WO2022054068A1 - Antibodies for the prevention, treatment and detection of coronavirus infection - Google Patents

Abstract

A human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus for use in preventing, treating or detecting Coronavirus infection in a subject in need thereof, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

Description

ANTIBODIES FOR THE PREVENTION, TREATMENT AND DETECTION OF CORONAVIRUS INFECTION

RELATED APPLICATIONS:

This application claims priority from U.S. Provisional Patent Application No. 63/077,826 filed September 14, 2020, which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING STATEMENT

The ASCII file, entitled 89016 Sequence Listing.txt, created on 13 September 2021, comprising 81,333 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to antibodies for the prevention, treatment and detection of a Coronavirus infection.

Severe acute respiratory syndrome Coronavirus 2 (SARS CoV-2) is the etiological cause of the Coronavirus disease 19 (COVID-19) that emerged in late 2019, causing a global pandemic (7). SARS CoV-2 belongs to the family Coronaviridae, alongside SARS-CoV that emerged in 2002 causing approximately 8,000 infections with a lethality of 10% (2). These viruses, along with another closely related Middle East Respiratory Syndrome Coronavirus (MERS CoV), are the result of a zoonotic transfer from an animal reservoir that causes major lung damage and lifethreatening respiratory illness in humans (2). Presently, no approved targeted therapeutics are available for COVID- 19.

Recent evidences indicate that some infected individuals have strong levels of anti-SARS- CoV-2 antibodicsL?). It was also reported that infected individuals are able to produce neutralizing antibodies following infection (4-9). Evidence indicates that antibodies play a crucial role in COVID- 19 progression; First, studies show that SARS CoV-2-infected macaques that produced specific anti-SARS CoV-2 antibodies are immune to re-infection (10). Second, plasma transfer was recently tested as treatment for severe patients and looks promising (11-13). Lastly, immunocompromised population is more susceptible to develop severe symptoms (14, 15). Moreover, it was recently reported that between 48-62 % of infected individuals who do not develop any symptoms of the disease have specific anti-SARS CoV-2 antibodies (16). A chimeric antibody that binds the receptor binding domain (RBD) of the SPIKE protein of SARS CoV-2 was reported in Wang et al. Nature Communications volume 11, Article number: 2251 (2020).

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus for use in preventing or treating Coronavirus infection in a subject in need thereof, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to an aspect of some embodiments of the present invention there is provided a method of preventing or treating Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5, thereby preventing or treating Coronavirus in the subject.

According to an aspect of some embodiments of the present invention there is provided a method of producing an antibody capable of binding an antigenic determinant of Coronavirus, the method comprising:

(a) expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus; and optionally,

(b) recovering the antibody from the host cell.

According to an aspect of some embodiments of the present invention there is provided a vaccine comprising an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to an aspect of some embodiments of the present invention there is provided a monoclonal antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein the antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to an aspect of some embodiments of the present invention there is provided a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus attached to a heterologous effector moiety or carrier, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to some embodiments of the invention, said antibody is a recombinant antibody.

According to some embodiments of the invention, said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2212, 1109, 2230 or 2189.

According to some embodiments of the invention, said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2212.

According to some embodiments of the invention, said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2230.

According to some embodiments of the invention, said human antibody comprising an antigen binding domain which binds said Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.

According to some embodiments of the invention, said plurality of different human antibodies comprise: 1109, and 2212; 2303 and 1109; 2230 and 2212; 2189 and 2212; 1145 and 2212; and/or 2303 and 2212.

According to an aspect of some embodiments of the present invention there is provided a method of detecting a Coronavirus infection, the method comprising contacting a biological sample suspected of being infected with Coronavirus with a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus under conditions which allow a specific immunocomplex formation between said antibody and said Spike, wherein a presence of said immunocomplex is indicative of Coronavirus infection, wherein an antigen binding domain of said antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5. According to some embodiments of the invention, an antigen binding domain of said antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2303 or 2310.

According to some embodiments of the invention, said antibody is labeled.

According to some embodiments of the invention, said contacting is effected in-vivo.

According to some embodiments of the invention, said contacting is effected ex- vivo.

According to an aspect of some embodiments of the present invention there is provided a diagnostic kit for detecting a Coronavirus infection, the kit comprising a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus which allow a specific immunocomplex formation between said antibody and said Spike, wherein an antigen binding domain of said antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to some embodiments of the invention, an antigen binding domain of said antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2303 or 2310.

According to some embodiments of the invention, said antibody is labeled.

According to some embodiments of the invention, said human antibody comprising an antigen binding domain which binds said Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.

According to some embodiments of the invention, said Coronavirus is SAR-CoV-2, Middle East respiratory syndrome Coronavirus (MERS-CoV) or severe acute respiratory syndrome Coronavirus (SARS-CoV).

According to some embodiments of the invention, said Coronavirus is SAR-CoV-2.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

In the drawings:

FIGs. 1A-B show expression of SARS-CoV-2 receptor binding domain. (A) schematic diagram of the RBD construct. A human HGF signal peptide (SEQ ID NO: 179) was added at the N-terminal to enhance protein secretion. At the C-terminal two tags were added - a His-tag for protein purification and an Avi-tag for specific biotinylation. (B) Representative western-blot of cell supernatant containing the RBD protein 3 days post transfection. The protein was detected via anti-Avitag antibody.

FIGs. 2A-I show serological responses towards SARS CoV-2 RBD. Each patient plasma underwent 2-fold serial dilutions, incubated with the RBD protein and detected via (A) anti-human IgG (B) anti-human IgM or (C) anti-human IgA antibodies conjugated to HRP. (D) Patient plasma was also tested for inhibition of RBD-ACE2 binding: ELISA plates were coated with human- ACE2 protein, while each patient plasma sample was pre-incubated with biotinylated RBD. The plasma-RBD mixture was then added to the plates and detected via strepavidin-HRP. (E-G) Patient plasma inhibition data was plotted against plasma reactivity of IgG, IgM and IgA antibodies. Correlation and p-value were calculated by GraphPad Prism version 8.0.0.

FIGs. 3A-J show monoclonal antibodies cloned from donors CoVl and CoV2. (a) Flow cytometry gating strategy for staining anti-SARS-CoV-2 RBD specific memory B cells (b) Graph presents the frequencies of anti-SARS-CoV-2 RBD specific memory B cells in COVID-19 donors CoV01-CoV17. No PBMCs were obtained for donor C0VI8, therefore this donor was not included. Symbol code is given on the right side of the panel, (c) Pie charts represent the total number of RBD-specific memory B cell sequences (heavy chains) obtained from donors CoVOl and CoV02. The numbers in the middle of the pies represent the total number of sequences and the pink shaded slices represent B cell clonal families. The white area represents single sequences that were not clonal, (d) Left panel: number of nucleotide substitutions in VH of the RBD-specific memory B cell sequences of CoVOl (red) and CoV2 (magenta). Statistical analysis was calculated using one-way Anova. Right panel: amino acid length of CDRH3 from RBD-specific memory B cell sequences isolated from CoVOl (red) and CoV2 (magenta), (e) and (f) AUC in ELISA for each one of the 22 cloned mAbs against SARS-CoV-2 RBD and Spike proteins, respectively, (g) and (h): % competition with mAb CR3022, and % of RBD:ACE2 inhibition, as tested in ELISA in the presence of the 22 mAbs isolated from CoVOl and CoV02. MGO.53 isotype control mAb was calculated as 0% inhibition / competition and the other mAbs were normalized to it. (i) and (j) : frequency and mean fluorescence intensity (MFI) of RBD-PE stained hACE2-expressing cells identified by flow cytometry in the presence of 22 mAbs isolated from CoVOl and CoV02. MAbs which reduced RBD-PE staining and MFI are marked with black arrows.

