WO2022162587A1

WO2022162587A1 - Anti-sars-cov-2 antibodies and use thereof in the treatment of sars-cov-2 infection

Info

Publication number: WO2022162587A1
Application number: PCT/IB2022/050731
Authority: WO
Inventors: Craig Fenwick; Giuseppe Pantaleo; Didier Trono; Priscilla TURELLI
Original assignee: Centre Hospitalier Universitaire Vaudois (C.H.U.V.); Ecole Polytechnique Federale De Lausanne (Epfl)
Priority date: 2021-01-27
Filing date: 2022-01-27
Publication date: 2022-08-04

Abstract

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

Description

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

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 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, 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); 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 P1O6); 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).

Another aspect of the present invention provides an anti-SARS-CoV-2 antibody, or an antigenbinding 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:3 and SEQ ID NO: 105 to SEQ ID NO: 126, 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: 13 (antibody P5C3).

Another aspect of the present invention provides an anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, comprising a human heavy chain variable region amino acid sequence that comprises or consists of an amino acid sequence selected from SEQ ID NO:3 and SEQ ID NO: 105 to SEQ ID NO: 126, and a human light chain variable region amino acid sequence that comprises or consists of SEQ ID NO: 13 (antibody P5C3).

Another aspect of the present invention provides an anti-SARS-CoV-2 antibody, or an antigenbinding 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 Tyr451, Leu452, Tyr453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, Tyr 489, Pro491, Leu492, Gln493, Ser494, Tyr495, and Gly496 in SEQ ID NO: 127.

Another aspect of the present invention provides a pharmaceutical composition comprising the anti-SARS-CoV-2 antibody, or an antigen-binding fragment 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 any one of claims 1-36 and detecting the antibody in the sample.

Another aspect of the present invention provides the anti-SARS-CoV-2 antibody, or an antigenbinding 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 antigenbinding 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 antigenbinding 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-2antibody, 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 effects 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 effects 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 Ml 531; 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 effects of select newly reported antibodies are shown for the 2019-nCoV -D614G mutant (A), B.l.1.7 UK variant (B), B.1.351 South African variant (C) and a mink variant (D).

Figure 6 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 7 shows the evaluated the neutralizing potency of P5C3 in vivo in a prophylactic hamster challenge model of SARS-CoV-2 infection.

Figure 8 shows neutralizing activity of P5C3 antibodies with (A) mutations N58, M74 and N100, and (B) mutations T28, G52, S53, G54 and R70.

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 SAR.S-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 "anti-SARS-CoV-2 antibody" means an immunoglobulin, antigenbinding 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. A "neutralizing antibody" is one that can neutralize, i.e., prevent, inhibit, reduce, impede or interfere with, the ability of a pathogen to initiate and/or perpetuate an infection in a host. The terms "neutralizing antibody" and "an antibody that neutralizes" or "antibodies that neutralize" are used interchangeably herein. 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".

SAR.S-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 invention provides a panel of antibodies that bind the SARS-CoV-2 RBD and/or Spike protein or fragment thereof. In some embodiments, the antibodies described herein have potent neutralizing activity against the SARS-CoV-2 virus. These antibodies could form the basis of a monotherapy or combination (cocktail) therapy comprising 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 innovation 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, 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); 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 P1O6); 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 invention provides an anti-SARS-CoV-2 antibody, or an antigenbinding 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 another embodiment, the invention provides an anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, wherein 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).

In another embodiment, the invention provides the 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: 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 P1O6). In another embodiment, the invention provides an anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof of claim 1, wherein the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; theLCDRl, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively (antibody MS35).

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 105-126, 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 105-126 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 105-126 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 105-126 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: 125 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: 126 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: 125 and SEQ ID NO: 126, 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 Pl 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: 125 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: 126 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:

125, 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:

126, 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: 125, 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: 126, and the human light chain variable region amino acid sequence comprises or consists of SEQ ID NO: 13. Anti-SARS-CoV-2 antibodies: Heavy chain amino acid sequences

SEQ ID No. 1: (P1G17)

QVQLVESGGGVVQPGGSLRLSCAASGFTFSMYGIHWVRQAPGKGLEWVAVISYDGS DNYFAD S VKGRF SISRDNSKNTL SLQMNSLRAEDTAVYYC AKQGP VYS S SWFQIRNY GMDVWGQGTT VTVS S

SEQ ID No. 2: (P7K18)

EVQLVESGGGLVQPGGSLRLSCAASGFIFSSYTMNWVRQAPGKGLEWISYISDSGTIY HTDSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARDPAVAAADYFDYWGQGT LVTVSS

SEQ ID No. 3: (P5C3)

QMQLVQSGPEVKKPGTSVKVSCKASGFTFTSSAVQWVRQARGQRLEWIGWIVVGSG NTDYAQQFQERVTITRDMSTSTAYMELSSLGSEDTAVYYCAAPNCSGGSCYDGFDLW GQGTMVTVSS

SEQ ID No. 4: (P2B11)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNTISWVRQAPGQGLEWMGRIIPILGITN YAQKFQGRVTITADRSTSTAYMELSSLRSEDTAVYFCARDGDTAMVFIGYFDLWGRGT LVTVSS

