the cell culture medium using standard techniques (e.g., a full-length IgGl-based antibody may be purified using a recombinant protein-A column (GE-Healthcare)). This protocol is described in further detail in Fenwick et al J Exp Med. 2019 Jul 1 ;216(7): 1525-1541. doi: 10.1084/jem.20182359.

Example 3

Binding characterization of anti-SARS-CoV-2 antibodies

Binding affinities of the purified anti-SARS-CoV-2 antibodies listed in Table 3 were evaluated for recombinant expressed Spike trimer and RBD proteins in Luminex binding assays. The P5C3, P2G3 and P6E16 antibodies exhibited the highest binding KD values for Spike (55.6, 46.3 & 43.4 ng/ml, respectively) and RBD protein (30.1, 15.7 & 10.4 ng/ml, respectively) relative to all antibodies tested, include REGN10933, REGN10987 (discovered by Regeneron) and S309 (discovered by Vir Biotechnology) reference antibodies (Table 3). The ability to block the interaction between the Spike trimer and the ACE-2 receptor was next evaluated in a Luminex competitive binding assay. To perform the Spike trimer /ACE-2 blocking assay, Spike beads were incubated with different dilutions of the test antibodies with agitation at 500 rpm for 30 minutes on a plate shaker. The ACE-2 mouse Fc fusion protein (Creative Biomart) was then added to each well at a final concentration of 1 μg/ml, re-sealed with adhesive film, protected from light and agitated at 500 rpm for 60 minutes on a plate shaker. Beads were then washed on the magnetic plate washer and anti-mouse IgG-PE secondary antibody (OneLambda ThermoFisher) was added at a 1/100 dilution with 50m1 per well. Following a 30-minute incubation with agitation, beads were washed then read directly on a Luminex FLEXMAP 3D plate reader (ThermoFisher). MFI for each of the beads alone wells were averaged and used as the 100% binding signal for the ACE-2 receptor to the bead coupled Spike trimer. MFI from the well containing the commercial anti-Spike blocking antibody was used as the maximum inhibition signal. In this binding assay, P5C3, P2G3 and P1H23 antibodies exhibited potent inhibition of the Spike /ACE-2 interaction with IC50 values of 55, 52 and 63 ng/ml, respectively (Table 3). These values are at a similar level to the REGN10933 and REGN10987 reference antibodies tested in parallel. All the other neutralizing antibodies described in this submission were capable of completely blocking the Spike/ ACE-2 interaction with the exception of P7K18 that only partially blocking and P2B11 that was non-blocking of the Spike/ ACE-2 interaction. Anti-SARS-CoV-2 antibodies were further evaluated for their ability to bind the Spike trimer protein from the 2002 SARS virus. Of the newly discovered anti-SARS-CoV-2 antibodies, P7K18 and P1L7 bound effectively to SARS Spike along with the S309 reference antibody that was isolate from a patient infected with the original SARS virus (Table 3).

Table 3: Binding studies of anti-SARS-CoV-2 neutralizing antibodies in biochemical assays

* ncomplete inhibition

Example 4

Neutralization characteristics of anti-SARS-CoV-2 antibodies

Antibodies discovered with binding properties for Spike, SI and/or RBD proteins were further characterized in neutralization assays using the SARS-CoV-2 Spike pseudotyped lentivirus or the live SARS-CoV-2 virus. The Spike pseudotyped lentivirus encoding the Luciferase reporter gene was incubated in a concentration response with each of the antibodies for 1 hour and the mixture was then added to 293T cells stably expressing the ACE-2 receptor in a 96-well plate. Following a 72-hour incubation at 37 °C with 5% CO2, cells infected with virus produced elevated levels of luciferase while the presence of neutralizing antibody inhibited viral infection and luciferase production. The inhibition IC50 values for each of the antibodies (Table 4) corresponds with the inhibition curves in Figure 2. In this assay, P5C3, P2G3, P6E16, P106 and P1H23 are the most potently antibodies identified in this application that are more potent than or have equivalent potency to REGN10933 and REGN 10987 antibodies tested in parallel. P1M12, MS35 and B2B11 have neutralizing IC50 values that are slightly higher than REGN10987, while P7K18, P1L7 and P1G17 antibodies are 5 to 8-fold less potent than the REGN10987 reference antibody.

Antiviral potency in the live virus SARS-CoV-2 assay was assessed by incubating different concentrations of antibody with virus for 1 hour followed by transferring the mixture of virus and antibody to Vero E6 cells in a 96-well plate. Three days later, plates were washed and live cells that remained adherent were stained with dye. Antibodies with neutralizing activity protected cells from infection and prevented cell lysis due to the cytopathic effect of the virus. Densitometry analysis of the stained plates corresponded with the presence of cells that were protected from infection and was used to calculate the IC50 values for the different antibodies tested (Table 4 and Figure 3). In these tests, P5C3 and P2G3 were the most potent neutralizing antibody discovered that was 6 to 9-fold more potent than REGN10933 and REGN10987 tested in parallel. P6E16 was also approximately 2 to 3-fold more potent than both Regeneron antibodies, while P1H23, MS42 and P106 antibodies were equipotent with the REGN10933 antibody. The P1M12, P2B11, P7K18, P1L7 and P1G17 antibody clones also inhibited the live SARS-CoV-2 virus at IC50 values that ranged between 596 to 8800 ng/ml.

Table 4: Activity of anti-SARS-CoV-2 antibodies in the Spike pseudoviral neutralization assay and the live virus SARS-CoV-2 cytopathic effect neutralization assay.

* SARS-CoV-2 virus with D614G mutation

Example 5

Comparative competitive binding studies of different antibodies to recombinant SARS- CoV-2 RBD protein

Competitive binding studies allowed for the mapping of competitive, partially competitive and non-overlapping binding of different anti-SARS-CoV-2 antibodies to the RBD protein. The competitive binding assay was performed in a similar manner to the Spike/ ACE-2 assay except that 20 μg/ml of the indicated competitor antibody was incubated with RBD coupled beads for 30 minutes followed by the addition of 0.5 to 2 μg/ml of the indicated biotinylated antibodies. Beads were washed and the RBD bound biotinylated antibody was detected with PE labeled Streptavidin (Sigma). Direct completion between the two antibodies tested resulted in low level PE fluorescence associated with the RBD beads while non-competitive binding gave fluorescence signals that were comparable to controls where biotinylated antibodies were incubated with RBD beads in the absence of competitor. Biotinylated antibodies were prepared using the EZ-link NHS-PEG biotinylation kit (Pierce ThermoFisher) according the manufacturers protocol. Based on these studies, P5C3, P6E16, P1H23, MS42 and P1M12 antibodies have overlapping binding epitopes with the REGN10933 antibody. Of these, P5C3, MS42 and P1H23 antibodies have non-overlapping or partially overlapping epitopes with the REGN10987 and S309 antibodies. The P2G3, P106, MS35 and P2Bll antibodies bound RBD at a non-overlapping epitope with the REGN10933 antibody and competitively with the REGN10987 and S309 antibodies. P2B11 binds a distinct epitope compared to P2G3, P106, MS35 and REGN10987 since it does not bind competitively with the ACE-2 protein (Table 5). The P7K18, MS31 and P1L7 antibodies bind dissimilar epitopes on RBD since these antibodies did not block the binding of REGN10933, REGN10987 and S309 antibodies. P1G17 exhibited a distinct binding pattern that overlapped primarily with REGN10933, partially with REGN10987 and was non-overlapping with S309.

Amongst the newly discovered anti-SARS-CoV-2 antibodies, the most potent neutralizing antibody discovered, P5C3, can bind the RBD protein concomitantly with P2G3, P106, MS35, MS3 1, P2B11, P7K18 and P1L7 antibodies. These tests indicate that P5C3 has the possibility to act in combination with these other antibody clones in neutralizing the SARS-CoV-2 virus. Neutralizing the virus through binding to different epitopes has the potential effect of greater neutralization potency, reduced chance of developing viruses with mutations that confer resistance and greater breadth in neutralizing viruses with polymorphism in the general population. Based on the neutralizing activity in the live virus SARS-CoV-2 CPE neutralization assay and competitive binding studies, the best antibody combinations identified is either P5C3 with P2G3, P5C3 with P106 or P5C3 with MS35, which are all predicted to be superior to the REGN10933 /REGN10987 combination discovered by Regeneron. The combinations of P5C3 with MS31, P5C3 with P2B11, P5C3 with P7K18 would also be anticipated to provide potent antiviral profiles against the SARS-CoV-2 virus with similar potency compared to the REGN10933 / REGN10987 combination. The binding epitope of P5C3 is also partially overlapping with P1G17.

P2G3 is the second most potent neutralizing antibody disclosed in this application and can bind the RBD protein concomitantly with P5C3, P6E16, P1H23, MS42 and P1M12. P2G3 binds RBD competitively with P106, P7K18 and P1G17. Based on the neutralizing activity in the live virus SARS-CoV-2 CPE neutralization assay and competitive binding studies, the best antibody combinations identified is either P2G3 with P5C3, P2G3 with P6E16, P2G3 with P1H23, P2G3 with MS42 or P2G3 and P1M12, which are all predicted to be superior to the REGN10933 / REGN10987 combination discovered by Regeneron. The combinations of P2G3 with MS31, P2G3 with P2B11, P2G3 with P7K18 would also be anticipated to provide potent antiviral profiles against the SARS-CoV-2 virus.