FIGs. 4A-D show activity of the 22 anti-SARS-CoV-2 mAbs in EFISA. (a-b) CoVOl mAbs (left) and CoV02 mAbs (right) binding to SARS-CoV-2 RBD (a) and Spike trimer (b). Antibodies were assayed at a starting concentration of 10 pg/ml with 7 additional consecutive 4- fold dilutions. The color-code is indicated to the right of each graph, (c) Antibody inhibition of biotinylated-RBD:ACE2 binding as measured in EEISA. Plates were coated with human ACE2 and biotinylated-RBD was detected via streptavidin conjugated to HRP. Antibodies were assayed at 300nM with 6 additional consecutive 4-fold dilutions. The y-axis is represented as log2 of the ODeso values. Lower OD indicates higher mAb inhibition, (d) Antibody competition with biotinylated-CR3022. Antibodies were pre-incubated with bound RBD before biotinylated- CR3022 was added and detected via streptavidin-HRP. Lower ODeso values indicate higher level of competition between the mAbs and CR3022.

FIGs. 5A-B predict the epitope of mAb TAU-2230 using phage-displayed affinity purified peptides, (a) Left panel: AUC binding in ELISA of the 16 affinity-selected phage-displayed peptides to mAb TAU-2230. Fth-1 serves as a negative control phage that does not display an insert peptide. The symbols corresponding to each peptide are shown in the right panel next to “Clone name”. Right Panel: amino acid sequences of 16 phage displayed affinity purified peptides. These peptides were used as input for the computer software “Mapitope”. (b) Left panel: ACE2:RBD complex, PDB ID 6M0J SARS-CoV-2 RBD is depicted in gray surface representation, while ACE2 is in yellow cartoon representation, the TAU-2230 predicted contact residues are shown in magenta. Right panel: magnified view of the ACE2:RBD interface from two angles, as well as the predicted TAU-2230 epitope (magenta) on the surface of the SARS-CoV-2 RBD. The in residues in the black squares are both ACE2 contact residues and predicted TAU- 2230 contacts.

FIGs. 6A-F show neutralization by anti-SARS-CoV-2 mAbs. (a) Upper panel: a table presenting IC50, IC80 and R squared for neutralization of Pseudo-typed GFP-reporter viral particles. Lower panel: pie chart presenting mAb classification based on ELISA mapping and pseudo-typed viral particle neutralization data. The number in the middle of the pie denotes the total number of mAbs tested and the slices represent neutralization. Color code is given below, (b) Confocal microscopy image of intracellular SARS-CoV-2 infection as detected by fluorescently labeled anti-nucleocapsid antibody, the blue staining indicated DAPLstained nuclei. Representative mAbs are shown along with “uninfected”, “no antibody”, and MGO.53, which serves as a human isotype mAb control, (c) quantification of (b). (d) - number of dead cells after 3 days of infection in culture in the presence of 0.1 - 100 pg/ml of the indicated mAbs. (e) Syncytia formation inhibition activity as measured by % fusion area in cell-to-cell fusion assay (left) and its quantification (right).

FIGs. 7A-C show neutralizing activity by combinations of anti-SARS-CoV-2 mAbs targeting different sites, (a) monotreatment, (b) double mix (c) triple mix

FIG. 8 is a table representation of antibody binding affinity to each RBD generated in this study. Green color indicates binding affinity of >75%, orange of 25-75% and red of <25% when compared to the wild type strain. The strain in which each mutation appears is indicated above the mutation. The antibodies are separated into ACE2bs and Non-ACE2bs mAbs.

FIGs. 9A-C show antibody binding to RBD of variants of concern (VOCs) compared to compared to wild type SARS-CoV-02 (W.T.). Binding was measured by ELISA for each mAb to (A) Alpha, Beta, Gamma and Delta VOCs, (B) single amino-acid mutations found in the VOC and other circulating strains and (C) double amino-acid mutations found in VOCs.

FIGs. 10A-C show inhibition of RBD:ACE2 interactions by the mAbs. (A) Flow cytometry analysis of antibody interference to RBD:ACE2 binding. Antibodies were added to Spike expressing cells before adding ACE2 conjugated to APC. mGO53 antibody was used as negative control. Unlabeled ACE2 (“Cold” ACE2) was used a positive control for inhibition. Fluorescence was read using CytoFLEX S4 (Beckman Coulter). (B) RBD:ACE2 inhibition potency by mAbs for each VOC Spike. Inhibition was calculated by the percent of ACE2 positive cells. Percentage of positive cells was normalized for each VOC and W.T. (C) Pie charts indicating the percentage of antibodies with strong, mediocre or no inhibition for each VOC and W.T.

FIGs. 11A-B are graphs showing the affinity of antibodies of some embodiments of the invention summarized in a table, (a) SPR sensograms showing binding of injected SARS-CoV-2 RBD at six different concentrations (15.6 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM and 500 nM) to immobilized anti-SARS-CoV-2 TAU mAbs (0.5 pg/ml). mGO53 was used as isotype control. SPR assays were performed on a Biacore T200 instrument at 25 °C. Three replicates were performed for each mAb and all samples were diluted in HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 0.003 M EDTA, 0.05% Tween-20, pH 7.4). Sensograms were fitted to 1:1 binding model using non-linear regression in the biaevaluation software. KD was calculated using the ratio of the kinetic constants Ko=Kd/K_a. (b) Rmax, KD, and U-value values. DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to antibodies for the prevention, treatment and detection of a Coronavirus infection.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

Whilst reducing embodiments of the invention to practice the present inventors isolated from Coronavirus (SARS-CoV-2)-infected human patients, antibodies that are physiologically active in combatting the disease. These antibodies were cloned into an IgGl scaffold.

Of those antibodies, eight bound soluble RBD and one mAb, TAU-2212, bound to the viral spike when it is expressed on viral particles or human cells. mAbs TAU-1145, -2189, -2230 and -2303 are able to inhibit binding of RBD to human ACE2 while mAbs 1109, 1145, 1115, 2189, 2212, 2230, 2310 and 2303 neutralized live W.T. virus.

Some of the antibodies were also able to bind and neutralize variants of SARS-CoV-2.

Being naturally occurring antibodies, the antibodies represent the most effective response of the human body towards the virus. Moreover, these are the safest antibodies to be used as drugs and passive vaccines. Combinations of antibodies can be used to overcome resistance and the ability to bind a conformational epitope in the case of 2212 is also promising in overcoming resistance which stems from mutations that affect linear epitopes. Indeed combinations of antibodies can lead to higher coverage against various variants of the virus.

Notwithstanding the above, these antibodies are also particularly effective as diagnostic tools. Usually neutralizing antibodies are targeting conserved sites on the virus, sites that have an important function during the viral life cycle and therefore substitutions due to random mutation in these epitopes are under negative selection pressure. In view of that, these antibodies can be used to detect different viral isolates. The fact that embodiments of the invention describe several mAbs that bind to non-overlapping epitopes on the Spike protein, pairs of mAbs binding nonoverlapping sites can be used in a sandwich ELISA, or lateral flow assay the presence of SARS- CoV-2 and effectively detect it.

Thus, according to an aspect of the invention, there is provided a monoclonal antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5. According to an alternative or an additional aspect, there is provided a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus attached to a heterologous effector moiety or carrier (e.g., protein, polymeric or lipid carrier, e.g., liposome), wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to an alternative or an additional aspect, there is provided a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus for use in preventing or treating Coronavirus infection in a subject in need thereof, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to an alternative or an additional aspect, there is provided a method of preventing or treating Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5, thereby preventing or treating Coronavirus in the subject.