SEQ ID No. 5: (P1H23)

QVTLRESGPALVKPTQTLTLTCTFSGFSLSTSGMCMNWIRQPPGKALEWLARIDWDDE KYYSTSLQTRLTISKDTSKNQVVLTMTNIDPVDTATYYCARDMAAAGFDSWGQGTLV TVSS

SEQ ID No. 6: (P6E16)

EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYSMNWVRQAPGKGLEWVSYIGRSSHT IYYADSVKGRFTISRDNAKNSLYLQMNSLRDEDTAVYYCARIDYYDSSGYPDYWGQG TLVTVSS

SEQ ID No. 7: (P1O6)

QVQLVESGGGVVQPGRSLRLSCAASGFTFSNYGMHWVRQAPGKGLEWVAVISFDGN NKYFADAVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKDRALRNWNDMYYY

GMDVWGQGTT VTVS S

SEQ ID No. 8: (P1M12)

QVQLVESGGGVVQPGRSLRLSCAASGFTFNTYGMHWVRQAPGKGLEWVAVISYDGS NTYYADSVKGRFTISRDNSKNRLYVQMNSLRAEDTAVYYCARGFFCSGDTCFQTYYY YGLD VWGQGTT VTVS S

SEQ ID No. 9: (P1L7)

EVQLVESGGGLVQPGGSLRLSCAASGFTVRSNYMTWVRQAPGKGLEWVSIIHTDGST FYANSVKGRFTISRHSSKNTLSLQMASLRAEDTAVYYCARVVTDAFDLWGQGTLVTV SS

SEQ ID No. 10: (P1L4)

EVQLVESGGGLVQPGGSLRLSCEASGFTFDSYWMNWVRQAPGKGLEWVANIRHDGG

EKNYLDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCARIKVDYDYVWGSYRY YYNLDVWGQGTTVTVSS

SEQ ID No. 81: (MS31)

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSNAMKWVRQAPGKGLEWVAVISYDGS NKYYADSVKGRFTISRDNSKKTLYLQMNSLRAEDTAVYYCARVVIPYCTNGVCYVDY WGLGTLVTVSSAS

SEQ ID No. 82: (MS35)

QVQLVQSGAEVKKPGSSVKVSCKASGGTFSNYAISWVRQAPGQGLEWMGRIISIPGIA DYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCATPTEDMVVVPADDSDAFD VWGQGTMVTVSSAS

SEQ ID No. 83: (MS42)

EVQLVESGGGLIQPGGSLRLSCVASGLTVSSNYMTWVRQAPGKGLEWVSLIYAGGST YYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARDLSYYGMD VWGQGTT VTVSSAS Anti-SARS-CoV-2 antibodies: Light chain amino acid sequences

SEQ ID No. 11: (P1G17)

QSALTQPASVSGSPGQSITISCTGTSSDVGSYNLVSWYQHHPGKAPKLMIYEASKRPSG

VSNRFSGSKSDNTASLTISGLQAEDEADYYCYSYAGSSTWVFGGGTKVTVLG

SEQ ID No. 12: (P7K18)

SYVLTQPPSVSVAPGKTARITCGGNDIGSKSVHWYQQKAGQAPVLVIYYDSDRPSGIP

ERFSGSNSGNTATLTISRVEAGDEADYYCQVWDTGRVFGGGTKLTVLG

SEQ ID No. 13: (P5C3)

EIVLTQSPGTLSLSPGERATLSCRGSQSVRSSYLGWYQQKPGQAPRLLIYGASSRATGI

PDRF SGSGSGTDFTLTISRLEPEDFAVYYCQQYGS SPWTFGQGTKVEIK

SEQ ID No. 14: (P1H23)

DIQMTQSPSSLSASVGDRVTITCRASHSISRYLNWYQQKPGKAPKLLIYIASTLQSGVP

SRFSGSGAGTDFTLTISSLQPEDFATYYCQQSFSTPRTFGQGTKVEIK

SEQ ID No. 15: (P6E16)

DIQMTQSPSSLSASVGDRVTITCRASQGISNYLAWYQQKPGKVPKLLIYAASTLQSGV

PSRFSGSGSGTDFTLTISSLQPEDVATYYCQKYNNALWTFGQGTKVEIK

SEQ ID No. 16: (P2B11)

DIQMTQSPSTLSASVGDRVTITCRASQSISSWLAWYQQKPGKAPNLLIYTASSLESGVP

SRFSGSGSGTEFTLTISSLQPDDFATYYCQQYHSYPWTFGQGTKVEIK

SEQ ID No. 17: (P1O6)

DIQMTQSPSSLSASVGDRVTITCQASQDINNYLNWFQQKPGKAPKLLIYGASTLETGV

PSRFSGSASGTDFTFTISSLQPEDIATYYCQQYDTLPLTFGGGTKVGVR

SEQ ID No. 18: (P1M12)

DIQMTQSPSSVSASVGDRVTISCRASQGINSWLAWYQQKPGKAPKLLIYDASSLQSGV

PSRFSGSGSGTDFTLTISSLQPEDFATYYCQQAHTFPITFGQGTRLEIK SEQ ID No. 19: (P1L7)