P6E16 is the third most potent neutralizing antibody disclosed in this application and binds competitively with REGN10933, P5C3, P106, P2B11 and P1G17. P6E16 binds RBD at a nonoverlapping epitope with REGN10987, P7K18 and P1L7, which may be considered as potential partner antibodies to be used in an anti-SARS-Cov-2 combination therapy.

The administration of a cocktail (a combination) of two or more antibodies binding to distinct epitopes on the Spike turner are expected to: 1) have a more potent effect at neutralizing the SARS-CoV-2 virus, 2) help to prevent the development of resistant virus to one of the neutralizing antibodies administered in the cocktail (the combination) and 3) have enhanced breadth overall in the neutralization of circulating strains of the SARS-CoV-2 virus that have mutations in the Spike protein that alter the binding and/or neutralization activity associated with one of the antibodies used in the cocktail.

Given that the best anti-SARS-CoV-2 antibody cocktail could come from the combination of neutralizing mAbs identified by different groups or companies, a more limited competitive binding analysis has been performed between some of the above-mentioned best mAbs and a panel of authorized and clinically advanced mAbs. These advanced mAbs include those identified by Regeneron (REGN10933 and REGN10987), AstraZeneca (AZD8895 and AZD1061), Adagio (ADG-2) and Vir/GSK (S309/Sotrovimab). These studies, outlines in Table 6, show that REGN10933, P5C3 and AZD8895 are strongly competitive with one another in binding, but are not competitive with REGN10987, AZD1061, P2G3, or Vir-S309. ADG2 has an intermediate profile that is overall blocking or partially blocking with REGN10933, REGN10987, P5C3 and AZD8895. Interestingly, adding ADG2 in excess as a competitor to the Spike RBD protein either completely or partially blocked binding of all other mAbs testing in this study. These studies are consistent with P5C3 having a complementary binding profile with REGN10987, AZD1061, P2G3 and Vir-S309 mAbs.

Focusing on the second highly potent anti-SARS-CoV-2 mAh candidate, P2G3 neither competes, nor blocks Class 1 or Class 2 mAbs (Greaney/Bloom, Nature; https://doi.org/10.1038/s41467-021-24435-8) P5C3, REGN10933 and AZD8895 for binding to the Spike RBD (Table 6). Similarly, P2G3 bounding to RBD is non-competitive with ADG2 mAh binding but bound ADG2 partially inhibits binding of P2G3. The remaining antibodies evaluated are all Class 3 mAbs that bind to the core region of RBD. P2G3 bound competitively with REGN10987, AZD1061 and Vir-S309/Sotrovimab. As a differentiating feature between Class 3 mAbs, REGN10987 bound RBD inhibited binding of AZD1061 but only showed partially blocking of P2G3 and Vir-S309/Sotrovimab mAbs. Additionally, Vir- S309/Sotrovimab bound RBD blocked binding of P2G3 but was only partially blocking of REGN10987 and AZD1061. The competitive binding profile of the different mAbs for the Spike/ACE2 interaction is also shown in Table 6, where REGN10933, P5C3, AZD8895, ADG2, REGN10987 and AZD1061 all show complete blocking activity. P2G3 is completely blocking of ACE2 binding to the 2019-nCoV Spike trimer protein but only partial blocking of ACE2 binding to Spike from Alpha, Gamma and Omicron variants of concern. In contrast, Vir-S309 binds Spike in a manner that is non-blocking of ACE2 binding. This suggests that at least with select variant including Omicron, virus inhibition by P2G3 may not be solely mediated through the Spike-ACE2 interaction, but rather a mechanism analogous with that of S309/Sotrovimab, reported to be through induction of Spike trimer cross-linking, steric hindrance, aggregation of virions and/or inhibiting viral membrane attachment through C-type lectin receptors. As such, P2G3 exhibited a unique profile relative to the mAbs tested with traits that overlapped with both AZD1061 and Vir-S309/Sotrovimab.

Table 5: Antibody competitive binding studies with SARS-CoV-2 RBD to define competitive, partially overlapping and non-overlapping binding epitopes between antibody pairs. Antibodies added in excess to the RBD are shown in the left had column while the staining biotinylated antibodies are displayed in the top row of the table. Competitive antibody pairs have percent binding less than 35% for the indicated biotinylated antibody are shown as dark boxes. Partially overlapping epitopes have percent binding between 36 to 70% for the indicated biotinylated antibodies and have white background. Non-competitive antibody pairs with non-overlapping epitopes that are able to co-bind to RBD have percent binding of greater that 70% and are displayed with grey boxes.

Table 6: Antibody competitive binding studies with SARS-CoV-2 RBD to define competitive, partially overlapping and non-overlapping binding epitopes between antibody pairs. Studies performed as outlined in Table 5 except that competitive is defined as <25% co-binding and partially competitive is defined as 25%-70% co-binding of antibody pairs. Box with strips indicates incomplete blockade of the ACE2 interaction with Spike trimer protein from Alpha, Gamma and Omicron variants of concern.

Competitive binding of mAbs for Spike RBD Example 6

Activity of anti-SARS-CoV-2 antibodies in blocking the Spike/ACE-2 interaction using Spike proteins with mutations found in circulating variants of the SARS-CoV-2 virus.

The wild type (WT) version of the trimeric Spike proteins or mutant versions expressing amino acid substitutions (M153I, N439K, S459Y, S477N, S477R, E484K, or N501T) reported for circulating variants of the SARS-CoV-2 virus were expressed as recombinant proteins in transiently transfected CHO cells and purified using Strep-Tactin affinity matrix. The Spike proteins were individually coupled to Luminex beads and stored at 4 °C until use. In the Spike/ACE-2 binding assay, a concentration response of the Fab fragment for a neutralizing antibody was incubated with Spike beads for 30 minutes followed by the addition of 1 μg/ml of human ACE-2 ectodomain fused to mouse Fc protein. Beads were then washed and a positive interaction of ACE-2 bound to Spike (Wild type or mutant forms) was detected with a PE-labeled anti-mouse secondary antibody. Neutralizing antibody Fabs capable of binding Spike or Spike mutants and inhibiting the Spike/ACE-2 interaction at a given concentration of Fab resulted in the concentration response curves shown in Figure 4 and Table 7. These data show that P5C3 and P6E16 are the most potent antibody Fabs in disrupting the Spike WT /ACE- 2 interaction. Importantly, P5C3 has the highest potency of all antibody Fabs tested with an IC50 of < 200 ng/ml against all of the Spike mutants in the Spike/ACE-2 interaction assay. In addition, P5C3 shows only minor losses in activity for the mutant forms of Spike relative to wild type protein with a maximum shift of 5-fold for Spike protein with the E484K substitution. In contrast, REGN10933 and REGN10987 show 16- to 24-fold losses in potency against Spike proteins with E484K and N439K substitutions, respectively. These data provide strong evidence that P5C3 is an ultrapotent SARS-CoV-2 neutralizing antibody that will maintain antiviral potency against many of the most prevalent variant strains with mutations in the viral Spike protein.

Table 7: Activity of anti-SARS-CoV-2 antibody Fab fragments in blocking the interaction between the ACE-2 protein and trimeric Spike proteins expressed as wild type or mutant versions with the indicated amino acid substitutions

Example 7

Binding characterization of anti-SARS-CoV-2 antibodies to 2019-nCoV and Spike mutations found in variants of concern

Affinities of the purified anti-SARS-CoV-2 antibodies listed in Table 8 were evaluated for binding to recombinant expressed Spike trimer and recombinant Spike trimer expressed with mutations found in SARS-CoV-2 variants of concern in Luminex binding assays as described above.

Table 8: Binding of anti-SARS-CoV-2 antibodies to 2019-nCoV and Spike mutations found in variants of concern in Luminex bead based assay

The P5C3 and P2G3 exhibited the highest overall binding to the different Spike trimer proteins that included mutations found in SARS-CoV-2 variants of concern including B.l.1.7 (UK variants) B.1.351 (South African variant), P.l (Brazilian variant) and the L452R mutation identified in the CAL.C20 (Californian variant) (Table 10). P5C3 has a binding ICso of 15 to 36 ng/ml and P2G3 ICxo of 29 to 61 ng/ml against all the Spike proteins tested, which are both superior to the benchmark control antibodies tested in parallel (i.e. REGN10933 with a range of 13 to 96 ng/ml, REGN10987 with a range of 15 to 5443 ng/ml and S309 with a range of 162 to 420 ng/ml). Apart from P5C3 and P2G3, the anti-SARS-CoV-2 antibodies P106, MS31, MS35 and MS42 exhibited distinct binding profiles against the different variant Spike proteins that were similar in affinity profile compared to the benchmark antibodies that are currently in clinical trial or approved for use in patients.

