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WO2022153212A1 - Anticorps neutralisant le sars-cov-2 - Google Patents

Anticorps neutralisant le sars-cov-2 Download PDF

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Publication number
WO2022153212A1
WO2022153212A1 PCT/IB2022/050265 IB2022050265W WO2022153212A1 WO 2022153212 A1 WO2022153212 A1 WO 2022153212A1 IB 2022050265 W IB2022050265 W IB 2022050265W WO 2022153212 A1 WO2022153212 A1 WO 2022153212A1
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Prior art keywords
antibody
antibodies
cov
sars
antigen
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Inventor
Branislav Kovacech
Eva Kontsekova
Rostislav SKRABANA
Norbert ZILKA
Peter Filipcik
Andrej KOVAC
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Axon Neuroscience SE
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Axon Neuroscience SE
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1002—Coronaviridae
    • C07K16/1003—Severe acute respiratory syndrome coronavirus 2 [SARS‐CoV‐2 or Covid-19]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • SARS severe acute respiratory syndrome
  • the coronaviral genome encodes four canonical structural proteins, the spike (S) protein, nucleocapsid (N) protein, membrane (M) protein, and the envelope (E) protein (Schoeman and Fielding, 2019).
  • the transmembrane S glycoprotein forms homotrimers and mediates receptor attachment and subsequent fusion between the viral and host cell membranes to facilitate viral entry into the host cell (Song et al, 2004).
  • SARS-CoV-2 stimulates a cellular response and production of specific antibodies. Within the first week after symptoms onset, IgA and IgM antibodies directed to the virus’ S and N proteins develop, then followed by the longer-lasting and more specific IgG. Almost 100% of individuals seroconvert by the end of the second week after symptom development (Long et al, 2020; Wolfel et al., 2020).
  • antibodies can neutralize the virus and simultaneously engage the host immune cells such as natural killers, neutrophils or macrophages through their Fc receptors.
  • antibodies can trigger several immune effector mechanisms including antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) (Jaworski, 2020).
  • ADCC antibody-dependent cellular cytotoxicity
  • ADCP antibody-dependent cellular phagocytosis
  • effector function can also lead to disease enhancement as was previously described in case of dengue virus or respiratory syncytial virus (Polack et al, 202; Dowd and Pierson, 2011).
  • the risks of antibody dependent enhancement (ADE) will need to be further evaluated for SARS-CoV-2 (Jaworski, 2020).
  • Antibodies can be engineered with reduced FcR binding ability to minimize potential ADE effects with a double mutation at the Fc region of the antibody (L234A/L235A) (Hessell et al, 2007). It has been shown that a SARS-CoV-2 antibody with the LALA double mutation within the Fc region showed that the antiviral efficacy was not affected in the preclinical study on macaques (Shi et al., 2020).
  • Neutralizing antibodies (NAbs) against SARS-CoV-2 are being evaluated for prophylaxis and as therapeutic agents for COVID-19 patients (Yang et al., 2020). Indeed, clinical trials suggest that antibody treatments can prevent deaths and hospitalizations among people with mild or moderate COVID-19. Some of therapeutic antibodies against COVID- 1 have been authorized for emergency use (Kaplon et al, 2021).
  • mutant variants are partially or completely resistant against therapeutic antibodies that were authorized for emergency use (Tada et al., 2021; Thomson et al., 2021; Jones et al., 2021; Kim et al., 2021).
  • the hybridoma technology and phage display are used less commonly in the development of SARS-CoV-2 antibodies.
  • the disclosure is directed, in part to antibodies against SARS-CoV-2 Spike protein and/or its RBD, compositions comprising those antibodies or nucleic acids encoding the same and methods of using the same.
  • hybridoma technology was used to develop second generation antibodies efficiently neutralizing the new variants of SARS-CoV-2.
  • Hybridoma screening yielded two monoclonal antibodies, AX290 and AX677, with non-overlapping epitopes exhibiting subnanomolar or nanomolar affinities to the receptor binding domain of Spike carrying amino acid substitutions N501Y, N439K, E484K, K417N and N501Y/E484K/K417N found in the virus variants.
  • the antibodies showed excellent neutralization potency of authentic wild type SARS-CoV-2 virus and its Variants of Concern B.l.1.7 (Alpha, South East England), B.l.351 (Beta, South Africa), and B.l.617.2 (Delta).
  • AX677 binds the Spike protein of the Omicron variant (B.l.1.529) equally well as the wild type Spike protein.
  • the antibody combination prevented appearance of escape mutations of the authentic SARS-CoV-2 virus in in vitro assay. This antibody combination is thus a promising tool for COVID-19 therapy.
  • An isolated antibody or antigen-binding fragment thereof that binds SARS-CoV-2 S protein and/or its RBD wherein said antibody comprises at least one heavy chain variable region complementarity determining region (CDR) and/or at least on light chain variable region CDR as those set forth in Table 3, or at least a CDR that is at least 80%, 85%, 90%, 95%, 98% or at least 99% identical to a CDR set forth in Table 3, as determined by IMGT, preferably wherein the antibody comprises the six CDRs of clone 290 or 677 of Table 3, preferably wherein the antibody is a pan-SARS-CoV-2 variant antibody whose neutralizing activity is resistant to all SARS-CoV-2 variants, preferably wherein the antibody binds a RBD of a coronavirus.
  • CDR heavy chain variable region complementarity determining region
  • the antibody or antigen-binding fragment thereof of embodiment 1, comprising three heavy chain CDRs and/or three light chain CDRs selected from the heavy chain and light chain CDRs as set forth in Table 3, or three heavy chains CDRs and/or three light chain CDRs that are at least 80%, 85%, 90%, 95%, 98% or at least 99% identical to those set forth in Table 3, as determined by IMGT, preferably an antibody comprising all CDRs of any one of the antibodies described in Table 3.
  • the antibody or antigen-binding fragment thereof of any one of embodiments 1 and 2 comprising a heavy chain variable region and/or a light chain variable region that is at least 80%, 85%, 90%, 95%, 98% or at least 99% identical to the variable regions encoded by any one of nucleic acids set forth in any one of Tables 1 and 2, respectively.
  • an immunoglobulin molecule a monoclonal antibody, a chimeric antibody, a CDR-grafted antibody, a humanized antibody, a Fab, a Fab', a F(ab')2, a Fv, a disulfide linked Fv, a scFv, a single domain antibody, a diabody, a multispecific antibody, a dual-specific antibody, and a
  • a vector comprising an isolated nucleic acid of embodiment 7.
  • a host cell comprising an isolated nucleic acid of embodiment 7 or a vector of embodiment 8.
  • composition for preventing or treating SARS-CoV-2 infection comprising the antibody or antigen-binding fragment thereof of any one of embodiments 1 through 6, or a nucleic acid of embodiment 7 and a pharmaceutically acceptable carrier, diluent, or excipient
  • a method of diagnosing, preventing, or treating a disease caused by SARS-CoV-2 infection by SARS-CoV-2 or a variant comprising administering to a subject in need thereof an effective amount of an antibody or antigen-binding fragment thereof of any one of embodiments 1 through 6 or a nucleic acid of embodiment 7, preferably wherein the SARS-CoV-2 variant is B.l.1.7 (Alpha), B.1.351 (Beta) and B.l.617.2 (Delta), and/or B.l.1.529 (Omicron).
  • a method of preventing, reducing, and/or treating SARS-CoV-2 infection by SARS-CoV- 2 or a variant in a subject in need thereof comprising administering to the subject an effective amount of an antibody or antigen-binding fragment thereof of any one of embodiments 1 through 6 or a nucleic acid of embodiment 7, preferably wherein the SARS- CoV-2 variant is B.l.1.7 (Alpha), B.1.351 (Beta) and B.l.617.2 (Delta), and/or B.l.1.529 (Omicron).
  • An article of manufacture comprising a container and a composition within the container, wherein the composition comprises the antibody or antigen-binding fragment thereof of any one of embodiments 1 through 6, the nucleic acid of embodiment 7 and instructions to administer an effective dose of the antibody or fragment thereof to a subject.
  • any one of embodiments 1 through 6, nucleic acid of embodiment 7, vector of embodiment 8, or host cell of embodiment 9 in the manufacture of a medicament for diagnosing, preventing, or treating a disease caused by SARS-CoV-2 infection, or treating SARS-CoV-2 infection, by SARS- CoV-2 or a variant, preferably wherein the SARS-CoV-2 variant is B.l.1.7 (Alpha), B.1.351 (Beta) and B.l.617.2 (Delta), and/or B.l.1.529 (Omicron). 19.
  • a composition comprising one or more antibodies or antigen-binding fragments thereof of embodiment 1, or one or more antibodies comprising the six CDRs of any one of the antibodies of Table 3, and a pharmaceutically acceptable carrier; preferably a pharmaceutical composition for preventing or treating a coronavirus infection, the composition comprising a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment, wherein the first antibody or antigen-binding fragment binds to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein and/or sterically blocks ACE2 binding, and wherein the second antibody or antigen-binding fragment does not bind to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein, does not sterically block ACE2 binding, and/or binds adjacent to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein.
  • composition of embodiment 20, wherein the one or more antibodies are selected from monoclonal antibodies AX96, AX290, AX266, AX677, AX99.
  • composition of embodiment 20, wherein the one or more antibodies are selected from monoclonal antibodies AX68, AX97, AX12, AX322 and AX352.