According to an alternative or an additional aspect, there is provided a vaccine comprising an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

As used herein “antigenic determinant” refers to a peptidic amino acid sequence which comprises an epitope that is recognized by an antigen binding domain of an antibody. Hence the antigenic determinant may comprise one or more epitopes. According to a specific embodiment, the antigenic determinant forms a portion of a viral protein with or without amino acid alterations with respect to the wild-type viral sequence.

According to the present invention, the antigenic determinant is of a Coronavirus.

It would be appreciated that some of the antibodies of the invention mimic the activity of Ace2 in binding the Spike protein, while others don’t e.g., 1109, 2212, 2220 and 2310. According to a specific embodiment, the antibody comprises the CDRs of the respective antibodies as listed in Table 5, each of which is considered as a separate embodiment. Table 5 is considered as an integral part of this section of the document not limited to the Examples section only.

According to a specific embodiment, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2212, 1109, 2230 or 2189.

According to a specific embodiment, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2212.

According to a specific embodiment, the antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2230.

As used herein, “Coronavirus” refers to enveloped positive- stranded RNA viruses that belong to the family Coronaviridae and the order Nidovirales.

Examples of Corona viruses which are contemplated herein include, but are not limited to, 229E, NL63, OC43, and HKU1 with the first two classified as antigenic group 1 and the latter two belonging to group 2, typically leading to an upper respiratory tract infection manifested by common cold symptoms.

However, Coronaviruses, which are zoonotic in origin, can evolve into a strain that can infect human beings leading to fatal illness. Thus particular examples of Coronaviruses contemplated herein are SARS-CoV, Middle East respiratory syndrome Coronavirus (MERS- CoV), and the recently identified SARS-CoV-2 [causing 2019-nCoV (also referred to as “COVID- 19”)].

It would be appreciated that any Corona virus strain is contemplated herein even though SARS-CoV-2 is emphasized in a detailed manner.

According to specific embodiments, the Corona virus is SARS-CoV-2.

As used herein the SARS-CoV-2 includes any variants and mutants thereof including, but not limited to, the B.1.1.7 (Alpha), B.1.351 (Beta), B.1.617.2 (Delta), P.l (Gamma), B.1.526 (Iota), B.1.427 (Epsilon), B.1.429 (Epsilon), B.1.617 (Kappa, Delta), B.1.525 (Eta) and P.2 (Zeta). The present inventors have synthesized proteins of some of these variants, referred herein as “variants of concern” or “VOCs” to support the use of the antibodies or combinations thereof of some embodiments of the invention is combating wild type viruses and variants thereof.

The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof (such as Fab, F(ab')2, Fv, scFv, dsFv, or single domain molecules such as VH and VL) that are capable of binding to an epitope of an antigen, in this case PstSl.

According to specific embodiments, the antibody is a whole or intact antibody. According to specific embodiments, the antibody is an antibody fragment.

Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide- stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2.

As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDRH1 or Hl; CDRH2 or H2; and CDRH3 or H3) and three in each of the VL (CDRL1 or LI; CDRL2 or L2; and CDR L3 or L3).

The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www(dot)bioinf-org(dot)uk/abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).

As used herein, the “variable regions” and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.

Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:

(i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;

(ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule. (iii) disulfide- stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond.

(iv) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CHI domains thereof;

(v) Fab’, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);

(vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds); and

(vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen.

According to specific embodiments the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE.

According to specific embodiments, the antibody is an IgG antibody.

According to a specific embodiment the antibody isotype is IgGl or IgG4.

According to a specific embodiment the antibody isotype is IgGl.

The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.

According to specific embodiments, the antibody comprises an Fc domain.

According to specific embodiments, the antibody is a naked antibody.

As used herein, the tern "naked antibody" refers to an antibody which does not comprise a heterologous effector moiety e.g. therapeutic moiety, detectable moiety.

As used herein “heterologous” means not occurring in nature in conjunction with the antibody.

According to specific embodiments, the antibody comprises a heterologous effector moiety e.g. e.g. therapeutic moiety, detectable moiety. The effector moiety can be proteinaceous or non- proteinaceous; the latter generally being generated using functional groups on the antibody and on the conjugate partner. The effector moiety may be any molecule, including small molecule chemical compounds and polypeptides. For example the effector moiety can be a known drug to Coronavirus infection. According to specific embodiments, the antibody is a monoclonal antibody.

Antibody fragments according to some embodiments of the invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent lightheavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VE chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nafl Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or crosslinked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97- 105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio/Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.

Another form of an antibody fragment is a peptide coding for a single complementaritydetermining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)]. It will be appreciated that for human therapy or diagnostics, humanized antibodies are preferably used.

According to specific embodiments, the antibody is a humanized antibody. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigenbinding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art. Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

According to preferred embodiments, the antibody is a human antibody. According to a specific embodiment, the human antibody carries human Vh,Dh, Jh, VI, J, gene segments such as in germ line antibodies or natural variants thereof. Although synthetic antibodies are also contemplated.

Present teachings also provide for a method of producing an antibody capable of binding an antigenic determinant of Coronavirus, the method comprising:

(a) expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus; and optionally

(b) recovering the antibody from the host cell.

Thus, a polynucleotide encoding an antibody of some embodiments of the invention is cloned into an expression construct selected according to the expression system used.

A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the antibody of some embodiments of the invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors, such as Ti plasmid, containing the coding sequence. Mammalian expression systems can also be used to express the antibodies of some embodiments of the invention.

Examples for mammalian expression vectors include, but are not limited to, pcDNA3, pcDNA3. (+/-), pGL3, pZeoSV2(+/-), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3., pSinRep5, DH26S, DHBB, pNMT, pNMT4, pNMT8, which are available from Invitrogen, pCI which is available from Promega, pMbac, pPbac, pBK-RSV and pBK-CMV which are available from Strategene, pTRES which is available from Clontech, and their derivatives.

Expression vectors containing regulatory elements from eukaryotic viruses such as retroviruses can be also used. SV40 vectors include pSVT7 and pMT2. Vectors derived from bovine papilloma virus include pBV-MTHA, and vectors derived from Epstein Bar virus include pHEBO, and p2O5. Other exemplary vectors include pMSG, pAV009/A⁺, pMTO0/A⁺, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV-40 early promoter, SV-40 later promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells. Examples of bacterial constructs include the pET series of E. coli expression vectors [Studier et al. (990) Methods in Enzymol. 85:60-89).

In yeast, a number of vectors containing constitutive or inducible promoters can be used, as disclosed in U.S. Pat. Application No: 5,932,447. Alternatively, vectors can be used which promote integration of foreign DNA sequences into the yeast chromosome.

In cases where plant expression vectors are used, the expression of the coding sequence can be driven by a number of promoters. For example, viral promoters such as the 35S RNA and 9S RNA promoters of CaMV [Brisson et al. (984) Nature 30:5-54], or the coat protein promoter to TMV [Takamatsu et al. (987) EMBO J. 6:307-3] can be used. Alternatively, plant promoters such as the small subunit of RUBISCO [Coruzzi et al. (984) EMBO J. 3:-680 and Brogli et al., (984) Science 224:838-843] or heat shock promoters, e.g., soybean hsp7.5-E or hsp7.3-B [Gurley et al. (986) Mol. Cell. Biol. 6:559-565] can be used. These constructs can be introduced into plant cells using Ti plasmid, Ri plasmid, plant viral vectors, direct DNA transformation, microinjection, electroporation and other techniques well known to the skilled artisan. See, for example, Weissbach & Weissbach, 988, Methods for Plant Molecular Biology, Academic Press, NY, Section VIII, pp 42-463.