Binding studies were then repeated and analyzed in parallel with our best mAbs and with a panel of authorized and clinically advanced mAbs from Regeneron (REGN10933 and REGN10987), AstraZeneca (AZD8895 and AZD1061), Adagio (ADG-2) and Vir/GSK

(S309/Sotrovimab). These studies show that P5C3 and P2G3 have the most potent and broadly active binding profile of all mAbs evaluated in parallel. These differences are strongly evident in mAh binding to Spike trimer from the Omicron BA.1 variant and sub-variants (BA.1.1 and BA.2) that encode up to 37 mutations, deletions or insertions relative to the original 2019-nCoV Spike protein (Table 10). The binding studies demonstrated that P2G3 and P5C3 mAbs exhibited the highest affinity across all the Spike VOCs with improved ICxo values of 2.5 to 9.5-fold relative to ADG-2, AZD1061, Vir-S309/Sotrovimab, and >60-fold improved affinity relative to AZD8895, REGN10933 and REGN10987 in binding the Omicron variant Spike (Table 9). As previously reported, REGN10933 showed a >10-fold loss in binding for Spike from Beta and Gamma VOCs relative to the original 2019-nCoV virus.

Table 9: Binding of anti-SARS-CoV-2 antibodies to 2019-nCoV and Spike mutations found in variants of concern in Luminex bead based assay

Table 10. Amino acid substitutions and deletions on SARS-CoV-2 variants of concern

* Indicates that variant has different sub-populations that may or may not include the indicated amino acid substitutions.

Example 8

Neutralization characteristics of select anti-SARS-CoV-2 antibodies against SARS-CoV- 2 and SARS-CoV-2 variants in a live virus cytopathic effect assay

Antiviral potency of select antibodies was evaluated in the live virus cytopathic effect assay (CPE) assays performed with SARS-CoV-2 viruses encoding the D614G mutation, the B.l.1.7 (UK) variant, the B.1.351 (South African) variant and a mink (var 16) variant (Table 10). The P5C3, P2G3 and P6E16 antibodies tested in this assay were produced as LS variants with M428L and N434S substitutions in the antibody IgGl Fc domain that is reported to confer an extended biological half-life in humans. In these tests, P5C3 and P2G3 are the most potent neutralizing antibody discovered with a broad potency neutralizing all viral variants with an ICso value less than 22 ng/ml. By comparison, REGN10933 exhibited an almost complete loss in activity against B.1.351 and mink viruses, REGN10987 was ~6-fold less potent against the most common D614G viral mutant in circulation and S309 was >30-fold less potent against all viral variants tested (Table 11 and Figure 5 A-D). Given that P5C3 and P2G3 bind distinct, non-competitive sites on the Spike protein RBD, these two antibodies represent ideal partners for combined administration as a combination therapy, exhibiting a more potent neutralizing activity against current viral variants and/or suppress the development of resistant variants of the SARS-CoV-2 virus. The MS35 antibody also exhibited a highly potent and broad neutralization profile inhibiting all viruses tested with ICxo values between 17 and 135 ng/ml, a profile highly similar to REGN10987. Additionally, MS35 binds an epitope on the Spike RBD that is non-competitive with P5C3. As such, these two antibodies could be administered as a combination therapy, exhibiting a more potent neutralizing activity against current viral variants and/or suppress the development of resistant variants of the SARS-CoV-2 virus.

Furthermore were evaluated neutralization profiles of individual and mAh combinations of the above-mentioned best mAbs and a panel of authorized and clinically advanced mAbs against the live Omicron BA.1, Omicron BA.2 and Delta variant viruses, which are currently the most common circulating SARS-CoV-2 viruses. While the authorized and clinically advanced mAbs all showed potent activity against the Delta variant (Figure 5E), P2G3 was the most potent mAh evaluated against the Omicron virus with ECxo values of 0.035 μg/ml and 0.014 μg/ml against BA.l and BA.2 sub-variants, respectively (Figure 5F and 5G). The P2G3/P5C3 combination similarly showed comparable neutralization activity to P2G3 alone against the Omicron sub-variants with an ECxo of 0.039 μg/ml and 0.027 μg/ml total mAh for BA.l and BA.2, respectively. These activities were superior to all comparator mAh and mAh combinations, including the AZD1061+AZD8895 mAh combination (AZD cocktail) that showed a 23-fold reduced activity (ECxo of 0.820 μg/ml), Sotrovimab with a 60-fold reduced activity (ECxo of 2.1 μg/ml), ADG2 with an 89-fold reduced activity (ECxo of 3.1 μg/ml) and >500-fold reduced activity of the REGN cocktail (REGN10933+REGN10987), relative to the P2G3 mAh monotherapy against Omicron BA.l (Table 12). Of note, the herein presented Omicron variant live virus neutralizing activities are consistent with recent publications for the same mAbs tested in inventor's panel.

In addition, P2G3 and P5C3 were evaluated either alone or combined compared with the panel of authorized and/or clinically advanced mAbs in a neutralization assay performed with lentivirus pseudotyped with the Omicron Spike protein. P2G3 showed the most potent neutralizing activity against lentiviruses pseudotyped with Omicron BA.l, BA.2.12.1 that encodes additional RBD mutations L452Q and BA.4 that encodes L452R, F486V and R493Q mutations with an ECso value of 0.038 μg/ml, 0.011 μg/ml and 0.012 μg/ml, respectively (Figure 5H-J). In studies performed in parallel, P2G3 exhibited the most potent neutralizing activity against Omicron BA.1 Spike pseudovirus and was >42-fold more potent than ADG-2, AZD1061, AZD8895, REGN10933 and REGN10987 mAbs and 19-fold more potent than Sotrovibmab (Figure 5H). P5C3, was the second most potent mAh with anECso value of 0.223 μg/ml. Unexpectedly, the P2G3/P5C3 combination provided a small-enhanced activity over P2G3 alone in neutralizing the Omicron Spike pseudovirus, showing an ECxo value of 0.024 μg/ml. This 2-fold improved activity was observed with only 0.0115 μg/ml of the more potent P2G3 mAh in the combination, providing evidence for cooperative inhibition of the Omicron Spike pseudovirus when combined with P5C3 (Figure 5H). This enhanced activity of the P2G3/P5C3 combination was also observed with the Omicron BA.2.12.1 sub-variant, showing ECxo value of 0.006 μg/ml for the combination, 0.011 μg/ml for P2G3 alone and 0.116 μg/ml for P5C3 alone (Figure 51). Together, this neutralization assay data demonstrates that P2G3 has the best overall neutralization profile across all past and current variants of concern. These studies also show that P2G3 + P5C3 is the mAh combination with the most potent and broad neutralization profile, displaying good complementarity in neutralizing all the strongly resistant SARS-CoV-2 variants of concern, including the Omicron sub-variants.

Table 11: Neutralizing activity of antibodies against different SARS-CoV-2 viral variants in the live virus cytopathic effect assay

Table 12: Neutralizing activity of antibodies against different SARS-CoV-2 viral variants in the live virus cytopathic effect assay or neutralizing assays using lentiviruses pseudotyped with Spike protein from the Omicron variant. Example 9

Evaluations of P2G3 and P5C3 mAbs synergistic activity in the Spike- ACE2 interaction assay, antibody dependent cellular phagocytosis (ADCP) assay and antibody dependent cellular cytotoxicity assays (ADCC)

Using the Spike-ACE2 biochemical interaction assay, compared the activity of P2G3, P5C3 and combinations thereof in blocking ACE2 binding to either 2019-nCoV or Omicron subvariant Spike trimer was compared. Although assays performed with 2019-nCoV Spike, showed additive ACE2 blocking activity for P2G3/P5C3 used in combination (Figure 6A), the mAh cocktail showed clear signs of synergy in blocking ACE2 binding to Omicron Spike, where P2G3/P5C3 had an ICso values 0.080 μg/ml, P2G3 0.099 μg/ml and P5C3 0.140 μg/ml (Figures 6B and 6E). Furthermore, with the Omicron BA.1, BA.1.1 (R346K) and BA.2, the P2G3/P5C3 combination reached -100% of Imax in blocking ACE2 binding to Omicron Spike at 1 μg/ml total mAh, while P2G3 alone only blocked 75% of ACE2 binding and P5C3 alone reached an Imax of 100% at >20 μg/ml (Figures 6B-D). This cooperative binding is also observed as a higher inhibition curve Hill slope value for the P2G3 + P5C3 combination as compared to P2G3 or P5C3 individual mAbs.