  • composition of embodiment 20, where the one or more antibodies comprise monoclonal antibodies AX290 and AX677, or chimeric or humanized versions thereof.
  • composition comprising one or more antibodies, or antigen-binding fragments thereof, binding one or more of the epitopes I, II, and in of FIG. 21 A.
  • SARS-CoV-2 polypeptide selected from the polypeptides carrying one or more of the escape mutations listed in FIG. 26B.
  • a pharmaceutical composition for preventing or treating a coronavirus infection comprising a first antibody or antigen-binding fragment and a second antibody or antigen-binding fragment, wherein the first antibody or antigen-binding fragment binds to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein and/or sterically blocks ACE2 binding, and wherein the second antibody or antigen-binding fragment does not bind to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein, does not sterically block ACE2 binding, and/or binds adjacent to the ACE2 receptor binding domain of the SARS-CoV-2 spike protein, wherein the first and second antibodies are antibodies of claim 1.
  • a method for isolating an escape mutant of a SARS coronavirus comprising:a) expressing a SARS-CoV-2 chimeric vesicular stomatitis virus (VSV-SARS-CoV-2) comprising a receptor binding domain of a SARS-CoV-2 virus; b) applying a neutralizing antibody having an affinity for an epitope on the RBD to the virus; c) incubating in replication competent conditions for a period of time; and d) isolating the escape mutant, wherein the escape mutant comprises at least one mutation relative to the wild-type, wherein the antibody is an antibody of claim 1.
  • VSV-SARS-CoV-2 chimeric vesicular stomatitis virus
  • a vaccine comprising a polypeptide comprising an amino acid sequence comprising at least about 70% identity to an epitope targeted by an antibody or antigen-binding fragment of claim 1, preferably wherein the vaccine provides an effective immunological response to a coronavirus when administered to a subject 35.
  • FIG. 1 ELISA immunoreactivity of positive hybridoma clones derived from mice immunized with the Spike (S) protein of SARS-CoV-2 as an immunogen. All tested clones produced monoclonal antibodies specific to S protein. RBD domain was recognized by all monoclonal antibodies, except for antibody produced by the clone no. 3.
  • FIG. 2 ELISA immunoreactivity of positive hybridoma clones derived from mice immunized with the receptor binding domain (RBD) as immunogen. All hybridoma clones produce monoclonal antibodies specific to RBD domain and most of them also recognize S protein of SARS-CoV-2.
  • RBD receptor binding domain
  • FIG. 3 Inhibition activity of monoclonal antibodies specific to S protein of SARS- CoV-2 and S protein receptor binding domain (RBD) in competitive ELISA. Results show that selected antibodies effectively inhibit interaction of RBD domain with ACE2, used as the solid phase. Serum of a mouse immunized with the S protein RBD domain, diluted 1:200, was used as a positive control.
  • FIG. 4 Inhibition activity of monoclonal antibodies specific to RBD and S protein of SARS-CoV-2 in cell-based S-ACE2 interaction inhibition assay. Results show that the selected antibodies effectively inhibit interaction of S protein with the ACE2 receptor expressed on cell surface and prevent its internalization. Mouse serum specific to RBD domain diluted 1:200 was used as a positive control.
  • FIG. 5 Inhibition activity of selected monoclonal antibodies in SARS-CoV-2 S- typed pseudovirus assay. Monoclonal antibodies were serially diluted (1:25-1:1600) and mixed with the pseudovirus. Results show the different capability of individual monoclonal antibodies to neutralize the S-typed pseudovirus of SARS-CoV-2.
  • FIG. 6A and 6B Inhibitory activity of monoclonal antibodies specific to the S protein of SARS-CoV-2 or its RBD in plaque reduction neutralization test (PRNT).
  • the assay was done on two different of SARS-CoV-2 virus isolates from Slovak Republic ( Figure 6A-BMC5; Figure 6B-BMC6). MAbs were serially diluted (1:100-1:8100). Cut off for virus BMC5 was 22 PFU; cut off for virus BMC6 was 30 PFU. The data show wide range of potencies of individual monoclonal antibodies to neutralize the SARS-CoV-2 virus isolates. All three subclones of the 68 clone have similar activities and turned out to be identical and produce the same monoclonal antibody.
  • FIG. 7 Summary table with characteristics of MAbs specific to Spike protein/RBD of SARS-CoV-2 with neutralization activity. Dilution of MAbs: cell inhibition test (1:6); ELISA inhibition test (1:50); pseudovirus neutralization test (1:25), plaque reduction neutralization neutralization test (PRNT) (1:25).
  • FIG. 8 Testing scheme for identification of monoclonal antibodies neutralizing SARS-CoV-2.
  • FIG. 9 DNA sequences of the variable regions of the heavy chains of the SARS- CoV-2 neutralizing antibodies.
  • FIG. 9 discloses SEQ ID NOS 36-40, 43, 42, and 41, respectively, in order of appearance.
  • FIG. 10 DNA sequences of the variable regions of the light chains of the SARS- CoV-2 neutralizing antibodies.
  • FIG. 10 discloses SEQ ID NOS 46-50, 53, 52, and 51, respectively, in order of appearance.
  • FIG. 11 Variable regions of the selected antibodies with neutralizing activities against SARS-CoV-2 virus.
  • the complementarity determining regions (CDR) are underlined. They were identified according to the ImMunoGeneTics (IMGT) numbering system (see, e.g. Lefranc MP. The IMGT unique numbering for immunoglobulins, T-cell receptors, and Ig-like domains. The Immunologist 7, 132-136, 1999).
  • FIG. 11 discloses SEQ ID NOS 1-5, 58, 57, 56, 6- 10, 63, 62, and 61, respectively, in order of appearance.
  • IMGT ImMunoGeneTics
  • FIG. 12 Comparison of the DNA sequence of the variable heavy and light chains of the clone 266 with their closest germlines.
  • FIG. 12 discloses SEQ ID NOS 36, 71, 46, and 72, respectively, in order of appearance.
  • FIG. 13 Comparison of the DNA sequence of the variable heavy and light chains of the clone 677 with their closest germlines.
  • FIG. 13 discloses SEQ ID NOS 37, 73, 47, and 74, respectively, in order of appearance.
  • FIG. 14 Comparison of the DNA sequence of the variable heavy and light chains of the clone 290 with their closest germlines.
  • FIG. 14 discloses SEQ ID NOS 38, 75, 48, and 76, respectively, in order of appearance.
  • FIG. 15 Comparison of the DNA sequence of the variable heavy and light chains of the clone 68 with their closest germlines.
  • FIG. 15 discloses SEQ ID NOS 39, 71, 49, and 72, respectively, in order of appearance
  • FIG. 16 Comparison of the DNA sequence of the variable heavy and light chains of the clone 96 with their closest germlines.
  • FIG. 16 discloses SEQ ID NOS 40, 75, 50, 76, 43, 77, 53, 78, 42, 71, 52, 72, 41, 79, 51, and 80, respectively, in order of appearance.
  • FIG. 17 Binding characteristics and inhibition effectivity of monoclonal antibodies specific to SARS-CoV-2 S/RBD.
  • FIG. 18 Immunocytochemistry of HEK293T/17 cells transfected with human ACE2-TMPRSS2 (A) vs. control un-transfected cells (B). Confocal image shows staining of membrane-bound ACE2 glycoprotein on HEK293T/17 cells. Cells were fixed with 4% paraformaldehyde and labelled with anti-human ACE2 antibody at 10ug/ml, followed by goat anti- rabbit IgG secondary antibody at 1/500 dilution (green), and DAPI staining (blue nuclei). Scale bar: 20pm.
  • FIG. 19 Functional analysis of monoclonal antibodies in the S protein internalization cell assay.
  • the S - ACE2 binding inhibition by the selected monoclonal antibodies was evaluated by flow cytometry.
  • Purified mAbs (two-fold serial dilutions, 800-0.39 ng/ml) were mixed with the fluorescently (Alexa FluorTM546) labelled S protein (40 ng/ml) before adding to HEK293T/17-hACE2 cells.
  • Mean fluorescence intensity (MFI) of internalized S protein was determined from the gate of singlet and Alexa-negative cells. Representative histograms (cell count on y-axis, fluorescent intensity on x-axis) are shown. Background represents cells not stained with fluorescently labelled S, negative control are cells incubated with fluorescent S pre-incubated with an antibody that does not bind S, a hybridoma supernatant containing a neutralizing antibody was used as technical positive control.
  • FIG. 20 Kinetic sensorgrams of RBD binding to antibodies by SPR. Monoclonal antibodies were immobilized on the sensor chip surface and two-fold serial dilutions of RBD starting from 3.125 nM were injected (time point 0 s). After 120 s of RBD binding (injection end) die RBD-antibody complex dissociation in running buffer (PBS) was recorded for 480 s.
  • PBS running buffer
  • FIG. 21 The panel of selected neutralization antibodies target three non-overlapping epitope groups on RBD.
  • A Competitive ELISA was used for determination of the epitopes on RBD by neutralizing Mabs. Three non-overlapping epitopes were defined. Signal reduction formore than 30 % was considered a positive competition.
  • B Difference plots depicting the changes in deuterium uptake in the RBD peptides upon binding to AX677 (left panels) and AX290 (right panels). The HDX reaction times are indicated. Numbers represent amino acid positions in the recombinant RBD protein (adding 316 aligns them with the S protein numbering). Each plot represents an average of three technical replicates.