DIQMTQSPSSLSASVGDRVTFTCRASQSITIYLNWYQQKPGKAPKLLIYAASSLQSGVP

SRF SGSGSGTDFTLTIS SLQPEDFATYFCHQSYSTPHTFGQGTRLEIK

SEQ ID No. 20: (P1L4)

EIVLTQSPGTLSLSPGERVTLSCRASQSISNSHLAWYQQKPGQAPRLVIYGTSRRATGIP

DRFSGSGSGTDFTLTISRLEPEDFVVYYCQQYGSPITFGQGTRLEIK

SEQ ID No. 84: (MS31)

NFMLTQPHSVSESPGKTVTISCTGSSGFIASNYVQWYQQRPGSPPTTVIYEDNQRPSGV PDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDGSHHWVFGGGTHLTVLGQPKAA PSVTLFPPS

SEQ ID No. 85: (MS35)

DIVMTQSPDSLAVSLGERATINCKSSQSVLHSSNNKNYLGWYQQKPGQPPKLLIYWAS

TRESGVPDRFSGSGSGTDFTLTIRSLQAEDVAVYYCQQYYSTPLTFGGGTKVEIKRT

SEQ ID No. 86: (MS42)

DIQLTQSPSSLSASVGDRVTITCRASQGISSYLAWYQQKPGKAPKLLIYAASTLQSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQLDSYPPYTFGQGTKLEIKRT

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 pg/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 invention provides a derivative of the neutralizing 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 -carb oxy-2, 7- dichlorofluorescein, 5-carboxyfluorescein, 5-FAM, hydroxy tryptamine, 5-hydroxy tryptamine (5-HAT), 6-carboxyfluorescein (6-FAM), FITC, 6-carboxy-l,4-dichloro-2’,7’- dichloro^fluorescein (TET), 6-carboxy-l,4-dichloro-2’,4’,5’,7’-tetra^_,chlorofluorescein (HEX), 6-carboxy-4’,5’-dichloro-2’,7’-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-carboxytetramethyHrhodamine (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 or 83 and/or SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 84, 85 or 86. 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 antigenbinding 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 have been substituted, inserted and/or deleted in SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 81, 82 or 83 and/or in SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 84, 85 or 86. 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 or 83 and/or SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 84, 85 or 86, 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 and 87 to 104. 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 have been substituted, inserted and/or deleted in one or more SEQ ID NOs: 21 to 80 and 87 to 104. 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 and 87 to 104, 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 0.42 nM between the P5C3 Fab and the Spike trimer with a Kon rate 8.6 e5 1/Ms and Koff rate of 3.7 e-4 1/s. The P6E16 Fab had a similarly tight binding affinity for the Spike trimer with a Kd of 0.67 nM, Kon of 5.2 e5 1/Ms and Koff rate of 3.5 e-4 1/s.

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. coli 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, S 1 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 bi specific 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-2antibody, 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 SAR.S-CoV-2 virus and the ACE-2 receptor (See e.g., Example 6).

In some embodiments, the anti-SARS-CoV-2 antibodies 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 antigenbinding fragments thereof, 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, of any one of claims 1-9 wherein the antibody, or an antigen-binding fragments thereof, 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/rnL, less than 5 ng/mL, or less than 2.5 ng/mL.

In some embodiments, the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention, exhibits an in vitro neutralization IC50 of a SARS-CoV-2 virus of between 2 ng/mL and 25 ng/mL, between 2 ng/mL and 22 ng/mL, between 2 ng/mL and 20 ng/mL, between 2 ng/mL and 17 ng/mL, between 2 ng/mL and 15 ng/mL, between 2 ng/mL and 10 ng/mL, or between 2 ng/mL and 8 ng/mL. In some embodiments, anti-SARS-CoV-2 antibodies, or an antigen-binding fragment thereof, described herein exhibit an in vitro neutralization IC50 of a SARS-CoV-2 virus of about 2 ng/mL, 2.5 ng/mL, 3 ng/mL, 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, 22 ng/mL, 23 ng/mL, 24 ng/mL, or 25 ng/mL.

In some embodiments, 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 antigen-binding fragments thereof, 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 30 ng/mL, between 10 ng/mL and 25 ng/mL, between 10 ng/mL and 20 ng/mL, or between 10 ng/mL and 15 ng/mL. In some embodiments, the anti-SARS-CoV-2 antibodies, or an antigen-binding fragments thereof, described herein exhibit an in vivo affinity IC80 for the SARS-CoV-2 spike protein of less than

25 ng/mL, less than 22 ng/mL, less than 20 ng/mL, less than 15 ng/mL, less than 10 ng/mL, or less than 8 ng/mL. In some embodiments, the in vivo IC80 is about 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, 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 B.1.1.7, B.1.351, P.l, Bl.617.2, B.1.1.529, CAL.C20, Mink variant 16, C.37, and B.1.621.