Synergistic binding of P2G3 and P5C3 to Spike was further studied in an ADCP assay. In this in vitro assay, Spike trimer coated fluorescent beads are mixed with different concentrations of P2G3 and/or P5C3 then incubated with U937 effector cells. This monocyte cell line expresses high levels of Fc-gamma receptors (yR) including FcyRI and FcyRII capable of inducing phagocytosis of opsonized viruses or beads coated with the Spike antigen as in the case of our assay. To fully assess synergistic activity between the antibody pair, ADCP activities were profiled in dose-response matrices of P2G3 LS and P5C3 LS. Evaluated alone, P2G3 and P5C3 mAbs showed ADCP ICxo activities of 0.074 and 0.010 μg/ml, respectively. However, when combined, ICso ADCP activity was achieved with 0.012 μg/ml of P2G3 mixed with 0.00015 μg/ml of P5C3 (Figure 6C). Dose-response matrices were further evaluated using MacSynergy II software where a strong and highly significant synergy volume of 271 [mAh] ² % was calculated for the P2G3 /P5C3 combination in the ADCP assay (Figure 6D).

Furthermore was evaluated the ADCC activity of P2G3 and P5C3 compared to advanced mAbs from Regeneron and AstraZeneca. ADCC experiments were performed with five replicates per condition using effector cells from five different healthy donors that were mixed with CEM NKR Spike target cells incubated with 300 ng/ml of the indicated human IgG mAbs (Figure 6E). The P2G3 LS mAb exhibited strong activity in the ADCC assay, exhibiting significantly superior killing of Spike positive cells compared to all other anti-Spike mAbs tested. No further improvement of ADCC activity was observed with the combination of P5C3 and P2G3 mAbs. It is also important to note that ADCP and ADCC Fc-mediated antibody functional activities were monitored using P5C3 LS N100Q and P2G3 LS N54S/N56Q mAbs described in Example 12 below.

Example 10

Cryo-electron microscopy structure of P5C3 Fab in complex with the Spike trimer

To understand the structural basis of P5C3 potent neutralization of SARS-CoV-2 variants of concern (VOC), the complex formed by stabilized SARS-CoV-2 Spike trimer (containing the D614G mutation in the ectodomain backbone) and P5C3 Fab fragments was characterized using single particle cryo-electron microscopy (Cryo-EM). Dose-fractionated images were recorded with a FEI Titan Krios (Thermo Fisher), operated at 300kV, and equipped with a Gatan Quantum-LS energy filter (20 eV zero-loss energy filtration) followed by a Gatan K2 Summit direct electron detector. The EM map was generated by performing non-uniform refinement followed by local refinement of the Fab-RBD interacting region and finally an atomic model was built by positioning the Ca chains for the Fab and Spike. An initial model was built in Coot using the coordinates of the SARS-CoV-2 Spike with three Fab molecules bound (PDB ID: 7K4N). The final model was validated using the comprehensive validation method in PHENIX.

This structure, resolved at a resolution of 3.7A, showed that the P5C3 is a class I neutralizing antibody with its target epitope overlapping with the ACE2 receptor-binding site of Spike (Figure 7A-B) with RBD in the open only conformation. Upon analysis of the paratope/epitope interaction, it was discovered that the P5C3-Spike interface covers a large region of about 600 A ² surface centred on Phe486 and involving 23 amino acids of P5C3 and 21 amino acids of the Spike RBD. This result is consistent with the strong measured affinity and potency of the mAb. Moreover, it could be determined that P5C3 binds its epitope through five of its complementarity-determining regions (CDRs), namely CDRs HI, H2 and H3 of the heavy chain and LI and L3 of the light chain (Figure 7C). Analysis of the CDR loop contacts revealed that for the light chain CDRs, Tyr32 in LI and Trp96 in L3 provide the major binding interactions by contacting Pro479 and Phe486, respectively, on the Spike surface. In the CDR H3, Pro95, GlylOO, SerlOOA, CyslOOB, AsplOOD and PhelOOF make multiple contacts with the RBD thumb region (residues 475-489), while in CDR H2, Trp50 provides the main paratope-epitope interaction. Moreover, CDRs form multiple contact points by hydrophobic interactions and aromatic contacts with residues Phe456, Tyr473, Phe486 and Tyr489 of Spike. This binding mode is unusual for paratope-epitope interactions owing to the broad spatial separation of CDRH3 and CDRL3. Interestingly, the mAh also makes several contacts with the antiparallel b5, b6 strands of Spike (residues 451-456 and 491-495), a domain that should not be subjected to the development of resistance mutations owing to its essential folding function. To understand further why P5C3 binding is not affected by mutations harboured by SARS- CoV-2 variants, structure of the inventors was superimposed with that solved of the ACE2- RBD interaction (PDB ID 6M0J). ACE2 covers around 860A ² on the RBD, compared with 600A ² for P5C3 where P5C3 interacts with the RBD ridge at a 90-degree angle compared with 130-degree for ACE2 (19) (Figure 7DE). Importantly, -70% (414 A ² out of 600 A ² ) of the P5C3 buried surface area is shared with the ACE2 site on RBD. P5C3 and ACE2 also share key interactions with Leu455, Phe456, Ala475, Gly476, Ser477, Glu484, Phe486, Asn487, Tyr489 and Gln493 of the RBD, which constitute a core for tight binding. Indeed, these residues form a hydrophobic patch surrounding Phe486 on the RBD with Phe486 forming interacts with Gln24, Leu79, Met82 and Tyr83 of ACE2 (Figure 7E). Furthermore, additional critical residues necessary for RBD interaction with ACE2s are blocked by P5C3, such as Phe456 and Gln493.

Further, the P5C3 binding mode was compared to that of leader mAh candidates currently in clinical trials REGN10933, REGN10987 (PDB ID 6XDG) and LY-C0VOI6 (PDB ID 7C01). It was recently demonstrated that the neutralizing activity of these three mAbs could be negatively affected by mutation identified in circulating variants including K417T/N, N439K, S477N, E484K and N501 Y have been reported to increase their affinity to ACE2 and/or render the mAbs LY-CoV555, REGN10933 and REGN10987 less efficient. These results suggest that the multiple contacts made by P5C3 with 21 Spike amino acids including the large cluster of interactions extending from Ala475 to Gly496 mitigate losses in affinity that would result from some individual changes. As well, mutations conferring resistance to some of these other mAh are distal to the RBD/ACE2 interaction site, with minimal effect on ACE2 binding and by extension on P5C3 recognition. Taken together, these observations suggest that virus variants, harbouring mutations in the P5C3 encoded epitope would suffer from an important fitness cost. The P5C3 heavy and light chain CDR residues outlined above that interact with the Spike RBD domain are furthermore identifies as being important for the tight binding affinity and anti- SARS-CoV-2 neutralization activity of the P5C3 antibody. As such, it is anticipated that many of the conservative amino acid substitutions at these residues will have comparable binding affinities and/or neutralization activity to the parent P5C3 antibody described within.

Example 11

Cryo-electron microscopy structure of P5C3 Fab and P2G3 Fab in complex with the Omicron variant Spike trimer

To fully understand the molecular features that endow P2G3 and P5C3 potent neutralization of Omicron Spike, single particle cryo-EM reconstruction of the Omicron Spike trimeric ectodomain bound to both Fabs, at a 3.04 A resolution, was performed (Figure 8A). Fabs bind simultaneously at distinct sites on the Omicron Spike with a majority of particles showing three P2G3 Fabs and one P5C3 Fab binding. The P5C3 interacting region with the Omicron RBD is identical to that described when bound to a wild-type RBD (Fenwick, Turelli et al. 2021; 109814. 10.1016/j.celrep.2021.109814.). The P2G3 binding mode is different and the mAh can be classified as a Class 3 neutralizing Ab recognizing an epitope on the SARS-CoV-2 RBD (Barnes, West et al. 2020), distinct from the receptor-binding motif. Together, this mAh cocktail buries a surface of greater than 1200 A ² (Figure 8B), a structural observation that may explain the enhanced activity when both P2G3 and P5C3 are applied against the Omicron variant (Figure 5E-G and Table 12). The structure also rationalizes the stoichiometric binding of P2G3 Fab to the trimer as it is able to bind both up and down states of Omicron Spike (Figure 8A). To characterize the P2G3 paratope and epitope interface in detail, it was performed local refinement of the P2G3 Fab-RBD interacting region and reached a resolution of 3.84 A with well-defined density, allowing clear interpretation of sidechains position. The P2G3 paratope is composed of four complementarity-determining region (CDR) loops that bury a surface area of around 700 A ² at the back of the RBD. The interactions are mediated through electrostatic and hydrophobic contacts (Figure 8 C-D) and involve sixteen residues of the RBD, mainly bound by the heavy chain of the P2G3 mAh. The 18-residue-long CDRH3 sits at the top of a loop that comprises residues 344-347, and also contacts the amino-acids at the limit of the 5- stranded b-sheet (residues 440-451), overall accounting for more than 60% of the buried surface area (431 A ² ) (Figure 8 C-D). The interactions between P2G3 and the Omicron RBD are conserved in both RBD-up and RBD-down states. CDRH2 extends the epitope by interacting with R346 that is engaged by residue W53 by a potential cation-pi interaction (Figure 8D). The only contact from the light chain derives from the CDRL1 Y32 forming van der Waals interactions with V445 of the RBD (Figure 8C). Moreover, P2G3 is only observed to contact RBD amino acid residues and the distance to the nearest atom of the glycan branch is ~10A from P2G3. Importantly, the epitope defined by our structural studies rationalizes the potent neutralizing activity of P2G3 against the Omicron variant relative to other Class 3 mAbs. Omicron mutations S371L, N440K, G446S and the minor R346K sub-variant are all situated outside of, adjacent to or have little effect on recognition of the P2G3 -binding epitope, whereas two or more of these mutations directly impinge on epitopes recognized by REGN10987, AZD1061 and S309/Sotrovimab (Figure 8E). Furthermore, P2G3 displays a unique binding orientation on the RBD with its Fab angling away from most of these Omicron mutations (Figure 8F). In modelling the observed angles of attack of various Class 3 mAbs, it is likely that REGN10987 only binds to the up-RBD form while AZD1061 binds one up- and one down- RBD form, with steric hindrance blocking the third RBD site on the Omicron Spike trimer. In contrast, P2G3 and S309/Sotrovimab likely binds both the up and down forms of Omicron RBD without clashes (Figure 8G), a characteristic that may contribute to the largely conserved and high potency of P2G3 across VOCs.