  • FIG. 23 HDX-MS sequence coverage of RBD following digestion by immobilized nepenthesin-2. The digestion of RBD resulted in 288 identified peptides, covering 89% of the sequence.
  • FIG. 23 discloses SEQ ID NO: 81.
  • FIG. 24 Effect of RBD mutations on immunoreactivity of monoclonal antibodies in ELISA and PRNT.
  • A, B Binding of the antibodies to the RBD carrying the individual mutations (N501Y, N439K, E484K, K417N) and RBD with triple mutation N501Y/E484K/K417N were analysed. Effectivity of binding for each antibody was expressed by EC50 value.
  • FIG. 25 Selected mouse monoclonal antibodies neutralize SARS-CoV-2 variants of concern.
  • Live authentic SARS-CoV-2 virus variants : Slovakia/SK-BMC5/2020 isolate (WT), B.l.1.7 (UK) and B.1.351 (SA), were pre-incubated with serial dilutions of Abs (250 ng/ml - 2 ng/ml) and then added to VERO 6 cells. Plaque reductions (%) relative to negative control were calculated 72 h post infection. Data are plotted as the mean from two wells of one experiment. PRNT 50 mAb concentrations are shown in the table.
  • FIG. 26 A combination of AX290 and AX677 prevents rapid mutational escape of authentic live SARS-CoV-2 virus.
  • A A schematic diagram of microplate well allocations in the SARS-CoV-2 escape mutants experiment. MAbs were serially diluted starting with 50 ⁇ g/mL for the 1 st run (middle panels), preincubated with live SARS-CoV-2 viruses (diluted to MOM).5) and added to VERO 6 cells. Culture medium from the first wells where CPE appeared (indicated with the yellow circles) were used for the 2 nd round of virus replication (right panels) mixed with antibody dilutions starting at 100 ⁇ g/mL. White squares highlight wells with CPE where virus genomes were sequenced.
  • FIG. 26B discloses "IACLV” as SEQ ID NO: 35.
  • FIG. 27 Functional characteristics of chimeric monoclonal antibodies.
  • A Binding of the chimeric antibodies AX290ch and AX677ch to the spike protein RBD and RBD carrying the individual point mutations: N501 Y, N439K, E484K, K417N and RBD with the triple mutation N501 Y/E484K/K417N were analysed by ELISA. Effectivity of binding for each antibody was expressed by EC50 value.
  • FIG. 28 Kinetic sensorgrams of RBD WT and mutants binding to chimeric antibodies AX290 and AX677 by SPR Monoclonal antibodies were immobilized on the sensor chip surface and two-fold serial dilutions of RBD starting from 3.125 nM were injected (time point 0 s). After 120 s of RBD binding (injection end) the RBD-antibody complex dissociation in running buffer (PBS) was recorded for 480 s.
  • PBS running buffer
  • FIG. 29 The complete list of virus nucleotide and amino acid substitutions identified in the virus escape experiment.
  • the sequenced viral genomes were compared with the prototype sequence of Wuhan-Hu- 1 (NC_045512).
  • CtrlA, B and C are controls without any antibody; they show mutations originally identified in the Slovak SARS-CoV-2 isolate Slovakia/SK-BMC5/2020. No changes occurred during passaging of the virus in VERO E6 cells. Nucleotide changes emerged under the antibody pressure are highlighted in bold. Nt, nucleotide; AA, amino acid.
  • FIG. 30 The positions of the amino acid residues that were mutated in the viruses that escaped from the neutralization by AX677 (T345N) and AX290 (S477R) are marked by arrows on the structure (surface rendering) of SARS-CoV-2 Spike receptor-binding domain (RBD) bound to ACE2 (reproduced form PDB 6M0J).
  • T345 is located close to the AX677 binding site (dark region on the RBD surface model on the left)
  • S477 is located in the binding site of AX290 (dark region on the RBD surface model on the right).
  • a part of human ACE2 that is in contact with the receptor binding domain is rendered in a cartoon style.
  • FIG. 33 Histology and F4/80 immunohistochemistry of mouse lung tissues on day 7 after infection. Hematoxylin/Eosin-stained sections of paraformaldehyde-fixed, paraffin- embedded lung tissues (top row of micrographs), one representative example per treatment group. Normal tissue, small areas with inflammatory infiltrates and some thickened septa are seen in the samples from animals treated with AX290 and AX677 (or their combination), while infiltrate-filled lungs with little normal tissue were seen in the sections from isotype controls.
  • FIG. 35 Recombinant ectodomain of wild type Spike protein and Spike containing mutations (A67V, HV69-70del, T95I, G142D, VYY143-145del, N211del, L212I, ins214EPE, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, T547K, D614G, H655Y, N679K, P681H, N764K, D796Y, N856K, Q954H, N969K, L981F) of the lineage B.1.1.529 Omicron (#SPN-C52Hz, ACROBiosystems Inc., Newark, DE, USA) were immobilized on ELISA plate, blocked and incubated
  • the bound antibodies were visualized by HRP- labelled anti-human antibodies.
  • the experiment was performed in technical duplicates and data were evaluated by fitting to a sigmoidal four-parameter logistic curve using GraphPad Prism Version 6.07 (GraphPad Software Inc.). ND, could not be determined.
  • nucleotides includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64,
  • consisting of is defined as "closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith.
  • a claim which depends from a claim which "consists of the recited elements or steps cannot add an element or step.
  • the terms “consisting essentially of’ or “consists essentially” likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.
  • the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system.
  • “about ’ or “comprising essentially of’ may mean within one or more than one standard deviation per the practice in the art “About” or “comprising essentially of’ may mean a range of up to 10% (i.e., ⁇ 10%).
  • “about” may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value.
  • about 5 mg may include any amount between 4.5 mg and 5.5 mg.
  • the terms may mean up to an order of magnitude or up to 5-fold of a value.
  • any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.
  • administering refers to the physical introduction of an agent to a subject, using any of the various methods and delivery systems known to those skilled in the art.
  • Antibodies, polypeptides, nucleic acids and host cells of the present description, and immunogenic compositions and vaccines thereof may be administered to a subject in need thereof by routes known in the art, and may vary depending on the use. Routes of administration include, for example, local administration (e.g., to the lungs, intranasal) and parenteral administration such as subcutaneous, intraperitoneal, intramuscular, intravenous, intraportal and intrahepatic.
  • antibodies, polypeptides, nucleic acids, compositions, vaccines or host cells of the present disclosure, or immunogenic compositions thereof are administered to a subject by local infusion, for example using an infusion pump and/or catheter system.
  • routes of administration for the formulations disclosed herein include intravenous, intramuscular, subcutaneous, intraperitoneal, spinal or other parenteral routes of administration, for example by injection or infusion.
  • parenteral administration means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intralymphatic, intralesional, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion, as well as in vivo electroporation.
  • the polypeptides, nucleic acids, compositions, vaccines or host cells of the present disclosure, or immunogenic compositions thereof is administered via a non-parenteral route, e.g., orally.
  • non-parenteral routes include a topical, epidermal, or mucosal route of administration, for example, intranasally, vaginally, rectally, sublingually or topically.
  • Administering may also be performed, for example, once, a plurality of times, and/or over one or more extended periods.
  • An "amino acid/s" or an "amino acid residue/s" may be a natural or non-natural amino acid residue/s linked by peptide bonds or bonds different from peptide bonds.
  • the amino acid residues may be in D- configuration or L -configuration (referred to herein as D- or L- enantiomers).
  • An amino acid residue comprises an amino terminal part (NIL) and a carboxy terminal part (COOH) separated by a central part (R group) comprising a carbon atom, or a chain of carbon atoms, at least one of which comprises at least one side chain or functional group.
  • NIL refers to the amino group present at the amino terminal end of an amino acid or peptide
  • COOH refers to the carboxy group present at the carboxy terminal end of an amino acid or peptide.
  • the generic term amino acid comprises both natural and non-natural amino acids. Natural amino acids of standard nomenclature are listed in 37 C.F.R 1.822(b)(2).
  • non-natural amino acids are also listed in 37 C.F.R 1.822(b)(4), other non-natural amino acid residues include, but are not limited to, modified amino acid residues, L-amino acid residues, and stereoisomers of D-amino acid residues.
  • Naturally occurring amino acids may be further modified, e.g. amidation, hydroxyproline, y-carboxygiutamate, and O-phosphoserine.
  • an antibody includes, without limitation, a glycoprotein immunoglobulin, which binds specifically to an antigen.
  • an antibody may comprise at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or an antigen-binding molecule thereof.
  • Each H chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region.
  • the heavy chain constant region comprises three constant domains, CHI, CH2, and CH3.
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region.
  • the light chain constant region is comprises one constant domain, CL.
  • the VH and VL regions may be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with an antigen.
  • the constant regions of the Abs may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system.