Other expression systems such as insects and mammalian host cell systems [(e.g., Expi293F cells (Thermo Fisher Scientific Inc.)], which are well known in the art and are further described hereinbelow can also be used by some embodiments of the invention.

It will be appreciated that antibodies can also be produced in in-vivo systems such as in mammals, e.g., goats, rabbits etc.

Recovery of the recombinant antibody is effected following an appropriate time (e.g., in culture). The phrase "recovering the antibody” refers to collecting the whole fermentation medium containing the antibody and need not imply additional steps of separation or purification. Notwithstanding the above, antibodies of some embodiments of the invention can be purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

Hence the present teachings in embodiments thereof provide for a recombinant antibody, an antibody which is prepared by recombinant DNA technology. It defers from the isolated human antibody by the presence of non-naturally occurring sequences in the context of the isolated human antibody either at the DNA (e.g., promoter or other regulatory region) and/or protein level (e.g., Fc region, framework region). Once antibodies are obtained, they may be tested for activity.

Thus, antibodies described herein may be tested and/or characterized using a variety of methods. Such methods may be used to determine a variety of characteristics that may include, but are not limited to, antibody affinity; specificity; and activity (e.g., RBD binding, viral neutralization). In vitro testing systems include but are not limited to Vero cells and lung organoids. Antibody testing may further include testing in vivo (e.g., in animal and/or human studies) for one or more of toxicity (though in this case since the antibodies are of human origin they are considered safe), therapeutic effect, pharmacodynamics, pharmacokinetics, absorption, deposition, metabolism, and excretion. Testing in animals may include, but is not limited to, hampsters and Ace2-humanized mouse.

As used herein “neutralize” or “neutralizing” refers to an antibody that binds surface expressed viral Spike and inhibits its interaction with ACE2, as can be determined by an ELISA assay, e.g., whereby antibodies are added to Spike expressing cells before or after adding ACE2 conjugated to APCs.

Assays for determining binding of an antibody to a target antigen include, but are not limited to, ELISA and surface plasmon resonance (SPR).

As used herein “binding” or “binds” refers to an antibody- antigen mode of binding, which is generally, in the range of KD below 500 nM, such as determined by ELISA.

According to another specific embodiment, the affinity of the antibody to its antigen is determined by Surface Plasmon Resonance (SPR).

Specific examples for determining antibody binding are provided in the Examples section which follows.

As used herein the term “KD” refers to the equilibrium dissociation constant between the antigen binding domain and its respective antigen.

According to a specific embodiment, the KD for binding the target (e.g., SPIKE) is typically in the range of 0.01-100 nM

For example between 1-10 nM, 1-50 nM, 0.1-10 nM, 0.1-50 nM, 0.01-100 nM.

High binders which are specifically contemplated herein include, but are not limited to, 1109, 1115, 2303, 2310.

The antibody may be soluble or non- soluble.

The target may be soluble or non-soluble (i.e., particle/cell bound).

Non-soluble antibodies may be a part of a particle (synthetic or non-synthetic, e.g., liposome) or a cell (e.g., CAR-T cells, in which the antibody is part of a chimeric antigen receptor (CAR) typically as a scFv fragment). Increasing the cytotoxic activity of an antibody where necessary can also be achieved such as by using an antibody-drug conjugate (ADC) concept. In such a configuration the antibody is attached to a heterologous effector moiety that can be used to increase its toxicity or to render it detectable.

In some embodiments, antibodies of the invention may be developed for antibody drug conjugate (ADC) therapeutic applications. ADCs are antibodies in which one or more cargo (e.g., therapeutic agents) are attached [e.g. directly or via linker (e.g. a cleavable linker or a non- cleavable linker)]. ADCs are useful for delivery of therapeutic agents (e.g., drugs or cytotoxic agents, some are listed below under “combination therapy”) to one or more target cells or tissues (Panowski, S. et al., 204. mAbs 6:, 34-45). In some cases, ADCs may be designed to bind to a surface antigen on a targeted cell. Upon binding, the entire antibody- antigen complex may be internalized and directed to a cellular lysosome. ADCs may then be degraded, releasing the bound cargo.

It will be appreciated that also polyclonal antibodies can be formulated as ADCs and as such are also envisaged herein.

The antibody-drug delivery system presents a robust candidate for the delivery of perspective COVID-19 therapeutics (Meta et al. Med Hypotheses. 2020 Nov; 144: 110254.). Antibody-drug conjugates (ADC) function by identifying the viral envelope proteins obligatory for the propagation of infection in healthy cells (in thie case Spike for instance). The conjugation of the viral cell-killing ‘highly powerful active pharmaceutical ingredient’ (HPAPI) with antibodies holds high potential for the development of a perspective COVID-19 drug delivery system combining the explicit synergistic effect of both the ingredients.

It will be appreciated that the subject can be treated or diagnosed with a plurality of antibodies to achieve maximal neutralization (inhibition of the virus) either as a treatment or as a vaccine. Also diagnosis may be benefited by the use of a plurality of antibodies.

As used herein “plurality” refers to at least 2 antibodies having different antibgen binding domains (at least one different CDR), e.g., 2-3, 2-4, 2-5, 2-6, 2-7. 2-9, 2-10, 3-4, 3-5, 3-6, 3-7. 3- 9, 3-10.

According to a specific embodiment, the plurality of antibodies bind different epitopes on the virus.

According to a specific embodiment, the plurality of antibodies are composed of an antibody that binds a linear epitope and an antibody that binds a conformational epitope. According to a specific embodiment, the human antibody comprising an antigen binding domain which binds said Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.

According to a specific embodiment, the plurality of antibodies bind identical epitopes on the virus, but may be different in their effector (Fc-mediate) functions.

According to a specific embodiment, the human antibody comprising an antigen binding domain which binds said antigenic determinant comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.

According to a specific embodiment, the antibody binds the SPIKE protein of a Coronavirus.

As used herein “receptor binding domain (RBD)” refers to the receptor (ACE2) binding domain of SARS-CoV-2 of SPIKE, residues Arg319-Phe541 of SPIKE (wild-type or mutant).

Binding can be qualified using various methods known in the art, such as ELISA (exemplified in the section which follows) and surface plasmon resonance (SPR).

According to a specific embodiment, the plurality of different human antibodies comprise: 1109, and 2212; 2303 and 1109; 2230 and 2212;

2189 and 2212; 1145 and 2212; and/or 2303 and 2212.

The present invention envisages immunization against-, and prevention or treatment of Coronavirus infection with any of the antibodies described herein.

The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology.

As used herein, the term “preventing” refers to keeping a disease, disorder or condition from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

Prevention can be done by means of immunization, in this case passive immunization, where the antibody is administered.

As used herein, the term “subject” includes mammals, preferably human beings, male or female, at any age or gender, which suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology (e.g., above 65 of age).

The term “treating” refers to inhibiting, preventing or arresting the development of a pathology (disease, disorder or condition) and/or causing the reduction, remission, or regression of a pathology. Those of skill in the art will understand that various methodologies and assays can be used to assess the development of a pathology, and similarly, various methodologies and assays may be used to assess the reduction, remission or regression of a pathology (i.e., Coronavirus infection, e.g., COVID19 or related complications).

As used herein, the term “preventing” refers to keeping a disease, disorder or condition (i.e., Coronavirus infection, e.g., COVID19 or related complications) from occurring in a subject who may be at risk for the disease, but has not yet been diagnosed as having the disease.

Prevention can be done by means of immunization, in an embodiment passive immunization, where the antibody is administered, or active where the peptide is administered.