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 293 T 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 lU/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 pl). 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(l l):3699-3715). In some embodiments, neutralization may be determined as a measure of the concentration (for example pg/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 “ICso” 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, I0’¹, 10°, 10¹, 10², or 10³ pg/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 ICso value is below 25 pg/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 pg/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, 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 Tyr 451, Leu 452, Tyr 453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro 479, Glu484, Phe486, Asn487, Tyr489, Pro 491, Leu 492, Gln493, Ser494, Tyr 495, and Gly496 in SEQ ID NO: 127. In some embodiments, the epitope comprises each of Tyr451, Leu 452, Tyr453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro 479, Glu484, Phe486, Asn487, Tyr489, Pro 491, Leu 492, Gln493, Ser494, Tyr 495, and Gly496 in SEQ ID NO: 127. In some embodiments, the epitope comprises Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, and Tyr489 of SEQ ID NO: 127. In some embodiments, the epitope comprises Pro479 and Phe486 of SEQ ID NO: 127. In some embodiments, the epitope comprises Phe456, Tyr473, Phe486, and Tyr489 of SEQ ID NO: 127. In some embodiments, the epitope comprises amino acids 451-456 and 491-495 of SEQ ID NO: 127. In some embodiments, the epitope comprises Leu455, Phe456, Ala475, Gly476, Ser477, Glu484, Phe486, Asn487, Tyr489, and Gln493 of SEQ ID NO: 127. In some embodiments, the epitope comprises Phe456 and Gln493 of SEQ ID NO: 127.

In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof 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 Tyr451, Leu452, Tyr453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, Tyr489, Pro491, Leu492, Gln493, Ser494, Tyr495, and Gly496 in SEQ ID NO: 127. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof 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 SAR.S-CoV-2 Spike protein that comprises each of Tyr451, Leu452, Tyr453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, Tyr489, Pro491, Leu492, Gln493, Ser494, Tyr495, and Gly496 in SEQ ID NO: 127.

In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof 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 Tyr451, Leu452, Tyr453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, Tyr489, Pro491, Leu492, Gln493, Ser494, Tyr495, and Gly496 in SEQ ID NO: 127. In some embodiments, the anti-SARS-CoV-2 antibody or antigen-binding fragment thereof 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 Tyr451, Leu452, Tyr453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, Tyr489, Pro491, Leu492, Gln493, Ser494, Tyr495, and Gly496 in SEQ ID NO: 127. 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, Vai, Leu, He;

(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-2antibody 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-2antibody 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- di oxolane, poly-1, 3, 6-tri oxane, 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/Q311V/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. Unless otherwise noted, amino acid positions in the Fc domain are numbered according to the EU Index. See Edelman et al., The covalent structure of an entire gammaG immunoglobulin molecule. Proc. Natl. Acad. Sci. USA 1969, 63, 78-85; and Kabat, E.A.; National Institutes of Health (U.S.) Office of the Director. Sequences of Proteins of Immunological Interest, 5th ed.; DIANE Publishing: Collingdale, PA, USA, 1991.

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).

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 and 81-83. In some embodiments, the isolated nucleic acid encodes a VL amino acid sequence selected from the group consisting of SEQ ID NOs: 11-20 and 84-86. 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 and 81-83. 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 and 84-86. 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 hostcells 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, Ipp, 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 2p 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-Dalgamo (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- phosphoglycerate kinase or other glycolytic enzymes, such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase, 3 -phosphoglycerate 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 GENT MYCIN™ 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. 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. In one embodiment, a method of making an 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 and 81-83. 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 and 84-86. 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 antibody, or an antigen-binding fragment thereof, of the invention or one or more derivative of the invention and detecting the anti-SARS-CoV-2 antibody, or an antigenbinding 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 B. l.1.7, B.1.351, P.l, B.1.617.2, B.1.1.529, CAL.C20, Mink variant 16, 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 one or more 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 one or more anti-SARS-CoV-2 antibody, or an antigenbinding 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, P- 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 invention 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 a neutralizing 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 lU/mL. In some embodiments, the viral load is at least 5xl0² copies per ml, 10³ copies per ml, 10⁴ copies per ml, 10⁵ copies per ml, 10⁶ copies per ml, 10⁷ copies per ml, or > 10⁷ 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-SAR.S-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 sample, 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-SAR.S-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 anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention or the derivative of the invention, and a pharmaceutically acceptable carrier.

In an embodiment, the pharmaceutical composition of the invention comprises the anti-SARS- CoV-2 antibody P5C3 of the invention and one or more neutralizing antibodies of the invention selected from the group consisting of P1O6, P2B11, P7K18, P1L7, P1G17, MS31 and MS35; preferably the anti-SARS-CoV-2 antibody P5C3 of the invention and the anti-SARS-CoV-2 antibody MS35 of the invention.

In another embodiment, the pharmaceutical composition of the invention comprises the anti- SARS-CoV-2 antibody P6E16 of the invention and one or more anti-SARS-CoV-2 antibodies of the invention selected from the group consisting of P7K18 and P1L7.

In a further embodiment, the pharmaceutical composition of the invention comprises the anti- SARS-CoV-2 antibody P5C6 of the invention and Pl 06 anti-SARS-CoV-2 antibody of the invention.