Based on these structural data and escape mutant analysis, K444T and K444Q substitutions were identified as potential mutants that escaping P2G3 neutralizing activity. However, analysis of the GISAID EpiCoV database indicates these mutations exist at low frequencies (<0.1). Nevertheless, as the P2G3 epitope does not overlap with the ACE2 -binding site, it was reasoned that a combination of P2G3 with P5C3, the above described highly potent class I neutralizing mAh, may provide additive effects and efficiently neutralize virus variants that could potentially escape P2G3 neutralization.

Example 12

P5C3 and P2G3 both confers strong in vivo prophylactic protection from SARS-CoV-2 infection in the hamster challenge model

The neutralizing potency of P5C3 was evaluated in vivo in a prophylactic hamster challenge model of SARS-CoV-2 infection. Animals were administered an intraperitoneal injection of 5.0, 1.0 or 0.5 mg/kg of P5C3 or 5 mg/kg of an IgGl isotype control and challenged two days later (Day 0) with an intranasal inoculation of SARS-CoV-2 virus (2.4xl0 ⁶ PFU dose) (Figure 9A). Four days later, lung from control animals contained between 10 ⁴ and 5xl0 ⁶ TCID50 per mg of tissue, whereas infectious virus was undetectable in lung from hamsters treated with 5.0 and 1.0 mg/kg of P5C3, which displayed antibody plasma levels >12 μg/ml (ranging from 12.2 to 16.4 μg/ml) at the time of viral inoculation (Figure 9 B-C). Animals administered 0.5 mg/kg P5C3 had median plasma antibody levels of 6.7 μg/ml, and 4 out of 7 also exhibited undetectable infectious virus in the lung, while the remaining 3 showed a ~2 log reduction in TCID5o/mg lung tissue compared to the isotope mAb-treated controls. Significant reduction of viral RNA levels was also observed in all P5C3-treated groups (p<0.001) with a ~4 log reduction in viral genome copies per mg of lung tissue compared to control animals.

In the P2G3 in vivo prophylactic hamster challenge model of SARS-CoV-2 infection, animals were administered intraperitoneally 5.0, 1.0 or 0.5 mg/kg of P2G3, 5 mg/kg of REGN10933 or 5 mg/kg of an IgGl isotype control (Figure 9A). Four days later, lung from control animals contained between 10 ⁴ and 10 ⁶ TCID50 per mg of tissue, whereas infectious virus was undetectable in lung from almost all P2G3 treated hamsters. Only 1 out of 6 hamsters in the 0.5 mg/kg group had detectable levels of infectious virus that none the less showed a -1.5 log reduction in infectious virus compared to the isotype mAb-treated control animals (Figure 9D). Complete prophylactic protection in these studies was observed with P2G3 mAh plasma levels >6.2 μg/ml at the time of viral inoculation. Furthermore, all P2G3 treatment groups showed a significant -4 log reduction of genomic viral RNA levels (p<0.009) compared to control animals (Figure 9E).

Example 13

Identification of amino acid substitutions in P5C3 and P2G3 that provide a non-inferior neutralizing activity

Although both P5C3 and P2G3 are fully human antibodies derived from memory B cells of COVID-19 patients and/or vaccinated donors, heavy and light chain germline residues and somatic mutations acquired in both CDR and frame regions during antibody optimization in vivo can sometimes pose potential risks to the large scale production and developability of a monoclonal antibody drug product. In order to minimize these risks, gene engineering to introduce individual or combinations of mutations were incorporated into mammalian expression vectors for chain antibody sequences that resulted in the desired amino acid substitution(s). Antibodies were produced through transient transfection of ExpiCHO cells and purified from the cell culture supernatants six days later through Protein A affinity chromatography using standard methods. P5C3 antibodies produced with heavy chain mutations at positions N58, M74 and N100 were evaluated for neutralizing activity in a SARS- CoV-2 Spike D614G pseudoviral assay in comparison with the WT P5C3. Representative inhibition curves in Figure 10A show that N57Y, N57V, N57Q, N57L N57H, N100Q and N100Y (Sequence ID No. 113, 114, 115, 116, 117, 133, 134) have equivalent potency compared to WT P5C3 while M74Y and M74L mutations (Sequence ID No. 122, 123) in P5C3 exhibit slightly reduced potency. A serious of additional mutations in P5C3 were evaluated at residues T30, G54, S55, G56 and R72 with most showing equivalent or reduced activity (Figure 10B and Table 13-16)

In a similar manner, amino acid substitutions in the heavy chain were generated in the P2G3 antibody at residues N54, N56, D103 and N110 (Table 17-19). The N54S, N54H (Sequence ID No. 167, 164; Figure 11A), N56Q, N56H and N56Y (Sequence ID No. 168, 169, 171; Figure 11B) substitutions in P2G3 all exhibited neutralizing activity that was equivalent to the WT antibody. In contrast, the remaining substitutions evaluated a N54 and N56 and all substitutions evaluated at residues D103 and N110 (Table 17-19; Figure 11) exhibited reduced neutralizing activity and/or incomplete inhibition as evident by a Cmax less than 80%.

Following these studies, the P5C3 LS N100Q (Sequence ID No. 133) and P2G3 LS N54S/N56Q IgGl (Sequence ID No. 167 / 168) antibodies with the LS extended half-life mutation in the IgGl Fc domain (M428L / N434S) were produced and compared to the WT antibodies in binding to 2019nCoV, Alpha, Beta, Gamma and Delta Spike trimers proteins in a Luminex beads based assay (Figure 12, A to E, respectively). These studies show that the amino acid substitutions in P5C3 and P2G3 do not detrimentally affect the binding affinity to the tested Spike protein variants. In a likewise fashion, P5C3 LS N100Q and P2G3 LS N54S/N56Q demonstrated equivalent neutralization of Spike 2019nCoV D614G (Figure 13A), Spike Beta variant (Figure 13B) and Spike Delta variant (Figure 13C) pseudotyped viruses compared to the WT P5C3 and P2G3 antibodies. Overall, these substitutions are shown to have non-inferior binding and neutralizing activity compared to the WT antibodies with reduced risk of post- translational modification during antibody manufacturing and storage. Potential advantages with the P5C3 LS N100Q antibody relative to the WT sequence is reduced risk glycosylation at the Asn-x-S/T/C) motif. Potential advantages for the P2G3 LS N54S/N56Q antibody relative to the WT sequence is reduced risk of deamidation at the Asn-G/S/N motif.

Table 13: P5C3 antibody mutation sequences evaluated

Table 14: P5C3 Heavy chain CDR1 sequences for anti-SARS-CoV-2 neutralizing antibodies

Table 15: P5C3 Heavy chain CDR2 sequences for anti-SARS-CoV-2 neutralizing antibodies

Table 16: Heavy chain FR3 and CDR3 sequences for anti-SARS-CoV-2 neutralizing antibodies Table 17: P2G3 antibody mutation sequences evaluated

Table 18: P2G3 Heavy chain CDR2 sequences for anti-SARS-CoV-2 neutralizing antibodies

Table 19: P2G3 Heavy chain CDR3 sequences for anti-SARS-CoV-2 neutralizing antibodies

Sequence of the ancestral Wuhan SARS-CoV-2 Spike protein (SEQ ID NO: 199) (RBD residues 333 to 527 in bold and P2G3 contact residues underlined and shown in grey tone):

MFVFLVLLPL VSSQCVNLTT RTQLPPAYTN SFTRGVYYPD KVFRSSVLHS TQDLFLPFFS NVTWFHAIHV SGTNGTKRFD NPVLPFNDGV YFASTEKSNI

IRGWIFGTTL DSKTQSLLIV NNATNW IKV CEFQFCNDPF LGVYYHKNNK SWMESEFRVY SSANNCTFEY VSQPFLMDLE GKQGNFKNLR EFVFKNIDGY FKIYSKHTPI NLVRDLPQGF SALEPLVDLP IGINITRFQT LLALHRSYLT PGDSSSGWTA GAAAYYVGYL QPRTFLLKYN ENGTITDAVD CALDPLSETK CTLKSFTVEK GIYQTSNFRV QPTESIVRFP NITNLCPFGE VF_ FASV