  • Antibodies may include, for example, monoclonal antibodies, recombinantly produced antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), human antibodies, engineered antibodies, humanized antibodies, chimeric antibodies, immunoglobulins, synthetic antibodies, tetrameric antibodies comprising two heavy chain and two light chain molecules, an antibody light chain monomer, an antibody heavy chain monomer, an antibody light chain dimer, an antibody heavy chain dimer, an antibody light chain- antibody heavy chain pair, intrabodies, antibody fusions (sometimes referred to herein as “antibody conjugates”), heteroconjugate antibodies, single domain antibodies, monovalent antibodies, single chain antibodies or single-chain Fvs (scFv), camelized antibodies, affybodies, Fab fragments, F(ab’)2 fragments, disulfide-linked Fvs (sdFv), anti-idiotypic (anti-Id) antibodies (including, e.g., anti- anti-Id antibodies), minibodies, domain antibodies, synthetic antibodies (sometimes
  • An immunoglobulin may derive from any of the commonly known isotypes, including but not limited to IgA, secretory IgA, IgG and IgM IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4.
  • Isotype refers to the Ab class or subclass (e.g., IgM or IgGl) that is encoded by the heavy chain constant region genes.
  • antibody includes, by way of example, both naturally occurring and non- naturally occurring Abs; monoclonal and polyclonal Abs; chimeric and humanized Abs; human or nonhuman Abs; wholly synthetic Abs; and single chain Abs.
  • a nonhuman Ab may be humanized by recombinant methods to reduce its immunogenicity in man.
  • the term “antibody” also includes an antigen-binding fragment or an antigen-binding portion of any of the aforementioned immunoglobulins, and includes a monovalent and a divalent fragment or portion, and a single chain Ab.
  • an “antigen binding molecule”, “antigen binding protein” “antigen binding portion,” or “antibody fragment” refers to any molecule that comprises the antigen binding parts (e.g., CDRs) of the antibody from which the molecule is derived.
  • An antigen binding molecule may include the antigenic complementarity determining regions (CDRs).
  • Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, dAb, linear antibodies, scFv antibodies, and multispecific antibodies formed from antigen binding molecules.
  • Peptibodies i.e., Fc fusion molecules comprising peptide binding domains are another example of suitable antigen binding molecules.
  • the antigen binding molecule binds to an epitope present in the surface of a viral particle. In some embodiments, the antigen binding molecule binds to an epitope in the SARS coronavirus S protein. In certain embodiments, the antigen binding molecule binds to any one of polypeptides of the disclosure. In further embodiments, the antigen binding molecule is an antibody fragment that specifically binds to the antigen, including one or more of the complementarity determining regions (CDRs) thereof. In further embodiments, the antigen binding molecule is a single chain variable fragment (scFv). In some embodiments, the antigen binding molecule comprises or consists of avimers.
  • the term "antigen-binding fragment” means any antigen- binding fragment of an antibody, including an intact antibody or antigen-binding fragment that has been modified, engineered or chemically conjugated.
  • antibodies that have been modified or engineered are chimeric antibodies, humanized antibodies, and multispecific antibodies (e.g., bispecific antibodies.
  • Antigen-binding fragments include, inter alia, Fab, F(ab'), F(ab')2, Fv, dAb, Fd, complementarity determining region (CDR) fragments, single-chain antibodies (scFv), bivalent single-chain antibodies, single-chain phage antibodies, diabodies, triabodies, tetrabodies, (poly)peptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the (poly)peptide, etc.). Regardless of structure, the antigen-binding fragment binds with the same antigen that is recognized by the intact immunoglobulin.
  • An antigen-binding fragment can comprise a peptide or polypeptide comprising an amino acid sequence of at least 2, 5, 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, or 250 contiguous amino acid residues of the amino acid sequence of the binding molecule.
  • the above fragments may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineered by recombinant DNA techniques. The methods of production are well known in the art
  • isolated antibody and “isolated peptide” refer to a protein or peptide produced from cDNA-, recombinant RNA-, or any other synthetic-origin, or some combination thereof; as well as to proteins and peptides that, by virtue of their origin, or source of derivation, either (1) are not associated with proteins found in nature, (2) are free of other proteins from the same source, e.g. free of murine proteins, (3) are expressed by a cell from a different species, or [4] do not occur in nature.
  • epitope is used here to refer to binding sites recognized by a binding protein or an antibody.
  • Epitopes can be any molecule or grouping thereof, including, but not limited to, amino acids, amino acid side chains, sugars, and lipids, and can have a specific three- dimensional structure or conformation.
  • an epitope can comprise any portion of a S protein/peptide or RBD molecule that includes primary, secondary, tertiary, or quaternary structure, as those terms are generally known in the art
  • a “linear epitope” is made up of a continuous sequence of amino acid residues.
  • a “conformational epitope” is an epitope to which the antibody or binding protein binds in a conformational-specific manner.
  • the binding can depend on the epitope-carrying-protein’s secondary, tertiary, or quaternary structure.
  • the antibody binds in a structure-specific manner, a tertiary- structure-specific manner, or a quaternary-structure-specific manner.
  • the terms "specifically binds,” “binds specifically,” and “specific to,” are interchangeable and mean that an antibody or antigen-binding fragment thereof (or other binding protein) forms a complex with an antigen or epitope that is relatively stable under physiologic conditions.
  • Specific binding can be characterized by a dissociation constant of about 1 x 10" 6 M or smaller, for example less than about 100 nM, and most for example less than 10 nM.
  • Methods for determining whether two molecules specifically bind are known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like.
  • an antibody or antigen-binding fragment thereof provided by the disclosure is a molecule that binds the antigen or an epitope with such a dissociation constant of at least about 1 x 10" 6 M or smaller, but does not bind other molecules with such a dissociation constant
  • the antibody preferentially binds or recognizes the binding partner even when the binding partner is present in a mixture of other molecules or organisms.
  • the binding may be mediated by covalent or non- covalent interactions or a combination of both.
  • the term “specifically binding” means immunospecifically binding to an antigenic determinant or epitope and not immunospecifically binding to other antigenic determinants or epitopes.
  • an antibody that immunospecifically binds to an antigen may bind to other peptides or polypeptides with lower affinity as determined by, e.g., radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), BIACORE, or other assays known in the art
  • RIA radioimmunoassays
  • ELISA enzyme-linked immunosorbent assays
  • BIACORE enzyme-linked immunosorbent assays
  • Antibodies or fragments thereof that immunospecifically bind to an antigen may be cross-reactive with related antigens, carrying the same epitope.
  • antibodies or fragments thereof that immunospecifically bind to an antigen do not cross-react with other antigen.
  • neutralizing refers to antibodies that inhibit a coronavirus from replication, in vitro and/or in vivo, regardless of the mechanism by which neutralization is achieved, or assay that is used to measure the neutralization activity.
  • variable region typically refers to a portion of an antibody, generally, a portion of a light or heavy chain, typically about the amino-terminal 110 to 120 amino acids in the mature heavy chain and about 90 to 115 amino acids in the mature light chain, which differ extensively in sequence among antibodies and are used in the binding and specificity of a particular antibody for its particular antigen.
  • the variability in sequence is concentrated in those regions called CDRs while the more highly conserved regions in the variable domain are called framework regions (FR).
  • variable region is a human variable region.
  • variable region comprises rodent or murine CDRs and human framework regions (FRs).
  • FRs human framework regions
  • the variable region is a primate (e.g., non-human primate) variable region.
  • the variable region comprises rodent or murine CDRs and primate (e.g., non-human primate) framework regions (FRs).
  • an antigen binding molecule, an antibody, or an antigen binding molecule thereof, or a polypeptide of the disclosure “cross-competes” with a reference antibody or an antigen binding molecule thereof or polypeptide of the disclosure if the interaction between an antigen and the first binding molecule, an antibody, or an antigen binding molecule or polypeptide of the disclosure blocks, limits, inhibits, or otherwise reduces the ability of the reference binding molecule, reference antibody, an antigen binding molecule thereof, or polypeptide of the disclosure to interact with the antigen or with the coronavirus S protein.
  • Cross competition may be complete, e.g., binding of the binding molecule to the antigen completely blocks the ability of the reference binding molecule to bind the antigen, or it may be partial, e.g., binding of the binding molecule to the antigen reduces the ability of the reference binding molecule to bind the antigen.
  • an antigen binding molecule that cross-competes with a reference antigen binding molecule binds the same or an overlapping epitope as the reference antigen binding molecule.
  • the antigen binding molecule that cross- competes with a reference antigen binding molecule binds a different epitope as the reference antigen binding molecule.
  • RIA solid phase direct or indirect radioimmunoassay
  • EIA solid phase direct or indirect enzyme immunoassay
  • sandwich competition assay Stahli et al., 1983, Methods in Enzymology 9:242-253
  • solid phase direct biotin-avidin EIA Karlin et al, 1986, J. Immunol.
  • solid phase direct labeled assay solid phase direct labeled sandwich assay (Harlow and Lane, 1988, Antibodies, A Laboratory Manual, Cold Spring Harbor Press); solid phase direct label RIA using 1-125 label (Morel et al., 1988, Molec. Immunol. 25:7-15), solid phase direct biotin-avidin EIA (Cheung, et al., 1990, Virology 176:546-552), and direct labeled RIA (Moldenhauer et al., 1990, Scand. J. Immunol. 32:77-82). These methods are also examples of methods for measuring the affinity of the peptides of the disclosure for the antibodies or coronaviral S protein of the disclosure.
  • an “antigen” refers to any molecule that provokes an immune response or is capable of being bound by an antibody or an antigen binding molecule (e.g., a TCR).
  • the immune response may involve either antibody production, or the activation of specific immunologically- competent cells, or both.
  • An antigen may be endogenously expressed, i.e. expressed by genomic DNA, or may be recombinantly expressed.