In a further embodiment, the pharmaceutical composition of the invention comprises the anti- SARS-CoV-2 antibody P1H23 of the invention and P1O6 anti-SARS-CoV-2 antibody of the invention. In an embodiment, the pharmaceutical composition comprises the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of the invention and a pharmaceutically acceptable carrier. In a further 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 P5C3 antibody of the invention and the second anti-SARS-CoV-2 antibody is selected from P1O6, P2B11, P7K18, P1L7, P1G17, MS31 and/or MS35.

Pharmaceutical compositions and formulations of an anti-SARS-CoV-2 antibody as described herein are prepared by mixing such antibody 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 chondr oitinases.

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 gelatinmicrocapsules 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-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides, copolymers of L-glutamic acid and ethyl-L-glutamate, non-degradable ethylenevinyl 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 antigenbinding 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 antigenbinding 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 anti-SARS-CoV-2 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 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 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 P5C3 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 Pl 06, P2B11, P7K18, P1L7, P1G17, MS31 and MS35. preferably the anti-SARS-CoV-2 antibody P5C3 of the invention is administered in combination with the anti-SARS-CoV-2 antibody MS35 of the invention. the anti-SARS-CoV-2 antibody P6E16 of the invention is administered in combination with one or more anti-SARS-CoV-2 antibodies of the invention selected from the group consisting ofP7K18 and PlL7.

- the anti-SARS-CoV-2 antibody P5C6 of the invention is administered in combination with Pl 06 anti-SARS-CoV-2 antibody of the invention.

- the anti-SARS-CoV-2 antibody P1H23 of the invention is administered in combination with Pl 06 anti-SARS-CoV-2 antibody of the invention.

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 P5C3 of the invention is administered in combination with one or more anti-SARS-CoV-2 antibodies of the invention selected from P1O6, P2B11, P7K18, P1L7, P1G17, MS31 or MS35.

In an embodiment of the method of prophylaxis, treatment, and/or attenuation of a SARS-CoV- 2 virus infection in a subject, the P5C3 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 P5C3 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 P5C3 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 anti-SARS-CoV-2 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 anti-SARS-CoV-2 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 discolouration 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 one or more anti-SARS-CoV-2 antibody, or antigen-binding fragment thereof, of the invention or one or more 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 one or more anti-SARS-CoV-2 antibody, or antigen-binding fragments thereof, of the invention or one or more 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 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 Pl 06, that bind to non-overlapping epitopes on the viral Spike protein 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-1 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 O.lmg/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 intermittently, 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 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 antibodies discovered against the SARS-CoV-2 virus. The P5C3 antibody has IC50 values of 5.1 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). This analysis resulted in the identification of ten (10) 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. 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 lU/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 pl 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 pl 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 50pl per well. Plates were agitated for 45 minutes, and then washed on the magnetic plate washer. Beads resuspended in 80 pl 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% CO2) before the addition to 293 T 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, P1O6, P2B11, P7K18, P1L7 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 and P6E16 antibodies exhibited the highest binding KD values for Spike (55.6 & 43.4 ng/ml, respectively) and RBD protein (30.1 & 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 pg/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 50pl 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 and P1H23 antibodies exhibited potent inhibition of the Spike /ACE-2 interaction with IC50 values of 55 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

* Incomplete 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, P6E16, P1O6 and P1H23 are the most potently antibodies identified in this application that are more potent than or have equivalent potency to REGN10933 and REGN10987 antibodies tested in parallel. P1M12 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 was 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 and P1O6 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.

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 pg/ml of the indicated competitor antibody was incubated with RBD coupled beads for 30 minutes followed by the addition of 0.5 to 2 pg/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 P1O6, MS35 and P2B11 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 P1O6, 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 P1O6, MS35, MS31, 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 P5C6 with P1O6 or P5C3 with MS35, which are both predicted to be superior to the REGN10933 / REGN10987 combination discovered by Regeneron. The combination of P1H23 with P1O6 would also be anticipated to provide a potent antiviral profile against the SARS-CoV-2 virus with equivalent potency compared to the REGN 10933 / REGN 10987 combination. The binding epitope of P5C3 is also partially overlapping with P1G17.

P6E16 is the second most potent neutralizing antibody disclosed in this application and binds competitively with REGN10933, P5C3, P1O6, P2B11 and P1G17. P6E16 binds RBD at a nonoverlapping epitope with REGN10987, P7K18 andPlL7, 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 trimer 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.

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 nonoverlapping epitopes that are able to co-bind to RBD have percent binding of greater that 70% and are displayed with grey boxes.

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 pg/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 6. 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 6: 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 7 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. The P5C3 exhibited the highest overall binding to the different Spike trimer proteins that included mutations found in SARS-CoV-2 variants of concern including B.1.1.7 (UK variants) B.1.351 (South African variant), P.l (Brazilian variant) and the L452R mutation identified in the CAL.C20 (Californian variant) (Table 8). P5C3 has a binding ICso of 15 to 36 ng/ml against all the Spike proteins tested which is 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, the anti-SARS- CoV-2 antibodies P1O6, 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.

Table 7: Binding of anti-SARS-CoV-2 antibodies to 2019-nCoV and Spike mutations found in variants of concern in Luminex bead based assay

Table 8. Amino acid substitutions and deletions on SARS-CoV-2 variants of concern

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 8). The P5C3 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 was 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 9 and Figure 5). The MS35 antibody also exhibited a highly potent and broad neutralization profde inhibiting all viruses tested with ICso 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.