YAWNRKRISN CVADYSVLYN SASFSTFKCY GVSPTKLNDL CFTNVYADSF VIRGDEVRQI APGQTGKIAD YNYKLPDDFT GCVIAWNSNN YLYRLFRKSN LKPFERDIST EIYQAGSTPC NGVEGFNCYF PLQSYGFQPT NGVGYQPYRV W LSFELLHA PATVCGPKKS TNLVKNKCVN FNFNGLTGTG VLTESNKKFL PFQQFGRDIA DTTDAVRDPQ TLEILDITPC SFGGVSVITP

GTNTSNQVAV LYQGVNCTEV PVAIHADQLT PTWRVYSTGS NVFQTRAGCL IGAEHVNNSY ECDIPIGAGI CASYQTQTNS PRRARSVASQ SIIAYTMSLG AENSVAYSNN SIAIPTNFTI SVTTEILPVS MTKTSVDCTM YICGDSTECS NLLLQYGSFC TQLNRALTGI AVEQDKNTQE VFAQVKQIYK TPPIKDFGGF NFSQILPDPS KPSKRSFIED LLFNKVTLAD AGFIKQYGDC LGDIAARDLI

CAQKFNGLTV LPPLLTDEMI AQYTSALLAG TITSGWTFGA GAALQIPFAM QMAYRFNGIG VTQNVLYENQ KLIANQFNSA IGKIQDSLSS TASALGKLQD W NQNAQALN TLVKQLSSNF GAISSVLNDI LSRLDKVEAE VQIDRLITGR LQSLQTYVTQ QLIRAAEIRA SANLAATKMS ECVLGQSKRV DFCGKGYHLM SFPQSAPHGV VFLHVTYVPA QEKNFTTAPA ICHDGKAHFP REGVFVSNGT HWFVTQRNFY EPQIITTDNT FVSGNCDW I GIVNNTVYDP LQPELDSFKE ELDKYFKNHT SPDVDLGDIS GINASW NIQ KEIDRLNEVA KNLNESLIDL QELGKYEQYI KWPWYIWLGF IAGLIAIVMV TIMLCCMTSC CSCLKGCCSC GSCCKFDEDD SEPVLKGVKL HYT

Example 14

Identification of P2G3, a highly potent SARS-CoV-2 neutralizing antibody

The presence of anti-Spike antibodies in serum samples from a cohort of >100 donors was screened and efforts focused on one post-infected donor that received two doses of the mRNA- 1273 vaccine and displayed among the highest serum antibody levels with excellent breadth against a panel of SARS-CoV-2 variants in a trimeric Spike-ACE2 surrogate neutralization assay ² . Screening of B cell clone supernatants for high affinity Spike binding led us to prioritize six clones for mAbs production via expression of paired heavy and light chains in ExpiCHO cells. Amongst these purified mAbs, P2G3 exhibited the strongest binding affinity for the original 2019-nCoV Spike trimer and a panel of Spike proteins encoding mutations found in Alpha, Beta, Gamma and Delta VOCs (IC50 of 0.008-0.015 μg/ml) while also displaying good affinity (Kd of 0.062 μg/ml) for Omicron Spike (Table 9).

Upon performing cross-competitive Spike binding studies, it was found that P2G3 neither competed, nor blocked Class 1 or Class 2 mAbs (Greaney/Bloom, Nature) such as P5C3, REGN10933 and AZD8895 (Fig. 14) and displayed non- to partial-competitive binding with Class 4 mAh ADG-2. In contrast, P2G3 bound competitively with the Class 3 mAbs REGN10987, AZD1061 and Vir-S309/Sotrovimab. The competitive binding profile also indicates that P2G3 binds an epitope that is unique albeit overlapping with those recognized by both AZD1061 and Vir-S309/Sotrovimab, a mAh that does not block the RBD/ACE2 interaction.

Using a biochemical trimeric Spike-ACE2 surrogate neutralization assay, it was further determined that P2G3 and P5C3 had the most potent and broad blocking activity compared to our panel of advanced benchmark mAbs (Fig. 15 a and b) with P2G3 displaying ICxo activity against Omicron Spike ~4-fold improved over AZD1061 and ADG-2 and >20-fold improved over REGN10933, REGN10987 and AZD8895. In this assay the P2G3/P5C3 combination with currently approved antibody cocktails was compared. When the 2019-nCoV Spike was used as substrate, P2G3/P5C3 displayed additive effects on ACE2 binding inhibition (Fig. 15a), with levels of activity comparable to the AZD1061/AZD8895 and REGN10933/REGN10987 mAbs cocktails. However, when Omicron Spike was the target, the P2G3/P5C3 mAh cocktail was >73 -fold more active than all other commercial combinations and our two antibodies exhibited clear signs of synergy with ICso values of 0.46 μg/ml for P2G3, 0.66 μg/ml for P5C3 and 0.23 μg/ml for P2G3/P5C3(Fig. 15b). Furthermore, the P2G3/P5C3 combination reached -100% of Imax in blocking ACE2 binding to Omicron Spike at 1 μg/ml total mAh, while P2G3 alone only blocked 75% of ACE2 binding and P5C3 alone reached a -100% Imax at >20 μg/ml. P2G3 alone or in combination with P5C3 was compared with all currently authorized and/or clinically advanced mAbs in pseudovirus and live virus neutralization assays. P2G3 had potent neutralizing activity against lentiviruses pseudotyped with Spike from initial D614G, Alpha, Beta and Delta VOCs (ICso value of 0.022, 0.051, 0.038 and 0.035 μg/ml, respectively). Most importantly, P2G3 strongly neutralized the Omicron Spike pseudovirus with an ICxo value of 0.038 μg/ml (Fig. 15c). In side-by-side comparisons, P2G3 was >42-fold more potent than ADG-2, AZD1061, AZD8895, REGN10933 and REGN10987 mAbs and 19-fold more potent than Sotrovibmab at neutralizing Omicron Spike pseudotyped lentiviral particles (Fig. 15d). Second most potent was P5C3, with an ICso value of 0.223 μg/ml, and the P2G3/P5C3 combination unexpectedly revealed a small -enhanced activity over P2G3 alone in this assay, with an ICxo value of 0.024 μg/ml. This 2-fold improved activity was observed with only 0.0115 μg/ml of the more potent P2G3 mAh in the mix, providing evidence for cooperative inhibition of Omicron Spike pseudovirions when combined with P5C3. Furthermore, P2G3 and P5C3 maintained full neutralizing activity against a R346K Spike pseudovirus (Fig. 28), a mutation present in a minority of Omicron variants (Liu 2022).

P2G3 was profiled using the initial D614G strain and all current VOCs in a live virus cytopathic effect assay. P2G3 demonstrated broad and potent neutralizing activity with ICxo values of 0.028, 0.010, 0.017, 0.021, 0.019 and 0.045 μg/ml against the 2019-nCoV strain, Alpha, Beta, Gamma, Delta and Omicron variants, respectively (ICxo ranging from 67-300 pM). P2G3 was compared with other mAbs alone or in combinations for their ability to block the currently prevalent Omicron and Delta SARS-CoV-2 variants (Fig. 15f and 15g, respectively). All tested mAbs displayed good activity against the Delta variant, although Sotrovimab was -5 times less potent than ADG-2 and >10 times less potent than P2G3, P5C3 and both the AZD and RGN cocktails against this virus. However and most importantly, P2G3 was by far the most active mAh against Omicron with an ICxo value of 0.045 μg/ml, that is -20-fold, 47-fold and 69-fold lower than those of AZD1061/AZD8895, Sotrovimab and ADG-2, respectively, the RGN combination being completely ineffective against this variant (Fig. 15f). Of note, while P5C3 displayed against Omicron an ICxo in the range of that of the AZD cocktail, the P2G3/P5C3 combination showed comparable neutralization activity to P2G3 alone with an ICxo of 0.057 μg/ml total mAb. Importantly, although the Spike-ACE2 surrogate neutralization assay is strongly correlative with cell based neutralization assays (Fenwick, Turelli et al. 2021), it was observed ~12-fold improved potency of P2G3 in the cell-based assays compared to the biochemical Spike-ACE2 assay. This suggests that at least with the Omicron virus, the primary mechanism of virus inhibition by P2G3 is not mediated through the Spike-ACE2 interaction, but rather a mechanism analogous with that of S309/Sotrovimab, reported to be through induction of Spike trimer cross-linking, steric hindrance, aggregation of virions(Pinto, Park et al. 2020) and/or inhibiting target cell membrane attachment through C-type lectin receptors (Lempp, Soriaga et al. 2021).