  • An antigen may be specific to a certain tissue, such as a cancer cell, or it may be broadly expressed.
  • fragments of larger molecules may act as antigens.
  • the antigens are any one of the polypeptides of the disclosure.
  • a “therapeutically effective amount,” “effective dose,” “effective amount,” or “therapeutically effective dosage” of a therapeutic agent, e.g., polypeptide or antibody of the disclosure, is any amount that, when used alone or in combination with another therapeutic agent, protects a subject against the onset of a disease or promotes disease regression evidenced by a decrease in severity of disease symptoms, an increase in frequency and duration of disease symptom-free periods, or a prevention of impairment or disability due to the disease affliction.
  • the ability of a therapeutic agent to promote disease regression may be evaluated using a variety of methods known to the skilled practitioner, such as in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or by assaying the activity of the agent in in vitro assays.
  • the present disclosure also includes prophylactic or preventative therapies.
  • Prophylactic treatment may be administered, for example, to a subject not yet exposed to or infected with SARS-CoV-2 but who is susceptible to, or otherwise at risk of exposure or (re-) infection with SARS-CoV-2.
  • a method to prevent transmission includes administering an effective amount of one of the compounds described herein to humans for a sufficient length of time prior to exposure to crowds that can be infected, including during travel or public events or meetings, including for example, up to 3, 5, 7, 10, 12, 14 or more days prior to a communicable situation, either because the human is infected or to prevent infection from an infected person in the communicable situation.
  • Dosages of the molecules of the present disclosure may vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated. A physician will ultimately determine appropriate dosages to be used.
  • dose refers to a specific quantity of an antibody therapeutic that is taken at a specified time or at specified intervals.
  • dosing refers to the administration of a composition, for example, a pharmaceutical composition that includes an antibody or an antibody fragment described herein, to achieve a therapeutic objective.
  • a “dosing schedule” refers to both the dose and the time interval at which the dose is administered.
  • the dosing schedule is part of a treatment cycle.
  • treatment cycle refers to the period in which the antibody is administered followed by a period with no administration of the antibody, wherein the beginning of the subsequent cycle is marked by re-initiation of administration of the antibody such that the treatment cycle allows for a period of rest between days of administration of antibody.
  • a treatment cycle may vary in number of days, for example, 7, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 days.
  • the antibody, or antigen-binding fragment thereof is administered on more than one day.
  • the antibody may be administered once per day for one day, or once per day on two or more consecutive days, for example, the antibody may be administered once per day for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days.
  • the same dose of the antibody is administered on each day.
  • a different dose of the antibody is administered on one or more days. For example, a patient may receive a higher dose of antibody on a day of administration, relative to the dose received on a previous day. Alternately, a patient may receive a lower dose of antibody on one day of administration, relative to the dose received on a previous day.
  • administration of the antibody may occur over one or more treatment cycles.
  • the same dosing schedule may be repeated in a subsequent treatment cycle, i.e., after a first treatment cycle is completed.
  • 3.250 mg, or 3,500 mg of anti-S protein/RBD antibody or antigen binding fragment thereof is administered to a patient.
  • 1.250 mg, 1,500 mg, 1,750 mg, 2,000 mg, 2,250 mg, 2,500 mg, 2,750 mg, 3,000 mg, 3,250 mg or 3,500 mg of anti-S protein/RBD antibody or antigen binding fragment thereof is administered to a patient.
  • 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 325 mg, 350 mg, 375 mg, 400 mg, 425 mg, 450 mg, 475 mg, 500 mg, 525 mg, 550 mg, 575 mg, 600 mg, 625 mg, 650 mg, 675 mg, 700 mg, 725 mg, 750 mg, 775 mg, 800 mg, 825 mg, 850 mg, 875 mg, 900 mg, 925 mg, 950 mg, 975 mg, 1,000 mg, 1,250 mg, 1,500 mg, 1,750 mg, 2,000 mg, 2,250 mg, 2,500 mg, 2,750 mg, 3,000 mg, 3,250 mg or 3,500 mg of anti-S protein/RBD antibody or antigen binding fragment thereof is administered to a patient.
  • the dosage comprises about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1, 10, 25, or 50 ⁇ g, or optionally a dosage that ranges from about 10 ⁇ g to about 500 ⁇ g, about 10 ⁇ g to about 400 ⁇ g, about 10 ⁇ g to about 300 ⁇ g, about 10 ⁇ g to about 200 ⁇ g, about 10 ⁇ g to about 100 ⁇ g, about 10 ⁇ g to about 50 ⁇ g, or about 20 ⁇ g to about 500 ⁇ g, about 20 ⁇ g to about 400 ⁇ g, about 20 ⁇ g to about 300 ⁇ g, about 20 ⁇ g to about 200 ⁇ g, about 20 ⁇ g to about 100 ⁇ g, about 20 ⁇ g to about 50 ⁇ g, or about 30 ⁇ g to about 500 ⁇ g, about 30 ⁇ g to about 400 ⁇ g, about 30 ⁇ g to about
  • the antibody/antibodies of the disclosure may be administered for at least one cycle, but more cycles of administration such as 2, 3, 4, 5, 6, 7, 8, 9 or 10 cycles are also envisaged.
  • an administration of 1-5 cycles is considered to be beneficial for a patient in need.
  • Each cycle may comprise one administration, or more than one administration over a period of days or weeks.
  • the dose provided herein is for administration to an adult of average body weight and other relevant biological characteristics.
  • the dose is for administration to an adult not of average body weight or other relevant biological characteristics (including, for example, an obese or pediatric patient), with the dose adjusted to compensate for such things as body weight or other relevant biological characteristics.
  • the dosage administered to a patient is between about 0.1 mg/kg to 100 mg/kg of the patient's body weight
  • the dose provided herein is for administration to an infant or child, with the dose adjusted to compensate for such things as body weight and other relevant biological characteristics.
  • a “patient’ or “subject’ as used herein includes any human or animal who is afflicted with coronavirus-related disease or disorder.
  • the terms “subject’ and “patient’ are used interchangeably herein.
  • an in vitro cell refers to any cell, which is cultured ex vivo.
  • an in vitro cell may include a T cell.
  • peptide refers to a compound comprised of amino acid residues covalently linked by peptide bonds.
  • a protein or peptide contains at least two amino acids, and no limitation is placed on the maximum number of amino acids that may comprise a protein or peptide's sequence.
  • Polypeptides include any peptide or protein comprising two or more amino acids joined to each other by peptide bonds.
  • the term refers to both short chains, which also commonly are referred to in the art as peptides, oligopeptides and oligomers, for example, and to longer chains, which generally are referred to in the art as proteins, of which there are many types.
  • Polypeptides include, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, fusion proteins, among others.
  • the polypeptides include natural peptides, recombinant peptides, synthetic peptides, or a combination thereof.
  • Treatment refers to any type of intervention or process performed on, or the administration of an active agent to, the subject with the objective of reversing, alleviating, ameliorating, inhibiting, slowing down or preventing the onset, progression, development, severity, or recurrence of a symptom, complication or condition, or biochemical indicia associated with a disease.
  • treatment or “treating” includes a partial remission. In another embodiment, “treatment” or “treating” includes a complete remission.
  • prevention refers to administration to a patient susceptible to, or otherwise at risk of, a particular disease.
  • anyone in the general population is at risk for viral infections. Some individuals have an increased, genetic risk for viral infections.
  • Prevention can eliminate or reduce the risk or delay the onset of disease. Delay of onset or progression can be measured based on standard times of disease progression in similar populations or individuals.
  • GCG program package which includes GAP (Devereux et al, 1984, Nucl. Acid Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, Wis.).
  • GAP is used to align the two polypeptides or polynucleotides for which the percent sequence identity is to be determined.
  • sequences are aligned for optimal matching of their respective amino acid or nucleotide (the “matched span,” as determined by the algorithm.)
  • a standard comparison matrix (see, Dayhoff et al., 1978, Atlas of Protein Sequence and Structure 5:345-352 for the PAM 250 comparison matrix; Henikoff et al, 1992, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919 for the BLOSUM 62 comparison matrix) is also used by the algorithm.
  • identity may be determined as percentage of identity using known computer algorithms such as the “PASTA” program, using for example, the default parameters as in Pearson et al. (1988) Proc. Natl. Acad. Sci.
  • similarity and “similar” and grammatical variations thereof, as used herein, mean that an amino acid sequence contains a limited number of conservative amino acid substitutions compared to a peptide reference sequence, e.g. the variant peptide versus the parent peptide as defined herein.
  • a variety of criteria can be used to indicate whether amino acids at a particular position in a peptide are similar.
  • substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like, and such substitutions may be assayed for their effect on the function of the peptide by routine testing.
  • substitutions may be conservative or non-conservative amino acid substitutions.
  • a "conservative substitution” is the replacement of one amino acid by a biologically, chemically or structurally similar residue.
  • Biological similarity means that the substitution does not destroy a biological activity, e.g. T cell reactivity or HLA coverage.
  • Structural similarity means that the amino acids have side chains with similar length, such as alanine, glycine and serine, or a similar size.
  • Chemical similarity means that the residues have the same charge, or are both either hydrophilic or hydrophobic.