As used herein, the term “subject” includes mammals, preferably human beings, male or female, at any age or gender, who suffer from the pathology. Preferably, this term encompasses individuals who are at risk to develop the pathology (e.g., above 60 or 65 of age) or exposed to the virus, e.g., healthcare personnel, education personnel etc.

The composition of matter comprising the antibodies of the present invention can be administered to the subject per se, or in a pharmaceutical composition where it is mixed with suitable carriers or excipients.

As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

Herein the term "active ingredient" refers to the composition of matter comprising the antibodies accountable for the biological effect.

Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference. Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intrapulmonary or intraocular injections.

Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport polypeptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin polypeptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continues infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water based solution, before use.

The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (composition of matter comprising the antibodies) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., Coronaviral infection) or prolong the survival of the subject being treated. According to an embodiment of the present invention, an effective amount of the composition of matter comprising the antibodies of some embodiments of the present invention is an amount selected to neutralize Coronaviruses and/or eliminate infected cells e.g. by initiating ADCC.

Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For example, any in vivo or in vitro method of evaluating Coronavirus viral load may be employed.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

Dosage amount and interval may be adjusted individually to provide the active ingredient at a sufficient amount to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

The present teachings further envisage treating with other anti-viral drugs or antiinflammatory drugs or anti-coagulants as separate treatments or in a co -formulation.

Without being limited to COVID19 but for the sake of example, according to a specific embodiment, the antiviral drug is selected from the group consisting of remdesivir, an interferon, ribavirin, adefovir, tenofovir, acyclovir, brivudin, cidofovir, fomivirsen, foscamet, ganciclovir, penciclovir, amantadine, rimantadine and zanamivir.

Also contemplated are plasma treatments from infected persons who survived and/or antiHIV drugs such as lopinavir and ritonavir, as well as chloroquine.

Specific examples for drugs that are routinely used for the treatment of COVID-19 include, but are not limited to, Lopinavir /Ritonavir, Nucleoside analogues, Neuraminidase inhibitors, Remdesivir, polypeptide (EK1), abidol, RNA synthesis inhibitors (such as TDF, 3TC), antiinflammatory drugs (such as hormones and other molecules), Chinese traditional medicine, such ShuFengJieDu Capsules and Lianhuaqingwen Capsule, could be the drug treatment options for 2019-nCoV.

As mentioned, the antibodies of some embodiments of the invention can be used to detect a Coronavirus and preferably used in diagnosis of Coronavirus infection.

Thus, according to an aspect of the invention there is provided a method of detecting a Coronavirus infection, the method comprising contacting a biological sample suspected of being infected with Coronavirus with a human / recombinant/ monoclonal antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus under conditions which allow a specific immunocomplex formation between said antibody and said Spike, wherein a presence of said immunocomplex is indicative of Coronavirus infection, wherein an antigen binding domain of said antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5. According to a specific embodiment, an antigen binding domain of the antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2303 or 2310.

According to a specific embodiment, the antibody is directly labeled to allow detection. Alternatively the antibody is indirectly labels such as by the use of a labels secondary antibody or by an Sandwich ELISA assay.

According to a specific embodiment, the contacting is effected in-vivo.

According to a specific embodiment, the contacting is effected ex-vivo.

According to an additional or an alternative aspect of the invention there is provided a diagnostic kit for detecting a Coronavirus infection, the kit comprising a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus which allow a specific immunocomplex formation between said antibody and said Spike, wherein an antigen binding domain of said antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

According to a specific embodiment aantigen binding domain of said antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2303 or 2310.

According to a specific embodiment, the human antibody comprising an antigen binding domain which binds said Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.

As used herein the term "diagnosing" refers to classifying a disease, determining a severity of a disease (grade or stage), monitoring progression, forecasting an outcome of the disease and/or prospects of recovery.

The subject may be a healthy subject (e.g., human) undergoing a routine well-being checkup. Alternatively, the subject may be at risk of the disease or infection. Yet alternatively, the method may be used to monitor treatment efficacy.

As used herein “biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, plasma, serum, spinal fluid, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, sputum, and also samples of in vivo cell culture constituents. It should be noted that a “biological sample obtained from the subject” may also optionally comprise a sample that has not been physically removed from the subject (in vivo as opposed to in vitro).

Numerous well known tissue or fluid collection methods can be utilized to collect the biological sample from the subject in order to determine the level of Coronaviruses or infected cells in the sample. Regardless of the procedure employed, once a biopsy/sample is obtained the level of the variant can be determined and a diagnosis can thus be made.

As mentioned, the method of the present invention is effected under conditions sufficient to form protein-protein interactions i.e., complex (e.g. a complex between). Such conditions (e.g., appropriate concentrations, buffers, temperatures, reaction times) as well as methods to optimize such conditions are known to those skilled in the art, and examples are disclosed herein below.

The antibody-SPIKE complex may comprise e.g., be attached, to an identifiable moiety. Alternatively or additionally, the complex may be identified indirectly such as by using a secondary antibody.

According to one embodiment, diagnosis is corroborated using any diagnostic method known in the art, such as by measuring the viral load or titer, by antigen level measurement, antibody level measurement, virus isolation and/or genomic detection by reverse transcriptase- polymerase chain reaction (RT-PCR), etc. For example, a higher viral load or titre often correlates with the severity of an active viral infection. The quantity of virus per mF can be calculated for example by estimating the live amount of virus in an involved body fluid (e.g. serum sample or whole blood).

As used herein the term “about” refers to ± 10 %.

The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

The term “consisting of’ means “including and limited to”.

The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging/ranges between” a first indicate number and a second indicate number and “ranging/ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Culture of Animal Cells - A Manual of Basic Technique" by Freshney, Wiley-Liss, N. Y. (1994), Third Edition; "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); “Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., eds. (1984); "Animal Cell Culture" Freshney, R. I., ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. MATERIALS AND METHODS

Cloning of antibodies

Antibody cloning was performed similarly to what was reported in [von Boehmer, L., Liu, C., Ackerman, S. et al. Sequencing and cloning of antigen- specific antibodies from mouse memory B cells. Nat Protoc 11, 1908-1923 (2016). www(dot)doi(dot)org/10(dot)1038/nprot(dot)2016(dot)102]. In brief, first round PCR products were used as a template for additional amplification with specific 5’ V and 3’ J primers containing restriction sites for subsequent cloning into Ig expression vectors containing the human Igyl constant region. PCR products were purified (MACHEREY-NAGEL, 740609.250) and digested with the appropriate restriction enzymes - Agel and Sall for Igyl, Agel and BsiWI for IgK, Agel and Xhol for IgA. (NEB). The digested products were purified before ligation into human Igyl, IgK and Ig/. expression vectors containing a murine Ig gene signal peptide sequence (accession no. DQ407610). Transcription is under the influence of the human cytomegalovirus (HCMV) promotor. Ligation was performed in a total volume of 20 pl using T4 DNA Ligase (M0202L, NEB) and contained 7.5 pl of digested and purified PCR product and ~25 ng linearized vector. Competent E. Coli DH5aF bacteria (NEB) were transformed at 42 °C with 5 pl of the ligation product. Plasmid DNA was isolated from 2 ml bacteria cultures grown for 18-22 h at 37 °C in LB containing 100 pg/mL ampicillin. Colonies were screened by sequencing using 5' Absense primer (GCTTCGTTAGAACGCGGCTAC).