Example 15

P2G3 confers strong in vivo prophylactic protection from SARS-CoV-2 infection

Having demonstrated the best-in-class in vitro neutralizing activity of P2G3, it was next evaluated the neutralizing potency of P2G3 in vivo in a prophylactic hamster challenge model of SARS-CoV-2 infection. Antibody treated animals were challenged two days later with an intranasal inoculation of SARS-CoV-2 virus (2.4 xlO ⁶ TCID50) (Fig. 16a) and then four days later, hamster lung tissue was monitored for infectious virus and viral RNA. Infectious virus was undetectable in lungs from almost all P2G3 treated hamsters, with only 1 of 6 hamsters in the 0.5 mg/kg group showing detectable, though -1.5 log reduced, levels of infectious virus compared to the isotype mAb-treated control animals (Fig. 16b). Complete prophylactic protection in these studies was observed with P2G3 mAb plasma levels >6.2 μg/ml at the time of viral inoculation. Furthermore, all P2G3 treatment groups showed a significant -4 log reduction of genomic viral RNA levels (p<0.009) compared to control animals (Fig. 16c).

Example 16

Structural analysis of P2G3 and P5C3 Fab bound to Omicron Spike.

To decipher the molecular features underlying P2G3 and P5C3 potent neutralization of Omicron Spike, single particle cryo-EM reconstruction of the Omicron Spike trimeric ectodomain bound to both Fabs, at a 3.04 A resolution, was performed (Fig. 17a, Fig. 21-22 and Table 20). It was found that the Fabs to bind simultaneously at distinct sites on the trimer with a majority of images revealing three P2G3 Fabs bound to either up- or down-RBD conformations and one P5C3 Fab bound to an up-RBD. The region of the Omicron RBD interacting with the Class 1 P5C3 mAb is identical to that previously described for the ancestral RBD (Fig. 21-23). P2G3 binds as a Class 3 neutralizing mAb, recognizing an epitope on the SARS-CoV-2 RBD distinct from the receptor-binding motif. Together, the two mAbs cover a surface of greater than 1200 A’ (Fig. 17b and Fig. 17a, 19a), which may explain the enhanced activity observed when they are used in combination against Omicron (Fig. 15d and f). To characterize the P2G3 paratope and epitope interface in detail, local refinement of the P2G3 Fab-RBD interacting region was performed and reached a resolution of 3.84 A with well- defined density, allowing clear interpretation of sidechain positions (Fig. 21, 22, 25 and Table 20) The P2G3 paratope i s composed of four complementarity-determining region (CDR) loops burying a surface area of around 700 A ² at the back of the RBD. The interactions are mediated through electrostatic and hydrophobic contacts (Fig. 19b, c) and involve sixteen residues of the RBD, mainly bound by the heavy chain of the P2G3 mAb The 18-residue-long CDRH3 sits at the top of a loop that comprises residues 344-347, and also contacts the amino-acids at the limit of the 5-stranded b-sheet (residues 440-451), overall accounting for more than 60% of the buried surface area (431 A ² ) (Fig, 24a, b). The interactions between P2G3 and the Omicron RBD are conserved in both RBD-up and RBD-down states (Fig. 24c). CDRH2 extends the epitope by interacting with R346 that is engaged by residue W53 by a potential cation-pi interaction (Fig. 16f and Fig. 24d), an interaction that is likely conserved with the R346K Spike substitution (Fig. 21). The only potential contact from the light chain derives from the CDRL1 ¥32 forming an interactions with V445 of RBD (Fig. 16e). Importantly, the epitope defined by our structural studies rationalizes the potent neutralizing activity of P2G3 against the Omicron variant relative to other Class 3 mAbs. Omicron mutations S371L, N440K, G446S and the minor R346K sub-variant are all situated outside or adjacent to or have little effect on P2G3 binding its epitope, whereas two or more mutations directly impinge on epitopes for REGN10987, AZD1061 and S309 (Fig. 26a). Furthermore, the distinct properties of P2G3 are highlighted by its binding orientation on the RBD with the Fab angled away from most of these Omicron mutations (Fig. 26b, c). In modeling the observed angles of attack of various Class 3 mAbs, it is likely that REGN 10987 only binds to the up-RBD form while AZD1061 binds one up- and one down-RBD form, with steric hindrance blocking the third RBD site on the Omicron Spike trimer (Fig. 18d-e). In contrast, P2G3 and S309/Sotrovimab likely binds both the up and down forms of Omicron RBD without clashes (Fig. 18a, f), a characteristic that may contribute to the largely conserved and high potency of P2G3 across VOCs. Table 20: Cryo-EM data collection, refinement and validation statistics Example 17

Fc-mediated functional activity of P2G3 and P5C3 mAbs with synergistic activity observed in an antibody dependent cellular phagocytosis assays

Given that the P2G3/P5C3 cocktail consistently exhibited enhanced neutralization over individual mAbs in the surrogate neutralization, pseudoviral and live virus Omicron variant assays, and based on the potential importance Fc-mediated antibody effector functions in more efficient virus clearance and control, the complementarity of this mAh combination in mediating effector functions was investigated.

The targeted killing of cells that display SARS-CoV-2 Spike protein at the membrane surface is an important effector function that can help clear infected cells in vivo. In our in vitro antibody-dependent cellular cytotoxicity ADCC evaluation, CEM-NKR-Spike cells stably expressing 2019-nCoV Spike were cultured with T cell depleted primary effector cells from healthy donors in the presence and absence of anti-Spike mAbs. P2G3 mAh exhibited a robust ADCC activity that was superior in killing Spike positive cells compared to all other anti-Spike mAbs tested (Fig. 19).

The ADCP functional activity of P2G3 and P5C3 was next evaluated. In this in vitro assay, Spike trimer coated fluorescent beads are mixed with different concentrations of P2G3 and/or P5C3 then incubated with U937 effector cells. This monocyte cell line expresses high levels of Fc-gamma receptors capable of inducing phagocytosis of opsonized viruses or beads coated with the Spike antigen as in our assay. Used alone, P2G3 and P5C3 mAbs showed ADCP ICso activities of 0.074 and 0.010 μg/ml, respectively with P5C3 showing ~7-fold greater potency. However, when combined, ICxo ADCP activity was achieved with 0.012 μg/ml of P2G3 mixed with 0.00015 μg/ml of P5C3, levels 6-fold lower than individual ICso _s (Fig. 20a). Synergistic ADCP activity between the antibody pair were profiled in dose-response matrices of P2G3 and P5C3, where MacSynergy II analysis software showed a strong and highly significant synergy volume of 271 [mAb] ² % for the P2G3/P5C3 combination (Fig. 20b). Furthermore, P2G3 exhibits a 14-fold improved activity over P5C3 in inducing phagocytosis of Omicron Spike coated beads and the P2G3/P5C3 mix shows enhanced ADCP activities compared to mAbs used individually (Fig. 29). Example 18

Cross-neutralization of P2G3 and P5C3 escape mutants.

Monoclonal antibodies, as other classes of antivirals, are typically used in combination to prevent the emergence of resistant viruses. To gain insight into the predicted clinical value of our mAbs, the emergence of mutants escaping their blockade in tissue culture was characterized. For this, SARS-CoV-2 Delta and Omicron BA.l variants were grew in the presence of sub-optimal neutralizing doses of either P2G3 or P5C3 for three passages to generate a heterogeneous viral population, before switching to stringent mAb concentrations in order to select bona fide escapees. Viral genome sequencing of these mAb-resistant mutants pointed to the importance of Spike substitutions G476D, F486S and N487K/D/S for escaping P5C3, and K444T for avoiding P2G3 neutralization (Figure 30A). It was thus tested the impact of these mutations on viral infectivity using lentivector pseudotypes. P5C3-escaping Spike proteins were significantly less infectious than the wild-type control in both the ancestral D614G and Delta backgrounds, correlating with a drop in affinity for the viral ACE2 receptor in an in vitro binding assay, whereas the P2G3 -escaping K444T substitution had a milder effect in both assays (Figure 30 A-B). Yet, an examination of the GISAID EpiCoV database revealed that mutations G476D, F486S or N487K/D/S found in P5C3 escapees are only exceptionally encountered in SARS-Cov2 isolates, representing together only 0.0087% of the 8’568’006 available sequences as of March 2022, and that the K444T mutation is equally rare (0.0024% of compiled sequences), strongly suggesting that the corresponding viruses have a markedly reduced fitness in the wild. Moreover, cross-neutralization studies demonstrated that P2G3 completely blocked the infectivity of P5C3-escaping Delta derivatives (Figure 30 C-H), and P5C3 and P2G3 efficiently cross-neutralized each other’s escape mutants in the Delta and Omicron BA.l (Figure 30 C-H). Cryo-EM structural data supports the strong crossneutralization of one another’s viral escapees where Figure 30 I shows the extensive distance and completely non-overlapping nature of resistance mutations to P2G3 and P5C3. These data strongly support the co-administration of P2G3 and P5C3 mAbs that have potent neutralization profiles against SARS-CoV-2 variants of concern and complementary resistance mutant profiles that are likely to both strongly suppress viral spread and create a high biological barrier to the development of resistant virus. Example 19

P2G3 confers strong in vivo prophylactic protection from ancestral 2019-nCoV and Omicron SARS-CoV-2 infection

Having demonstrated the superior in vitro neutralizing activity of P2G3, it was next evaluated the neutralizing potency of P2G3 in vivo in mediating protection from SARS-CoV-2 Omicron BA.l infection in a cynomolgus macaques pre-exposure challenge study. Monkeys (n=2) were administered 10 mg/kg of P2G3 LS intravenously and challenged 72 hrs later via combined intranasal and intratracheal routes with lxlO ⁵ TCID50 of SARS-CoV-2 B.1.1.519 Omicron BA.l virus (Figure 31 A). Following viral challenge, control animals (n=4; data plotted to include two historical control animals MF5* and MF6*) showed similar genomic (g)RNA levels and kinetics with median peak viral loads (VL) of 6.9- and 6.6-loglO copies/ml gRNA at 2-3 days post challenge in tracheal swabs and bronchoalveolar lavage (BAL) samples, respectively (Figure 31 B-D). Nasopharyngeal swabs showed a higher-level variability in VL between control animals but still showed median peaks of 6.9-loglO copies/ml for gRNA. In comparison, the two P2G3 LS treated monkeys had a strong median peak AVL reduction of 3.8-, 2.5- and 3.9-loglO copies/ml gRNA for tracheal, nasopharyngeal and BAL samples, respectively.