  • a conservative amino acid substitution is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain, for example amino acids with basic side chains (e.g., lysine, arginine, histidine) ; acidic side chains (e.g., aspartic acid, glutamic acid) ; uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, histidine) ; nonpolar side chains
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan
  • Particular examples include the substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or the substitution of oonnee polar residue for another, such as the substitution of arginine for lysine, glutamic acid for aspartic acid, or glutamine for asparagine, serine for threonine, and the like.
  • Proline which is considered more difficult to classify, shares properties with amino acids that have aliphatic side chains (e.g., Leu, Vai, He, and Ala).
  • substitution of glutamine for glutamic acid or asparagine for aspartic acid may be considered aa similar substitution in that glutamine and asparagine are amide derivatives of glutamic acid and aspartic acid, respectively.
  • the disclosure provides hybridomas and antibodies against Spike protein (S) or Spike Receptor Binding Domain (RBD) of the SARS-CoV-2 virus. Accordingly, in one embodiment, the disclosure provides antibodies that bind Spike protein. In one embodiment, the antibodies bind to the RBD of the S protein. In one embodiment, the binding site or epitope of the antibodies lies in the S protein RBD. Accordingly, in one embodiment, the antibodies targeting the S protein and RBD can interfere with its binding to the receptor ACE2. In one embodiment, the antibodies may block the virus entry into the host cell.
  • S Spike protein
  • RBD Spike Receptor Binding Domain
  • the antibodies have a high affinity for the S protein or S protein RBD. In one embodiment, the antibodies have a weak binding to the S protein and may have decreased capability to inhibit virus sufficiently effectively. In one embodiment, the antibodies may have nanomolar affinity to RBD. In one embodiment, the antibodies may have subnanomolar affinity to RBD.
  • the affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method; see, for example, Pope ME, Soste MV, Eyford BA, Anderson NL, Pearson TW. (2009) J Immunol Methods. 341(l-2):86-96 and methods described herein.
  • affinity and other antigen-binding parameters are for example made with standardized solutions of antibody and antigen, and a standardized buffer.
  • affinity is determined by a method of the Examples.
  • antibodies of the present disclosure, including binding fragments or variants thereof, may also be described or specified in terms of their binding affinity for SARS-CoV-2 virus S protein or its RBD.
  • antibodies with high affinity have KD of less than 10 -7 M.
  • antibodies or binding fragments thereof bind SARS-CoV-2 virus S protein or RBD, or fragments or variants thereof, with a dissociation constant or KD of less than or equal to 5x10 -7 M, 10 -7 M, 5X10 -8 M, 10 -8 M, 5x 10 -9 M, 10 -9 M, 5X10 -10 M, 10 -10 M, 5X10 -11 M, 10 -11 M, 5X10 -12 M, 10 -12 M, 5X10 -13 M, 10 -13 M, 5X10 -14 M, 10 -14 M, 5x10 -15 M or 10 -15 M.
  • antibodies or binding fragments thereof bind SARS-CoV-2 virus S protein or RBD, or fragments or variants thereof, with a dissociation constant or KD of less than or equal to 5x10 -10 M, 10 -10 M, 5 x 10 -11 M, 10 -13 M, 5X10 -12 M or 10 -12 M.
  • the disclosure encompasses antibodies that bind S ARS- CoV-2 virus S protein or RBD with a dissociation constant or KD that is within a range between any of the individual recited values.
  • antibodies or binding fragments thereof bind SARS-CoV-2 virus S protein or RBD or fragments or variants thereof with an off rate (koff) of less than or equal to 5x10 -2 sec -1 , 10 -2 sec -1 , 5x10 -3 sec -1 or 10 -3 sec -1 , 5x10 -4 sec -1 , 10 -4 sec -1 , 5 x 10 -5 sec -1 , or 10 -5 sec -1 , 5 x 10 -5 sec -1 , 10 -6 sec -1 , 5x10 -7 sec -1 or 10 -7 sec -1 .
  • antibodies or binding fragments thereof bind SARS-CoV-2 virus S protein or RBD or fragments or variants thereof with an off rate (koff) less than or equal to 5x10 -4 sec -1 , 10 -4 sec -1 , 5x10 -5 sec -1 , or 10 -5 sec -1 , 5x10 -6 sec -1 , 10 -6 sec -1 , 5x10 -7 sec -1 or 10 -7 sec -1 .
  • the disclosure also encompasses antibodies that bind SARS-CoV-2 virus S protein or RBD with an off rate (koff) that is within a range between any of the individual recited values.
  • antibodies or binding fragments thereof bind SARS-CoV-2 virus S protein or RBD or fragments or variants thereof with an on rate (kon) of greater than or equal to 1CPM -1 sec -1 , 5x 10 3 M -1 sec -1 , 10 4 M -1 sec -1 , 5x10 -4 M -1 ,sec -1 , 10 5 M -1 sec -1 , 5x10 5 sec -1 , 10 6 M -1 sec-1, 5x10 6 M -1 sec -1 , 10 7 M -1 sec-1, or 5x10 7 M -1 sec -1 .
  • antibodies or binding fragments thereof bind SARS-CoV-2 virus S protein or RBD or fragments or variants thereof with an on rate (kon) greater than or equal to 10 5 M -1 sec -1 , 5x 10 5 M -1 sec -1 , 10 6 M -1 sec-1, 5x10 6 M -1 sec -1 , 10 7 M -1 sec -1 or 5x10 7 M -1 sec -1 .
  • the disclosure encompasses antibodies that bind SARS-CoV-2 virus S protein or RBD with on rate (kon) that is within a range between any of the individual recited values.
  • Binding affinity generally refers to the strength of the sum total of the noncovalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen).
  • binding affinity refers to intrinsic binding affinity which reflects a 1:1 interaction between members of a binding pair (e.g., antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the equilibrium dissociation constant (Kd), which is calculated as the ratio k off /k on .
  • Kd equilibrium dissociation constant
  • Affinity can be measured by common methods known in the art, including those described and exemplified herein.
  • An example of a commercially available system for kinetic characterization includes the OCTET® family of instruments. Low-affinity antibodies generally bind antigen slowly and tend to dissociate readily, whereas high-affinity antibodies generally bind antigen faster and tend to remain bound longer. A variety of methods of measuring binding affinity are known in the art, any of which can be used for purposes of the present disclosure.
  • the affinity of the antibody of the disclosure is measured as described in Example 2.
  • Other methods and reagents suitable for determination of binding characteristics of an antibody of the present disclosure, or an altered/mutant derivative thereof are known in the art and/or are commercially available (U.S. Pat Nos. 6,849,425; 6,632,926; 6,294,391; 6,143,574).
  • equipment and software designed for such kinetic analyses are commercially available (e.g. Biacore® Al 00, and Biacore® 2000 instruments; Biacore International AB, Uppsala, Sweden).
  • the antibodies of the disclosure may induce cell death.
  • An antibody which “induces cell death” is one which causes a viable cell to become nonviable.
  • Cell death in vitro may be determined in the absence of complement and immune effector cells to distinguish cell death induced by antibody-dependent cell-mediated cytotoxicity (ADCC) or complement dependent cytotoxicity (CDC).
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • CDC complement dependent cytotoxicity
  • the assay for cell death may be performed using heat inactivated serum (i.e., in the absence of complement) and in the absence of immune effector cells.
  • PI propidium iodide
  • trypan blue see Moore et al.
  • the antibodies of the disclosure may induce cell death via apoptosis.
  • An antibody which “induces apoptosis” is one which induces programmed cell death as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dilation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies).
  • apoptotic bodies Various methods are available for evaluating the cellular events associated with apoptosis.
  • phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chromatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells.
  • the antibody which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexin binding relative to untreated cell in an annexin binding assay.
  • the antibodies may be selected from monoclonal antibodies (Mab) 96, 290, 266, 677, 68, or antigen-binding fragments thereof. In one embodiment, these antibodies effectively inhibited interaction of RBD domain with ACE2. In one embodiment, the inhibition activity of the antibodies may be relatively high and ranged from 59 % up to 96 %. In one embodiment, inhibition activity of the antibodies may be between 1-5, 5-10, 10-15, 15-20, 20- 25, 25-30, 30-35, 35-40, 40-45, 45-50, 50-55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100 percent.
  • the antibodies inhibit the interaction of the RBD domain with ACE2 in vitro. In one embodiment, the antibodies inhibit the interaction of the RBD domain with ACE2 in vivo. Various methods are known to one of ordinary skill in the art to test this activity, including those used in the Examples.
  • the antibody or antigen binding fragment thereof immunospecifically binds to SARS-CoV-2 virus S protein or its RBD and is capable of neutralizing SARS-CoV-2 virus infection.
  • Neutralization assays can be performed using methods known in the art.
  • the term “inhibitory concentration 50%” (abbreviated as “ICso”) represents the concentration of an inhibitor (e.g., an antibody) that is required for 50% neutralization of SARS- CoV-2 virus. It will be understood by one of ordinary skill in the art that a lower ICso value corresponds to a more potent inhibitor.
  • an antibody or antigen binding fragment thereof according to the disclosure has a neutralizing potency expressed as 50% inhibitory concentration (ICso ug/ml) in the range of from about 0.01 ug/ml to about 50 ug/ml, or in the range of from about 0.01 ug/ml to about 5 ug/ml of antibody, or in the range of from about 0.01 ug/ml to about 0.1 ug/ml of antibody for neutralization of SARS-CoV-2 virus in a microneutralization assay.
  • the neutralization activity may be determined as described in EXAMPLE 4.