Cloned mAb vectors for IgGl heavy chain and Kappa or Lambda light chains were cotransfected at a ratio of 1:3 (H:K/L) into Expi293F cells (Thermo Fisher Scientific Inc.) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific Inc.). Seven days post transfection, the cell supernatant was collected, filtered (0.22 pm) and incubated with protein A coated agarose beads (GE Life Sciences, 17519901) for 2 h at RT. The beads were then loaded onto chromatography columns, washed, and eluted using 50 mM sodium phosphate (pH 3.0) into 1 M Tris-HCl (pH 8.0). Antibodies were buffer exchanged to PBS xl, aliquoted, and stored at -80 °C.

Pseudoparticles preparation and neutralization assays - were done as follows. SARS- CoV-2-Spike pseudoparticles were obtained by co-transfection of Expi293F™ cells with pCMV delta R8.2, pLenti-GFP (Genecopoeia), and pCDNA3.1 SAC19 according to manufacturer’s instructions (ThermoFisher Scientific) at a ratio of 1:2:1, respectively. The supernatant was harvested 72 hours post transfection, centrifuged at 1500 x g for 10 minutes to remove cell debris and passed through 0.45 pm filter (LIFEGENE, Israel). Next, pseudoparticles-containing supernatant was concentrated to 5 % of its original volume using Amicon Ultra with 100 KDa cutoff at 16°C (Merck Millipore). HEK-293 cells stably expressing hACE2 were seeded into 0.1 % Gelatin-coated 96-well plates (Greiner) at an initial density of 0.75xl0⁵ cells per well. The following day, concentrated pseudoparticles with serial dilution of antibodies, were incubated for 1 hour at 37 °C and then added to the 96 well pre-seeded plates.

After 48 hours, cells media was replaced with fresh DMEM media excluding Phenol Red, and 24 hours later the 96-well plates were imaged utilizing the IncuCyte ZOOM system (Essen BioScience). Cells were imaged with a lOx objective using the default IncuCyte software settings, which was used to calculate number of GFP-positive cells from four 488 nm-channel images in each well (Data for each antibody concentration was collected in triplicate).

Number of GFP-positive cells were normalized and converted to neutralization percentage. IC50 was calculated by PRISM software fitting to a non-linear regression model.

Construction of variant SARS-CoV-2 RBDs

To generate RBD constructs harboring single amino acid mutations the present inventors used the previously reported W.T. plasmid [Mor et al. Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors. PLoS Pathog 17, el009165, doi:10.1371/joumal.ppat.l009165 (2021)] as a template for PCR mutagenesis s. Pairs of overlapping DNA primers containing one or two base pair substitutions flanked by 20 bases on each side were designed and synthesized by Syntezza-Israel. PCR reactions were performed using KAPA HiFi HotStart ReadyMix (Roche) DNA polymerase. Each PCR reaction contained 10 pL KAPA HiFi HotStart ReadyMix, 0.5 pM of each primer, 1 ng template DNA and the volumes were adjusted to 20 pL with DNase/RNase free water (Bio-Lab). The PCR conditions were as follows: 95 °C for 3 min, 16 cycles of 98 °C for 20 sec and 72 °C for 90 sec. D ouble and triple amino acid mutants were generated similarly with appropriate template and primers.

Expression and purification of soluble SARS-CoV-2 RBDs

Each construct was used to transiently transfect Expi293F cells (Thermo Fisher) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher). Seven days post transfection, the cell supernatant was collected, filtered (0.22 pm), and incubated with Ni²⁺-NTA agarose beads (GE Life Sciences) for 2 h at room temperature (RT). Proteins were eluted by 200 mM imidazole, buffer-exchanged to PBS xl, aliquoted and stored at -80 °C.

ELISAs

High-binding 96 well ELISA plates (Corning #9018) were coated with 1 pg/mL RBD in PBS xl overnight at 4 °C. The following day, the coating was discarded, the wells were washed with “washing buffer” containing PBS xl 0.05 % Tween20 and blocked for 2 h at RT with 200 pL of “blocking buffer” containing PBS xl 3% BSA (MP Biomedicals) 20 mM EDTA and 0.05% Tween20 (Sigma). Antibodies were added at a starting concentration of 4 |ig/mL with seven additional 4-fold dilutions in blocking buffer, and incubated for 1 h at RT. The plates were then washed 3 times with washing buffer before adding a secondary anti-IgG (Jackson ImmmunoResearch) antibody conjugated to horseradish peroxidase (HRP) diluted 1:5000 in blocking buffer, and incubated for 1 h at RT. Following four additional washes, 100 pL of TMB (abeam) was added to each well and the absorbance at 650 nm was read after 20 min (BioTek 800 TS).

Antibody binding to cell-expressed spike

Codon optimized sequences encoding the SARS-CoV-2 Alpha and Beta variants Spike proteins were downloaded from the NCBI data base, synthesized by Syntezza-Israel, and cloned into the pCMV3 mammalian expression vector. Each construct was individually used to transiently transfect Expi293F cells (Thermo Fisher Scientific Inc.) using the ExpiFectamine 293 Transfection Kit (Thermo Fisher Scientific Inc.). 24 h post transfection the cells were centrifuged and resuspended in FACS buffer (PBS xl 2% FBS 2 mM EDTA). Approximately 3xl0⁶ cells were individually incubated with 20 pg of each mAb or unlabeled ACE2 for 30 min at 37 °C, 8 % CO2. Next, 200 ng of ACE2 conjugated to APC was added and the cells were incubated for an additional 20 min at 4 °C. Finally, unbound antibodies and ACE2 were washed and the Fluorescence was read using CytoFEEX S4 (Beckman Coulter).

Surface Plasmon Resonance

SPR was done by injecting SARS-CoV-2 RBD at six different concentrations (15.6 nM, 31.25 nM, 62.5 nM, 125 nM, 250 nM and 500 nM) to immobilized anti-SARS-CoV-2 TAU mAbs (0.5 pg/ml). mGO53 was used as isotype control. SPR assays were performed on a Biacore T200 instrument at 25 °C. Three replicates were performed for each mAb and all samples were diluted in HBS-EP buffer (0.01 M HEPES, 0.15 M NaCl, 0.003 M EDTA, 0.05% Tween-20, pH 7.4). Sensograms were fitted to 1:1 binding model using non-linear regression in the biaevaluation software. KD was calculated using the ratio of the kinetic constants Ko=Kd/K_a. (b) Rmax, KD, and U- value values.

EXAMPLE 1

Characterization of the antibody response in Co vid- 19 patients

Sixteen Covid- 19 convalescent donors were recruited for this study (Table 1). The donors were divided into three groups; “Hospitalized” - patients experiencing severe Covid-19 following SARS CoV-2 infection that required hospitalization and “Mild” - asymptomatic SARS CoV-2 infected donors or donors experiencing a mild disease. In addition, three additional subjects that were termed “Contacts” -donors who were in close contact with SARS CoV-2 positive subjects, were recruited, and tested SARS CoV-2 negative for three consecutive tests, and “Unexposed donors” - samples from subjects that were collected prior to November 2019. Each SARS CoV-2 positive subject donated 150 ml whole blood, from which PBMCs were isolated. Table 1: cohort of SARS CoV-2 positive donors used for this study

Next, the present inventors asked whether the unique immune signatures in Hospitalized versus Mild donors correspond to differential serum responses towards SARS CoV-2. For this, all serum samples were tested in ELISA for IgG, IgA and IgM antibodies against SARS CoV-2 Spike protein receptor binding domain (RBD) that was produced in house (Figures 1A-B). Samples from Hospitalized donors contained higher anti-RBD IgG levels compared to all the other groups, including SARS CoV-2 donors who had mild disease (p=0.019, Figure 2A). This was not repeated for the IgM and IgA, where all groups exhibited low titers of anti-RBD antibodies that did not differ statistically from Unexposed controls (Figures 2B and 2C). Patient plasma was also investigated for inhibition of RBD-ACE2 binding: For

ACE2:RBD inhibition ELISA, high-binding 96 well plates were coated with 2 pg/ml human ACE2 in PBS xl overnight at 4 °C. The next day, plates were washed and blocked with blocking buffer for 2 hours at RT. Concurrently, biotinylated RBD was mixed with of 4-fold serial dilutions of plasma or mAbs. The RBD-plasma/mAb mix was then applied to the ACE2 coated plates and incubated for 30 min. Biotinylated RBD was detected via streptavidin conjugated to HRP (Jackson ImmmunoResearch 016-030-084).