Active viral replication, as assessed by subgenomic (sg)RNA levels, peaked 2-3 days postchallenge with tracheal swabs and BAL showing median values of 4.8- and 4.5-loglO copies per ml, respectively and nasopharyngeal samples showing variable responses from undetectable (<2.9-logl0) to 5.4-loglO copies/ml (Figure 31 E-G). P2G3 LS treated monkeys had sgRNA levels that were at or below the limit of detection, exhibiting 1.9-, 2.2- and 1.6-loglO reduced levels in tracheal, nasopharyngeal and BAL samples, respectively. Consistent with viral protection resulting in reduced detection of gRNA and sgRNA, flow cytometry analysis of blood samples from NHPs shows P2G3 treated monkeys exhibited stable lymphocyte levels throughout the study, whereas strong lymphopenia, determined by lymphocyte levels below 2. lx 10 ³ cells/mΐ, was observed in all control animals challenged with the Omicron variant of SARS-COV-2 (Figure 31 H). Example 20

The P2G3/P5G3 combination shows strong in vivo therapeutic efficacy in the non-human primate Omicron SARS-CoV-2 challenge model

In a second study to evaluate mAb therapeutic efficacy, monkeys were challenged with SARS- CoV-2 B.1.1.519 Omicron BA.l virus as above and then 24 hours post challenge, animals in three groups were either left untreated (n=4) or administered a P2G3 LS + P5C3 LS combination at 5+5 mg/kg (n=6) or 2.5+2.5 mg/kg (n=6) through a single intravenous injection (Figure 32 A). Tracheal, nasopharyngeal and BAL samples collected longitudinally for each monkey were evaluated for both gRNA (Figure 32 B-L) and sgRNA (Figure 32 M-O) viral copies per ml. Consistent with the prophylactic challenge study, control animals showed comparable gRNA levels and kinetic profiles with median peak VL of 6.7- and 6.1-loglO copies/ml at 2-3 days post challenge in tracheal swabs and BAL samples, and elevated but more variable nasopharyngeal VL levels of 6.5-loglO copies/ml gRNA (Figure 32 H-J). Importantly, monkeys in the 5+5 mg/kg P2G3 LS + P5C3 LS combination treatment arm showed strong, significantly reduced median peak AVL of 2.1- and 1.5-logl0 for tracheal and BAL samples (p= 0.0095 for each), respectively and 1.2-loglO copies/ml gRNA for nasopharyngeal samples (Figure 32 H-J). Similarly, monkeys treated with the 2.5+2.5 mg/kg lower dose mAb combination showed median peak AVL reduction of 0.85- 1.1- and 1.2-loglO copies/ml gRNA for tracheal, BAL and nasopharyngeal samples, respectively, with BAL samples showing significantly reduced levels (p=0.0095) (Figure 32 H-J). Therapeutic efficacy was furthermore demonstrated by monitoring area under the curve (AUC) levels of gRNA, where the high and low dosed P2G3 LS + P5C3 LS combination exerted significant 4- and 2.5-fold reduction in virus detected in the trachea (p = 0.0095 and 0.0190, respectively) and 2.5- and 2.2-fold reduced gRNA AUC levels in nasopharyngeal fluids relative to the untreated control monkeys (Figure 32 K-L)

Active viral replication was also significantly inhibited in the high and low dose treatment arms with tracheal swabs showing 41- and 8.8-fold reduction in sgRNA on Days 2 / 3 post challenge (p=0.0005 and 0.0062), respectively (Figure 32 M-N). P2G3 LS + P5C3 LS treatment combinations furthermore reduced sgRNA in BAL samples by 6.2- and 8.8-fold compared to the untreated control monkeys (p=0.0095 for both) (Figure 32 O). Overall, these studies strongly support the therapeutic efficacy of the P2G3 LS + P5C3 LS combination in clearing virus and inhibiting active viral replication of the highly relevant Omicron BA.1 variant. Discussion, Conclusion

In sum, it is herein reported the discovery of P2G3, an anti-SARS-CoV-2 mAh with best-inclass breadth and potency for neutralization of all VOCs, including the recently identified Omicron variant. Indeed, the unique binding properties allows P2G3 to overcome all the Omicron mutations that abolished or impaired the neutralizing activity of almost the totality of the authorized or clinically advanced mAbs. Structural and competitive binding studies demonstrate that P2G3 is a Class 3 mAh recognizing an epitope on the RBD different from those bound by therapeutic mAbs currently in the clinics, and sequence analysis revealed an important HCDR3 diversity, with only 55% identity to any of the 4897 anti-Spike HCDR3 described thus far. Cryo-EM studies further show that P2G3 can bind both the open and closed conformations of the Spike trimer, burying a large surface area of around 700 A ² in a highly conserved region of RBD. Importantly, the binding epitope is largely non-overlapping with mutated residues present in the Omicron variant, a feature that is unique among almost all approved and clinically advanced anti-SARS-CoV-2 mAbs. The S371L Omicron mutation alone remarkably reduces neutralization activity of multiple potent mAbs of different binding Classes despite not being included in their footprint (Liu 2022). Local conformational changes in the 370-375 loop that impact the up- and down-RBD states and/or interference with the critical gate opener N343 glycan positioning (Sztain, Ahn et al. 2021) are possible justifications for this broad inhibitory effect (Liu 2022). However, the unique angle of attack on the RBD domain that is predicted to maintain P2G3 binding in both up- and down-RBD positions of the Spike trimer may explain the maintained potency of P2G3 against the Omicron variant. Interestingly, it is predicted that S309/Sotrovimab is also sterically free in binding all RBDs within the Omicron trimer and shows a reduced loss in activity relative to other advanced mAbs. As such, this region may be particularly important for neutralizing current and future VOCs. Although S309/Sotrovimab binds an overlapping epitope with P2G3, it does not block RBD/ACE2 interaction and acts solely by an alternate mechanism that is proposed to involve factors including the inhibition of cell adhesion through C-type lectins. Therefore, the improved binding affinity and additional activity in blocking RBD/ACE2 interaction, confers on P2G3 a 10 to 60-fold improved activity across all VOCs as compared to S309/Sotrovimab. Apart from the in vitro neutralization activity, it was also demonstrated that P2G3 confers an excellent in vivo prophylactic protection in the hamster challenge model at doses as low as 0.5 mg/kg. Despite the exceptional neutralization profile of P2G3 against currently circulating variants, development of resistance is almost inevitable when a virus is under selective pressure. It is thus noteworthy that it was further demonstrated that P5C3, a previously described, potent and broadly active neutralizing mAh whose mechanism of action is through blocking the Spike- ACE2 interaction, can bind Omicron Spike concomitantly with P2G3 and targets a highly conserved region of the RBD. Because of the cooperation between two different mechanisms of action, Spike-ACE2 interaction and a potential mechanism that inhibits vial cell attachment, these two antibodies may partner up in a very strong combination to ward off the occurrence of viral resistance.

Many of the spectacular advances gained in the fight against the pandemics, including COVID- 19 vaccines and potent neutralizing mAbs, were erased in a matter of months following the emergence of the Delta and Omicron variants. The Omicron variant exhibits markedly reduced sensitivity to vaccine-induced immunity in healthy donors but, more importantly, immunocompromised individual are now almost completely unprotected due to their inability to mount a protective humoral immune response following vaccination. For these vulnerable individuals, it is proposed that passive immunization through two-to-three injections per year with the extended half-life P2G3 and P5C3 LS (Zalevsky, Chamberlain et al. 2010) represents a very attractive prophylaxis option (Mohammed, Blebil et al. 2020). With the additive to synergistic properties in neutralization, binding and ADCP functional activity, this broadly active combination represents a superior anti-SARS-CoV-2 mAh cocktail for prophylactic protection and therapeutic interventions against all current VOCs and potentially future VOCs arising after Delta and Omicron.