  • the disclosure provides that the antibodies of the disclosure block or inhibit the entry of the pseudovirus into the cells in a dose-depended manner.
  • the neutralization effectivity of the tested antibodies differs from one another. The highest inhibition activity was shown by MAb68 and then a group of four antibodies (MAb96, MAb290, MA266 and MAb352).
  • the disclosure provides antibodies that target the critical epitopes on SARS-CoV-2 S protein that are essential for the infection of the recipient cells.
  • the disclosure provides that five of the MAbs (MAb96, MAb290, MAb266, MAb677 and MAb68) displayed strong neutralizing activity against SARS-CoV-2.
  • MAb68 displayed the most potent neutralizing activity against SARS-CoV-2, with a very high neutralization titer.
  • the disclosure provides antibodies that have neutralization activity against SARS-CoV-2.
  • the neutralization activity of the antibodies may be between 1-5, 5-10, 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50, 50- 55, 55-60, 60-65, 65-70, 70-75, 75-80, 80-85, 85-90, 90-95, 95-100 percent
  • Antibodies of the disclosure may be used as therapeutic agents. Such agents will generally be employed to treat or prevent a SARS-CoV-2 -related disease or pathology in a subject
  • An antibody preparation preferably one having high specificity and high affinity for its target antigen, is administered to the subject and will generally have an effect due to its binding with the target
  • Administration of the antibody may abrogate or inhibit or interfere with the internalization of the virus into a cell. In this case, the antibody binds to the target and prevents SARS-CoV-2 binding the ACE2 receptor.
  • the antibodies may be used to prevent SARS- CoV-2 virus activity in a subject in need thereof. In one embodiment, the antibodies decrease the infectivity of the virus in a subject in need thereof. In one embodiment, the antibodies may be used to prevent or treat COVID-19 or other SARS-CoV-2 -mediated diseases (e.g., 2019 coronavirus disease (COVID-19) caused by SARS-CoV-2 virus) or disorders in a subject in need thereof. In one embodiment, the antibodies may be used to decrease the signs and/or symptoms of SARS- CoV-2 -associated diseases or disorders in a subject in need thereof. In one embodiment, the antibodies may be used as a passive vaccine. In one embodiment, the disease is COVID-19.
  • COVID-19 2019 coronavirus disease 2019 coronavirus disease
  • the signs or symptoms of SARS-CoV-2 -associated diseases or disorders are selected from cold-like symptoms, including fever or chills, cough, fatigue, shortness of breath or difficulty breathing, fatigue, muscle or body aches, headache, sore throat, congestion or runny nose, nausea or vomiting, diarrhoea, aches, and loss of taste and/or smell, trouble breathing, persistent pain or pressure in the chest, new confusion, inability to wake or stay awake, bluish lips or face, or any one of those listed in www.cdc.gov/coronavirus/2019-ncov/symptoms-testing/symptoms.html. These symptoms may resolve with minimal medical care in a few weeks in otherwise healthy subjects. However, occasionally, symptoms persist for months.
  • the virus may cause long-term damage to the lungs, heart, and brain. Furthermore, in some patients, especially older adults, immunocompromised individuals, or those with underlying conditions, the virus can cause severe symptoms that result in hospitalization, ventilation, and/or death.
  • the antibodies of the disclosure may be effective at reducing one or more of these symptoms.
  • the disclosure provides a method of treatment of COVID-19, including drug resistant and multidrug resistant forms of the virus and related disease states, conditions, or complications of the viral infection, including pneumonia, such as 2019 novel coronavirus-infected pneumonia (NCIP), acute lung injury (ALI), and acute respiratory distress syndrome (ARDS). Additional non-limiting complications include hypoxemic respiratory failure, acute respiratory failure (ARF), acute liver injury, acute cardiac injury, acute kidney injury, septic shock, disseminated intravascular coagulation, blood clots, multisystem inflammatory syndrome, chronic fatigue, rhabdomyolysis, and cytokine storm.
  • NIP 2019 novel coronavirus-infected pneumonia
  • ALI acute lung injury
  • ARDS acute respiratory distress syndrome
  • Additional non-limiting complications include hypoxemic respiratory failure, acute respiratory failure (ARF), acute liver injury, acute cardiac injury, acute kidney injury, septic shock, disseminated intravascular coagulation, blood clots, multisystem inflammatory syndrome, chronic fatigue, rhabdomy
  • a method to prevent transmission includes administering an effective amount of one of the antibodies described herein to humans for a sufficient length of time prior to exposure to crowds that can be infected, including during travel or public events or meetings, including for example, up to 3, 5, 7, 10, 12, 14 or more days prior to a communicable situation, either because the human is infected or to prevent infection from an infected person in the communicable situation.
  • the disclosure provides a method of delaying the onset of one or more symptoms of a SARS-CoV-2 infection, comprising administering to a person at risk of suffering from said infection, a therapeutically effective amount of an antibody or antigen-binding fragment thereof, wherein said antibody or antigen-binding fragment thereof binds to an epitope in the spike protein or in the receptor binding domain (RBD) of the spike protein of a SARS-CoV-2 virus, and neutralizes SARS-CoV-2, and wherein the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence encoded by a nucleic acid selected from Table 1 and/or a VL amino acid sequence encoded by a nucleic acid selected from Table 2, or an amino acid sequence of Table 3.
  • the disclosure provides a method of delaying the onset of one or more symptoms of a SARS-CoV-2 infection, comprising administering to a person at risk of suffering from said infection, a therapeutically effective amount of an antibody or antigen-binding fragment thereof, and wherein the antibody or antigen-binding fragment thereof comprises a heavy chain with three CDRs comprising the amino acid sequences of the CDRs of Table 3 and/or a light chain with three CDRs comprising the amino acid sequences of the CDRs of Table 3.
  • the disclosure provides a method of neutralizing SARS-CoV- 2, comprising contacting an epitope in spike protein or the receptor binding domain (RBD) of the spike protein of a SARS-CoV-2 with an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain with three CDRs comprising the amino acid sequences of the CDRs of Table 3 and/or a light chain with three CDRs comprising the amino acid sequences of the CDRs of Table 3, wherein the antibody or antigen- binding fragment thereof neutralizes SARS-CoV-2.
  • RBD receptor binding domain
  • the disclosure provides SARS-CoV-2, comprising contacting an epitope in the spike protein or in the receptor binding domain (RBD) of the spike protein of a SARS-CoV-2 with an antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises a VL amino acid sequence encoded by a nucleic acid selected from Table 2, or an amino acid sequence of Table 3.
  • the subject is a child. In one embodiment, the subject is an adult. In one embodiment, the subject is more than 65 years old. In one embodiment, the subject is immunocompromised. [00113] In one embodiment, the patient/subject is administered one antibody of the disclosure, such as an antibody having the CDRs of the antibodies of Table 3. In one embodiment, one or more antibodies are administered in combination. In one embodiment, the antibodies, alone or in combination, prevent the generation of viral escape mutations. In one embodiment, the antibodies are administered concurrently. In one embodiment, the antibodies are administered sequentially.
  • a method for generating an antibody selective for an escape mutant isolated as described above comprising either: a) immunizing an animal with the escape mutant isolated above and isolating the antibody, or b) recombinantly expressing the antibody by transfecting a recombinant organism with a nucleic acid encoding an amino acid sequence of the antibody, wherein the amino acid sequence comprises at least one mutation relative to a reference antibody, the mutation increasing the affinity of the antibody to the escape mutant as compared to the reference antibody.
  • the reference antibody can comprise an anti SARS- CoV-2 antibody described in the art and selected from the group consisting of: 2B04, 2H04, 2-4, B38, CB6, C105, CC12.1, CC12.3, COVA2-04, CV30, REGN10933, COVA2-39, BD23, P2B-2F6, S309, and REGN10987.
  • the antibody or antigen binding fragment thereof is substantially purified (i.e., substantially free from substances that limit its effect or produce undesired side-effects).
  • the antibody or antigen binding fragment thereof is administered post-exposure, or after the subject has been exposed to SARS-CoV-2 virus or is infected with SARS-CoV-2 virus.
  • the antibody or antigen binding fragment thereof is administered pre-exposure, or to a subject that has not yet been exposed to SARS-CoV-2 virus or is not yet infected with SARS-CoV-2 virus.
  • the antibody or antigen binding fragment thereof is administered to a subject that is sero-negative for one or more SARS-CoV-2 virus.
  • the antibody or antigen binding fragment thereof is administered to a subject that is sero-positive for one or more SARS-CoV-2 virus subtypes.
  • the serostatus of the patient is unknown.
  • the antibody or antigen binding fragment thereof is administered to a subject within 1, 2, 3, 4, 5, 6 or 7 days of exposure, infection or symptom onset.
  • the antibody or antigen binding fragment thereof can be administered to a subject after 1, 2, 3, 4, 5, 6, 7, 10, 15, 20, 25, 30 or any number of days there between after exposure, infection or symptom onset [00116]
  • the disclosure provides antibodies with heavy chain variable regions as represented in Table 1 and FIG. 9:
  • the disclosure provides antibodies with light chain variable regions with nucleic acid sequences as represented in Table 2 and FIG. 10.
  • the disclosure provides antibodies with variable regions of amino acid sequences as represented in Table 3 and FIG. 11.
  • Table 3 Amino Acid Sequences of Variable Regions of Exemplary Antibodies of the Disclosure.