Table 9: Neutralizing activity of antibodies against different SARS-CoV-2 viral variants in the live virus cytopathic effect assay

Example 9: 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 the 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 6A-B) with RBD in the open only conformation. See Barnes et al. Structures of Human Antibodies Bound to SARS-CoV-2 Spike Reveal Common Epitopes and Recurrent Features of Antibodies. Cell (2020) 182:828-42; and Barnes et al SARS-CoV-2 Neutralizing Antibody Structures Inform Therapeutic Strategies. Nature (2020) 588:682-7 for classification system of anti-SARS-CoV-2 antibodies. Upon analysis of the paratope/epitope interaction, it was discovered that the P5C3-Spike interface covers a large region of about 600A² 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 Hl, H2 and H3 of the heavy chain and LI and L3 of the light chain (Figure 6C). 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 mAb also makes several contacts with the antiparallel 5, 06 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, the structure was superimposed with that solved of the ACE2-RBD interaction (PDB ID 6M0J). ACE2 covers around 860A² on the RBD, compared with 600A² for P5C3 where P5C3 interacts with the RBD ridge at a 90-degree angle compared with 130-degree for ACE2 (19) (Figure 6D). Importantly, ~70% (414 A² out of 600 A²) 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 6E). Furthermore, additional critical residues necessary for RBD interaction with ACE2s are blocked by P5C3, such as Phe456 and Gln493.

The P5C3 binding mode was compared to that of leader mAb 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 N501Y 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 mAb 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 10: P5C3 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 (2.3xl0⁴ PFU dose) (Figure 7A). Four days later, lung from control animals contained between 10⁴ and 5xl0⁶ 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 pg/ml (ranging from 12.2 to 16.4 pg/ml) at the time of viral inoculation (Figure 7 B-C). Animals administered 0.5 mg/kg P5C3 had median plasma antibody levels of 6.7 pg/ml, and 4 out of 7 also exhibited undetectable infectious virus in the lung, while the remaining 3 showed a ~2 log reduction in TCIDso/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.

Example 11: Identification of amino acid substitutions in P5C3 that provide a non-inferior neutralizing activity

Although P5C3 is fully human antibody derived from memory B cells of COVID-19 patients, 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 8A show that N58Y, N58V, N58Q, N58L N58H, N100Q and N100Y (Sequence ID No. 105, 106, 107, 108, 109, 125, 126) have equivalent potency compared to WT P5C3 while M74Y and M74L mutations (Sequence ID No. 114, 115) 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 8B and Table 10).

Following these studies, the P5C3 LS N100Q (Sequence ID No. 125) antibody with the LS extended half-life mutation in the IgGl Fc domain (M428L / N434S) was 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. These studies show that the amino acid substitutions in P5C3 do not detrimentally affect the binding affinity to the tested Spike protein variants. In a likewise fashion, P5C3 LS N100Q demonstrated equivalent neutralization of Spike 2019nCoV D614G, Spike Beta variant and Spike Delta variant pseudotyped viruses compared to the WT P5C3 antibodies. Overall, these substitutions are shown to have noninferior 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.

Table 10: P5C3 antibody mutation sequences evaluated

Claims

93 CLAIMS

1. 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: a) 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); 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 94 set forth in SEQ ID NO: 69, SEQ ID NO: 70, and SEQ ID NO: 71, respectively (antibody P1O6); 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).

2. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 1, 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).

3. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 1, wherein 95 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).

4. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 1, wherein 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 P1O6).

5. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 1, wherein the HCDR1, HCDR2, and HCDR3 sequences as set forth in SEQ ID NO: 90, SEQ ID NO: 91, and SEQ ID NO: 92, respectively; theLCDRl, LCDR2, and LCDR3 sequences as set forth in SEQ ID NO: 99, SEQ ID NO: 100, and SEQ ID NO: 101, respectively (antibody MS35).

6. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 1, 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: 1-10, 81-83, and 105-126 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: 11- 20 and 84-86.

7. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 6, wherein: a. 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 the amino acid sequence of SEQ ID NO: 3 and 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 the amino acid sequence of SEQ ID NO: 13; b. 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 the amino acid sequence of SEQ ID NO: 125 and the light chain variable region comprises an amino acid sequence that 96 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; c. 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 the amino acid sequence of SEQ ID NO: 126 and 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 the amino acid sequence of SEQ ID NO: 13; d. 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 the amino acid sequence of SEQ ID NO: 1 and 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 the amino acid sequence of SEQ ID NO: 11 ; e. 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 the amino acid sequence of SEQ ID NO: 2 and 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 the amino acid sequence of SEQ ID NO: 12; f. 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 the amino acid sequence of SEQ ID NO: 4 and 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 the amino acid sequence of SEQ ID NO: 16; g. 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 the amino acid sequence of SEQ ID NO: 5 and 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 the amino acid sequence of SEQ ID NO: 14; h. 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 the amino acid sequence of SEQ ID NO: 6 and 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 the amino acid sequence of SEQ ID NO: 15; i. 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 the amino acid sequence 97 of SEQ ID NO: 7 and 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 the amino acid sequence of SEQ ID NO: 17; j . 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 the amino acid sequence of SEQ ID NO: 8 and 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 the amino acid sequence of SEQ ID NO: 18; k. 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 the amino acid sequence of SEQ ID NO: 9 and 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 the amino acid sequence of SEQ ID NO: 19; l. 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 the amino acid sequence of SEQ ID NO: 10 and 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 the amino acid sequence of SEQ ID NO: 20; m. 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 the amino acid sequence of SEQ ID NO: 81 and 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 the amino acid sequence of SEQ ID NO: 84; n. 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 the amino acid sequence of SEQ ID NO: 82 and 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 the amino acid sequence of SEQ ID NO: 85; or o. 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 the amino acid sequence of SEQ ID NO: 83 and 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 the amino acid sequence of SEQ ID NO: 86. 98

8. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 6 or 7, 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: 125 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: 126 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; 99 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.

9. An 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:3 and SEQ ID NO: 105 to SEQ ID NO: 126, 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: 13 (antibody P5C3).

10. An anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, comprising a human heavy chain variable region amino acid sequence that comprises or consists of an amino acid sequence selected from SEQ ID NO:3 and SEQ ID NO: 105 to SEQ ID NO: 126, and a human light chain variable region amino acid sequence that comprises or consists of SEQ ID NO: 13 (antibody P5C3).

11. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 9-10, wherein 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 an amino acid sequence selected from SEQ ID NO:3, SEQ ID NO: 125 and SEQ ID NO: 126, and the human light chain variable region comprises an amino acid sequence that is at least 90%, 91%, 100

92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence of SEQ

ID NO: 13.

12. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 9-10, 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:

13. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof of claim 12, 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: 125, 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: 126, and the human light chain variable region amino acid sequence comprises or consists of SEQ ID NO: 13.

14. 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 Tyr451, Leu452, Tyr453, 101

Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, Tyr 489, Pro491, Leu492, Gln493, Ser494, Tyr495, and Gly496 in SEQ ID NO: 127.

15. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 14, wherein the epitope comprises each of Tyr451, Leu 452, Tyr453, Arg454, Leu455, Phe456, Tyr473, Ala475, Gly476, Ser477, Pro 479, Glu484, Phe486, Asn487, Tyr489, Pro 491, Leu 492, Gln493, Ser494, Tyr 495, and Gly496 in SEQ ID NO: 127.

16. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 14, wherein the epitope comprises Ala475, Gly476, Ser477, Pro479, Glu484, Phe486, Asn487, and Tyr489 of SEQ ID NO: 127.

17. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 14, wherein the epitope comprises Pro479 and Phe486 of SEQ ID NO: 127.

18. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 14, wherein the epitope comprises Phe456, Tyr473, Phe486, and Tyr489 of SEQ ID NO: 127.

19. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 14, wherein the epitope comprises amino acids 451-456 and 491-495 of SEQ ID NO: 127.

20. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 14, wherein the epitope comprises Leu455, Phe456, Ala475, Gly476, Ser477, Glu484, Phe486, Asn487, Tyr489, and Gln493 of SEQ ID NO: 127.

21. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 14, wherein the epitope comprises Phe456 and Gln493 of SEQ ID NO: 127.

22. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-21, wherein the antibody is an isolated monoclonal antibody.

23. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-22, wherein the antibody, or an antigen-binding fragment thereof, exhibits an in vitro neutralization IC50 of a SARS-CoV-2 virus at a concentration less than 10 pg/mL.

24. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-23 wherein the antibody, or an antigen-binding fragment thereof, exhibits 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, less than 5 ng/mL, or less than 2.5 ng/mL.

25. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-23 wherein the antibody, or an antigen-binding fragment thereof, exhibits an in vitro neutralization IC50 of a SARS-CoV-2 virus of between 2 ng/mL and 22 ng/mL, between 2 ng/mL and 17 ng/mL, or between 2 ng/mL and 8 ng/mL.

26. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-23 wherein the antibody, or an antigen-binding fragment thereof, exhibits an in vitro neutralization IC50 of a SARS-CoV-2 virus of about 2.5 ng/mL, 5 ng/mL, 8 ng/mL, 10 ng/mL 15 ng/L, 20 ng/mL, or 22 ng/mL.

27. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-26 wherein the antibody, or an antigen-binding fragment thereof, exhibits an in vitro affinity IC80 for the SARS-CoV-2 spike protein of between 10 and 40 ng/mL.

28. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-26 wherein the antibody, or an antigen-binding fragment thereof, exhibits an in vivo affinity IC80 for the SARS-CoV-2 spike protein of less than 22 ng/mL.

29. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 23-28, wherein the SARS-CoV-2 virus is a SARS-CoV-2 Spike protein pseudotyped lentivirus or a SARS-CoV-2 live virus.

30. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 29, wherein the SARS-CoV-2 live virus is selected from wild type SARS-CoV-2 or a variant of SARS-CoV-2 selected from B.1.1.7, B.1.351, P.l, Bl.617.2, B.1.1.529, CAL.C20, Mink variant 16, C.37, and B.1.621.

31. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-30, wherein the antibody 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.

32. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of claim 31, wherein: 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.

33. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1 - 32, wherein the antibody is a mono-specific antibody, a bispecific antibody, a trimeric antibody, a multi-specific antibody, or a multivalent antibody.

34. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1 - 33, wherein the antibody 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.

35. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-34, wherein 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.

36. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-35, further comprising a detectable label fixably attached thereto, wherein the detectable label is selected from the group consisting of fluorescein, DyLight, Cy3, Cy5, FITC, 104

HiLyte Fluor 555, HiLyte Fluor 647, 5-carboxy-2,7-dichlorofluorescein, 5-carboxyfluorescein,

5-FAM, hydroxy tryptamine, 5-hydroxy tryptamine (5-HAT), 6-carboxyfluorescein (6-FAM),

FITC, 6-carboxy-l,4-dichloro-2’,7’-dichloro^_,fluorescein (TET), 6-carboxy-l,4-dichloro- 2 ’ ,4 ’ , 5 ’ , 7 ’ -tetra^_,chlorofluorescein (HEX), 6-carboxy-4 ’ , 5 ’ -di chi oro-2 ’ , 7 ’ - dimethoxyfluorescein (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-carboxytetramethyHrhodamine (TAMRA), tetramethylrhodamine (TRITC), rhodamine WT, Texas Red, and Texas Red-X.

37. A pharmaceutical composition comprising the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-36 and a pharmaceutically acceptable carrier.

38. The pharmaceutical composition of claim 37, comprising a first and a second anti- SARS-CoV-2 antibody, wherein the first anti-SARS-CoV-2 antibody is the P5C3 antibody set forth in claim la, claim 7a, claim 7b, claim 7c, claim 9, claim 10, claim 11, claim 12 and/or claim 13 and the second anti-SARS-CoV-2 antibody is selected from those set forth in claim 1g (P1O6), claim Id (P2B11), claim 1c (P7K18), claim li (P1L7), claim lb (P1G17), claim Ik (MS31) or claim 11 (MS35).

39. 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 any one of claims 1-36 and detecting the antibody in the sample. 105

40. The method of claim 39, further comprising 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.

41. The method of claim 39 or 40, wherein the SARS-CoV-2 virus is selected from a wild type SARS-CoV-2 virus or a variant selected from B. l.1.7, B.1.351, P.l, B.1.617.2, B.1.1.529, CAL.C20, Mink variant 16, C.37, and B.1.621.

42. The method of any one of claims 39-41, wherein 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.

43. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-36 for use as a pharmaceutical.

44. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-36 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 any one of claims 1-36.

45. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, for use according to claim 44, wherein the anti-SARS-CoV-2 antibody P5C3 set forth in claim la, claim 7a, claim 7b, claim 7c, claim 9, claim 10, claim 11, claim 12, and/or claim 13 is administered in combination with one or more anti-SARS-CoV-2 antibodies selected from those set forth in claim 1g (P1O6), claim Id (P2B11), claim 1c (P7K18), claim li (P1L7), claim lb (P1G17), claim Ik (MS31) or claim 11 (MS35).

46. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, for use according to claim 45, wherein the P5C3 antibody set forth in claim la, claim 7a, claim 7b, claim 7c, claim 9, claim 10, claim 11, claim 12, and/or claim 13 and the one or more additional anti-SARS-CoV-2 antibodies are administered as part of the same composition. 106

47. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, for use according to claim 45, wherein the P5C3 antibody set forth in claim la, claim 7a, claim 7b, claim 7c, claim 9, claim 10, claim 11, claim 12, and/or claim 13 and the one or more additional anti-SARS-CoV-2 antibodies are administered as separate compositions.

48. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, for use according to claim 45 or 47, wherein the P5C3 antibody set forth in claim la, claim 7a, claim 7b, claim 7c, claim 9, claim 10, claim 11, claim 12, and/or claim 13 and the one or more additional anti-SARS-CoV-2 antibodies are administered sequentially or simultaneously.

49. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, for use according to any one of claims 44-48, wherein the subject has been diagnosed with a SARS- CoV-2 infection.

50. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, for use according to any one of claims 44-48, wherein the subject does not have a SARS-CoV-2 infection.

51. The anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, for use according to any one of claims 44-49, wherein treating and/or attenuating the SARS-CoV-2 virus infection comprises reducing viral load.

52. The antibody, or an antigen-binding fragment thereof, for use according to any one of claims 44-51, further comprising administering an antiviral agent.

53. An isolated nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigenbinding fragment thereof, of any one of claims 1-35.

54. A vector comprising a nucleic acid encoding the anti-SAR.S-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-35.

55. The vector of claim 54, wherein the vector is an expression vector. 107

56. A host cell comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-35 or comprising the vector of claim 54 or 55.

57. The host cell of claim 56, wherein the host cell is prokaryotic or eukaryotic.

58. A method of producing the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-35 comprising culturing a host cell comprising a nucleic acid encoding the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-35 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.

59. The method of claim 58, further comprising purifying the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof.

60. 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 any one of claims 1- 36 and instructions for use.

61. The kit of claim 60 wherein the anti-SARS-CoV-2 antibody, or an antigen-binding fragment thereof, of any one of claims 1-36 is in lyophilized form.