Next, the ability of the serum to inhibit RBD binding to its cellular receptor Angiotensin converting enzyme 2 (ACE2) was tested. Here again, all Hospitalized donors in the cohort were able to inhibit RBD:ACE2 interaction, as opposed to Mild and Contacts that had various level of serum functionality. Once again, the Hospitalized donors had significantly higher ability to block RBD:ACE2 interaction in ELISA compared to Mild donors group (p=0.042). As expected, the levels of anti-RBD IgG, but not IgM or IgA, directly correlated (r2= 0.9367, p<0.0001) with the ability of the serum to inhibit the RBD:ACE2 binding (Figures 2E-G).

It can be concluded that in the cohort, donors that experienced a severe Covid-19 have higher titers of anti-RBD antibodies compared to mildly sick and asymptomatic donors, and that their antibodies have more anti-viral activity.

EXAMPLE 2

Clonal characterization of the antibodies and sequence characterization

Next, the molecular properties of B cells directed against SARS Co-V-2 RBD in infected donors from both Hospitalized and Mild groups were explored. Two Hospitalized donors CoVOl and CoV02 and two Mild donors CoV03 and CoV05 were at focus of the study, all of whom exhibited average IgG binding and inhibition abilities compared to the other donors in their groups (Figures 2A and 2D). RBD-specific IgG positive memory B cells (Figure 3 A) were single cell sorted as previously described and the variable domains of their BCR were amplified by RT-PCR and sequences revealed by Sanger sequencing. Overall, a total of 231 RBD-specific B cells, 39, 125, 38 and 29 from donors CoVOl, CoV02, CoV03 and CoV05 respectively, were sequences (Figure 3B). All the donors had 19-36% percent of clonal expansion, with the exception of CoVOl, who had higher frequency of mutated sequences, but no clonally related B cells (Figures 3B and 3C) were identified. All donors had similar CDRH3 lengths (Figure 3D).

EXAMPLE 3

Functional characterization of the antibodies

Donors CoVOl and CoV02 were taken for further analysis. Twenty two monoclonal antibodies were produced from donors CoVOl and CoV02 (Table 2). Eight mAbs exhibited strong binding to SARS CoV-2 RBD (Figure 4A and 4B). Amongst these mAbs only four mAbs, TAU- 1145, TAU-2189, TAU-2230 and TAU-2303, were able to inhibit RBD:ACE2 interaction in

ELISA (Figure 4D).

The functional ability of the antibodies to prevent viral attachment to the host cells was further tested. For that a genetically engineered human ACE2 expressing cells were used. These cells were stained with RBD-biotinylated and conjugated to PE. Here, again, mAbs TAU-2189, TAU-2230 and TAU-2303 were found to completely inhibit RBD staining by 97-100%, and mAb T AU-1145 was able to inhibit by 90% both the number of cells and MFI (Figures 6E and 6F). It can be concluded that amongst 22 anti-SARS CoV-2 mAbs, four mAbs are strong Ace2 binders (Ace2bs).

The results are presented in Figures 6A-F and 7A-C.

EXAMPLE 4

Antibodies bind and neutralize SARS-CoV-02 variants

The ability of the antibodies to bind and neutralize variants of concern (VOC) Alpha (UK, B.l.1.7)², Beta (South African, B.1.351)³, Gamma (Brazilian, P.l)⁴ and the Delta variants was tested.

Monoclonal antibody binding to RBD from VOC

As mentioned above, RBD was used as a probe to fish-out anti-RBD B cells and clone antibodies from two infected donors. Hence, RBDs were produced from the emerging VOCs and tested for the binding of the mAbs by ELISA. The majority of previously isolated anti-RBD mAbs demonstrated strong binding to VOC-RBDs (Figure 9A); all mAbs bound to RBD from the alpha variant, five out of eight to Beta variant, seven out of eight to the Gamma variant (although some mAbs had weaker binding), and five out of eight to the Delta variants. The mAbs being most sensitive to VOC substitutions were ACE2 binding site (ACE2bs) mAbs T AU-1145, -2189, -2230, -2303. These mAbs were also the most potent neutralizers against the original SARS-CoV-2. In contrast, non-ACE2bs mAbs, TAU-1109, and TAU-2310 kept their binding to all variants, with similar affinity as to the original wild type strain.

E484K and L452R mutations reduce the binding of ACE2 mAbs, except TAU-2303 mAb

To investigate the contribution of each mutation separately, in variants with more than one amino acid substitution in the RBD (Beta, Gamma and Delta strains), the present inventors generated RBDs which comprise single or double amino acid substitutions correlating to each variant (Figures 9B and C). Additionally produced were three RBDs containing circulating mutations N439K⁵, the Danish Mink SARS-CoV-2 variant Y453F^6,7 and the A475V⁸ mutation (Figure 8). The greatest impact on binding was of the E484K and L452R mutations, that knocked out the binding of most ACE2bs mAbs, with the exception of mAb TAU-2303. Interestingly, this mAb that was not sensitive to either 501Y, the 417T single substitutions, was highly affected by the double mutant (Figure 9C, left panel). It is therefore concluded that mAb TAU-2303 binds ACE2 in a different manner than classical ACE2bs mAbs.

EXAMPLE 5

Inhibition of ACE2: Spike VOC interaction by anti-RBD neutralizing mAbs

Many of the amino acid substitutions appearing in VOCs are outside the RBD. Even though these residues are outside the surface that contacts ACE2 directly they can affect the conformation of the Spike trimer and the ability of the virus to interact with ACE2 and with antibodies [Alenquer, M. et al. Signatures in SARS-CoV-2 spike protein conferring escape to neutralizing antibodies. PLoS Pathog 17, el009772, doi: 10.1371/joumal.ppat.1009772 (2021); Gobeil, S. M. et al. Effect of natural mutations of SARS-CoV-2 on spike structure, conformation, and antigenicity. Science 373, doi:10.1126/science.abi6226 (2021)]. Therefore, the present inventors next wanted to measure the effect of signature VOC mutations on the ability of the previously isolated neutralizing anti-SARS-CoV-2 mAbs to bind the surface expressed viral Spike, and to inhibit its interaction with ACE2.

To examine the effect of mutations outside the RBD, as well as the binding of neutralizing mAbs that are directed against conformational quaternary epitopes, the present inventors expressed the Spike proteins of Alpha, Beta and Delta on HEK-293 cells and using flow cytometry assayed the ability of the mAbs to prevent the binding of ACE2-APC to the cells. When tested for binding to hACE2, the present inventors observed increased binding to ACE2 of the Alpha, Beta and Delta Spikes over the W.T. Spike, aligning with previously published data that the affinity of these variants to hACE2 is much higher compared to W.T. As expected and as can be seen in Figures 10A-C, all ACE2bs mAbs exhibited strong inhibition of the W.T. Spike. Interestingly, mAbs that bind the RBD outside the ACE2bs, TAU-1109 and TAU-2212, exhibited as well some inhibitory activity in this system. In agreement with the RBD ELISA results, the Beta VOC was resistant to all ACE2bs mAbs, with the exception of mAb TAU-2230. MAb TAU-2212, that exhibits an extremely potent neutralizing activity, yet does not bind a soluble RBD exhibited approximately 50 % inhibition against the W.T. Spike protein as well as the Alpha and the Delta variants, but was not active against the Beta variant. In contrast, the non-ACE2bs mAb TAU-1109 exhibited an increase in inhibition against Alpha and Beta strains compared to W.T. It can be concluded that amongst out mAbs some exhibit increased and other decreased activity against the VOC.

EXAMPLE 6 Affinity measurements for some of the antibodies of the invention

The affinity of some of the cloned antibodies was determined by SPR (conditions are described hereinanbove and in the Figure legends). Figures 11A-B show a very good affinity to the target in the nanomolar and sub -nanomolar range (0.1 nM). Table 2: 22 mAbs cloned from donors CoVOl and CoV02. Neutralizing mAbs are highlighted in bold

Table 3. complete amino add sequences of the antibodies

Table 4

Table 5*: Selected Antibodies

*-this table should be considered as an integral part of the specification and not merely affiliated to the Examples section. Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims. All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is/are hereby incorporated herein by reference in its/their entirety.

REFERENCES

(other references are listed in the document) P. Zhou et al., A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 579, 270-273 (2020). F. Wu et al., A new coronavirus associated with human respiratory disease in China. Nature 579, 265-269 (2020). F. Amanat et al., A serological assay to detect SARS-CoV-2 seroconversion in humans. NatMed l , 1033-1036 (2020). Y. Cao et al., Potent Neutralizing Antibodies against SARS-CoV-2 Identified by High- Throughput Single-Cell Sequencing of Convalescent Patients' B Cells. Cell 182, 73-84 el6 (2020). D. Pinto et al., Cross-neutralization of SARS-CoV-2 by a human monoclonal SARS-CoV antibody. Nature 583, 290-295 (2020). D. F. Robbiani et al., Convergent antibody responses to SARS-CoV-2 in convalescent individuals. Nature, (2020). T. F. Rogers et al., Isolation of potent SARS-CoV-2 neutralizing antibodies and protection from disease in a small animal model. Science, (2020). X. Wang et al., Neutralizing Antibodies Responses to SARS-CoV-2 in COVID-19 Inpatients and Convalescent Patients. Clin Infect Dis, (2020). A. Z. Wee et al., Broad neutralization of SARS-related viruses by human monoclonal antibodies. Science, (2020). W. D. Linlin Bao, Hong Gao, Chong Xiao, Jiayi Liu, Jing Xue, Qi Lv, Jiangning Liu, Pin

Yu, Yanfeng Xu, Feifei Qi, Yajin Qu, Fengdi Li, Zhiguang Xiang, Haisheng Yu, Shuran Gong, Mingya Liu, Guanpeng Wang, Shunyi Wang, Zhiqi Song, Wenjie Zhao, Yunlin Han, Linna Zhao, Xing Liu, Qiang Wei, Chuan Qin, Reinfection could not occur in

SARS-CoV-2 infected rhesus macaques. bioRxiv doi: w w w(dot)doi(dot)org/ 10(dot) 1101 /2020(dot)03 (dot) 13 (dot) 990226 , (2020) . J. Y. Ahn et al., Use of Convalescent Plasma Therapy in Two COVID- 19 Patients with Acute Respiratory Distress Syndrome in Korea. J Korean Med Sci 35, el49 (2020). L. Chen, J. Xiong, L. Bao, Y. Shi, Convalescent plasma as a potential therapy for COVID-19. Lancet Infect Dis 20, 398-400 (2020). K. Duan et al., Effectiveness of convalescent plasma therapy in severe COVID-19 patients. Proc Natl Acad Sci U SA, (2020). X. Li et al., Risk factors for severity and mortality in adult COVID- 19 inpatients in Wuhan. J Allergy Clin Immunol, (2020). H. O. Al-Shamsi et al., A Practical Approach to the Management of Cancer Patients During the Novel Coronavirus Disease 2019 (COVID-19) Pandemic: An International Collaborative Group. Oncologist, (2020). J. A. Al-Tawfiq, Asymptomatic coronavirus infection: MERS-CoV and SARS-CoV-2 (COVID-19). Travel Med Infect Dis, 101608 (2020).

Claims

47 WHAT IS CLAIMED IS:

1. A human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus for use in preventing or treating Coronavirus infection in a subject in need thereof, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

2. A method of preventing or treating Coronavirus infection in a subject in need thereof, the method comprising administering to the subject an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5, thereby preventing or treating Coronavirus in the subject.

3. A method of producing an antibody capable of binding an antigenic determinant of Coronavirus, the method comprising:

(a) expressing in a host cell a heterologous polynucleotide encoding a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus; and optionally

(b) recovering the antibody from the host cell.

4. A vaccine comprising an effective amount of a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

5. A monoclonal antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus and an excipient, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5. 48

6. A human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus attached to a heterologous effector moiety or carrier, wherein said antigen binding domain comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

7. The antibody, method or vaccine of any one of claims 1-6, wherein said antibody is a recombinant antibody.

8. The antibody, method or vaccine of any one of claims 1-7, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2212, 1109, 2230 or 2189.

9. The antibody, method or vaccine of any one of claims 1-7, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2212.

10. The antibody, method or vaccine of any one of claims 1-7, wherein said antigen binding domain comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2230.

11. The antibody, method or vaccine of any one of claims 1-10, wherein said human antibody comprising an antigen binding domain which binds said Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.

12. The antibody, method or vaccine of claim 11, wherein said plurality of different human antibodies comprise: 1109, and 2212; 2303 and 1109; 2230 and 2212;

2189 and 2212; 1145 and 2212; and/or 2303 and 2212.

13. A method of detecting a Coronavirus infection, the method comprising contacting a biological sample suspected of being infected with Coronavirus with a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus under conditions which allow a specific immunocomplex formation between said antibody and said Spike, wherein a presence of said immunocomplex is indicative of Coronavirus infection, wherein an antigen binding domain of said antibody comprises the complementarity determining 49 regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

14. The method of claim 13, wherein an antigen binding domain of said antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2303 or 2310.

15. The method of claim 13, wherein said antibody is labeled.

16. The method of any one of claims 13-15, wherein said contacting is effected in- vivo.

17. The method of any one of claims 13-15, wherein said contacting is effected ex- vivo.

18. A diagnostic kit for detecting a Coronavirus infection, the kit comprising a human antibody comprising an antigen binding domain which binds an antigenic determinant of Coronavirus which allow a specific immunocomplex formation between said antibody and said Spike, wherein an antigen binding domain of said antibody comprises the complementarity determining regions (CDRs) CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 or the heavy chain and light chain of an antibody selected from the group listed in Table 5.

19. The kit of claim 18, wherein an antigen binding domain of said antibody comprises CDRH1, CDRH2, CDRH3, CDRL1, CDRL2 and CDRL3 of 2303 or 2310.

20. The kit of any one of claims 18-19, wherein said antibody is labeled.

21. The method or kit any one of claims 13-20, wherein said human antibody comprising an antigen binding domain which binds said Coronavirus comprises a plurality of different human antibodies each comprising an antigen binding domain which binds a Coronavirus.

22. The antibody, method, vaccine or kit of any one of claims 1-21, wherein said Coronavirus is SAR-CoV-2, Middle East respiratory syndrome Coronavirus (MERS-CoV) or severe acute respiratory syndrome Coronavirus (SARS-CoV). 50

23. The antibody, method, vaccine or kit of any one of claims 1-22, wherein said Coronavirus is SAR-CoV-2.