  • the CDRs are underlined, as determined by IMGT.
  • LD variable mouse regions
  • ITALICS leader/signal peptide (cleaved off in the secretory pathway, not present in the final form)
  • the disclosure provides antibodies whose variable regions are as described in Table 3. In some embodiments, the disclosure provides antibodies comprising a light chain and/or heavy chain variable region identical to any one of the variable regions of Tables
  • a subject antibody comprises a light chain and/or heavy chain comprising an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to any one of the above listed heavy chain and light chain variable regions (see Table 3).
  • a subject antibody comprises a light chain and/or heavy chain comprising a nucleic acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, or at least about 99% amino acid sequence identity to any one of the above listed heavy chain and light chain variable regions (see Tables 1 and 2).
  • a subject antibody comprises a light chain comprising an amino acid sequence that differs from any one of the above listed heavy chain and light chain variable regions by only one, two, three, four, five, six, seven, eight, nine, or ten amino acids.
  • Those of ordinary skill in the art can determine which amino acids in a light chain and/or heavy chain variable region can be altered. For example, by comparing the amino acid sequences of light chain and/or heavy chain variable regions of antibodies with the same specificity, those skilled in the art can determine which amino acids can be altered without altering the specificity for S protein and/or RBD.
  • Antibodies of the disclosure may comprise a VH amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or having 100% identity to the VH amino acid sequences described in Table 3.
  • the antibodies may have a VH amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or having 100% identity to the amino acid sequence of the VH amino acid sequences described in Table 3.
  • Antibodies of the disclosure may comprise a VL amino acid sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95% or having 100% identity to the VL amino acid sequences described in Table 3.
  • the antibodies may have a VL amino acid sequence having at least, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or having 100% identity to the VL amino acid sequences described in Table 3.
  • the antibodies many have about 25% to about 95% sequence identity to the amino acid sequence of either the heavy or light chain variable domain of an antibody as described in Table 3.
  • a modified antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of an antibody as described in Table 3.
  • an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain CDR1, CDR2, or CDR3 of an antibody as described in Table 3.
  • an altered antibody may have an amino acid sequence having at least 25%, 35%, 45%, 55%, 65%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity or similarity with the amino acid sequence of the heavy or light chain framework regions FR1, FR2, FR3 or FR4 of an antibody as described in Table 3.
  • altered antibodies are generated by one or more amino acid alterations (e.g., substitutions, deletion and/or additions) introduced in one or more of the variable regions of the antibody.
  • amino acid alterations are introduced in the framework regions.
  • One or more alterations of framework region residues may result in an improvement in the binding affinity of the antibody for the antigen. This may be especially true when these changes are made to humanized antibodies wherein the framework region may be from a different species than the CDR regions.
  • framework region residues to modify include those which non-covalently bind antigen directly (Amit et al., Science, 233:747-753 (1986)); interact with/effect the conformation of a CDR (Chothia et al., J. Mol. Biol., 196:901-917 (1987)); and/or participate in the VL-VH interface (U.S. Pat. Nos. 5,225,539 and 6,548,640).
  • from about one to about five framework residues may be altered. Sometimes, this may be sufficient to yield an antibody mutant suitable for use in preclinical trials, even where none of the hypervariable region residues have been altered. Normally, however, an altered antibody will comprise additional hypervariable region alteration(s).
  • the antibody can comprise at least one amino acid substitution, deletion, or insertion in a variable region, a hinge region or an Fc region t relative to the sequence of a wild-type variable region, hinge region or a wild-type Fc region.
  • the antibody can comprise an Fc region that contains at least one amino acid substitution, deletion, or insertion relative to the sequence of a wild-type Fc region. In various embodiments, this substitution, deletion or insertion can prevent or reduce recycling of the antibody (e.g., in vivo).
  • the antibodies or antigen-binding fragments described herein can be expressed recombinantly (e.g., using a recombinant cell line or recombinant organism).
  • the antibodies or antigen-binding fragments may comprise post-translational modifications (e.g., glycosylation profiles, methylation) that differs from naturally occurring antibodies. Binding and Function of the Antibodies and Antigen-Binding Fragments.
  • the antibodies and antigen-binding fragments thereof described herein have some measure of binding affinity to a coronavirus. Most preferably, the antibody or antigen- binding fragment binds SARS-CoV-2 (that is, the coronavirus comprises SARS-CoV-2).
  • the antibodies and antigen-binding fragments thereof described herein can bind a receptor binding domain (RBD) expressed by the coronavirus (e.g., SARS-CoV- 2).
  • RBD receptor binding domain
  • the antibodies and antigen-binding fragments herein may have a certain affinity for a specific epitope on the coronavirus (e.g., an epitope on the receptor binding domain, RBD).
  • the binding of the antibody or antigen-binding fragment can neutralize the coronavirus (e.g., SARS-CoV-2).
  • the antibodies and/or binding fragment neutralize the coronavirus with an IC50 of about 0.0001 ⁇ g/ml to about 100 ⁇ g/ml.
  • One useful procedure for generating altered antibodies is called “alanine scanning mutagenesis” (Cunningham and Wells, Science, 244:1081-1085 (1989)).
  • alanine scanning mutagenesis (Cunningham and Wells, Science, 244:1081-1085 (1989)).
  • one or more of the hypervariable region residue(s) are replaced by alanine or polyalanine residue(s) to alter the interaction of the amino acids with the target antigen.
  • Those hypervariable region residue(s) demonstrating functional sensitivity to the substitutions then are refined by introducing additional or other mutations at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • the Ala-mutants produced this way are screened for their biological activity as described herein.
  • the substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g. a humanized or human antibody).
  • a parent antibody e.g. a humanized or human antibody.
  • the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they are generated.
  • a convenient way for generating such substitutional variants involves affinity maturation using phage display (Hawkins et al., J. Mol. Biol, 254:889-896 (1992) and Lowman et al, Biochemistry, 30(45): 10832-10837 (1991)). Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site.
  • the antibody mutants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene m product of Ml 3 packaged within each particle.
  • the phage-displayed mutants are then screened for their biological activity (e.g., binding affinity) as herein disclosed.
  • Mutations in antibody sequences may include substitutions, deletions, including internal deletions, additions, including additions yielding fusion proteins, or conservative substitutions of amino acid residues within and/or adjacent to the amino acid sequence, but that result in a “silent” change, in that the change produces a functionally-equivalent antibody
  • the antibodies according to the disclosure include, in addition, such antibodies having "conservative sequence modifications," nucleotide and amino acid sequence modifications which do not affect or alter the above-mentioned characteristics of the listed exemplary antibodies according to the disclosure. Modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis.
  • Conservative amino acid substitutions include ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.
  • glycine asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine
  • beta- branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • amino acids with similar properties can be Aliphatic amino acids (e.g., Glycine, Alanine, Valine, Leucine, Isoleucine); Hydroxyl or sulfur/selenium-containing amino acids (e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine); Cyclic amino acids (e.g., Proline); Aromatic amino acids (e.g., Phenylalanine, Tyrosine, Tryptophan); Basic amino acids (e.g., Histidine, Lysine, Arginine); or Acidic and their Amide (e.g., Aspartate, Glutamate, Asparagine, Glutamine).
  • Aliphatic amino acids e.g., Glycine, Alanine, Valine, Leucine, Isoleucine
  • Hydroxyl or sulfur/selenium-containing amino acids e.g., Serine, Cysteine, Selenocysteine, Threonine, Methionine
  • a predicted nonessential amino acid residue in any one of the listed exemplary antibodies can be for example replaced with another amino acid residue from the same side chain family.
  • Deletion is the replacement of an amino acid by a direct bond.
  • Positions for deletions include the termini of a polypeptide and linkages between individual protein domains.
  • Insertions are introductions of amino acids into the polypeptide chain, a direct bond formally being replaced by one or more amino acids.
  • Amino acid sequence can be modulated with the help of art-known computer simulation programs that can produce a polypeptide with, for example, improved activity or altered regulation.
  • a corresponding nucleic acid molecule coding for such a modulated polypeptide can be synthesized in-vitro using the specific codon-usage of the desired host cell.
  • a second way to generate a functional peptide/polypeptide or protein based on the sequences provided herein is through the use of computational, "in-silico" design.
  • computationally designed antibodies or antigen-binding fragments may be designed using standard methods of the art
  • an antibody or antibody binding fragment thereof is provided that binds a coronavirus (e.g., SARS-CoV-2) and is structurally similar to any of the antibodies described herein.
  • the disclosure provides derivatives of the antibodies of Table 3, which, are peptides, polypeptides and/or proteins derived from any of the antibodies or antibody binding fragments described herein.
  • the derivatives provided here are substantially similar to the antibodies or antibody binding fragments described herein. For example, they may contain one or more conservative substitutions in their amino acid sequences or may contain a chemical modification.
  • the derivatives and modified peptides/polypeptides/proteins all are considered "structurally similar" which means they retain the structure (e.g., the secondary, tertiary or quarternary structure) of the parent molecule and are expected to interact with the antigen in the same way as the parent molecule.
  • a class of synthetically derived antibodies or antigen-binding moieties can be generated by conservatively mutating resides on the parent molecule to generate a peptide, polypeptide or protein maintaining the same activity as the parent molecule. Representative conservative substitutions are known in the art and are also summarized here.
  • antibody fragments such as fragments of the listed exemplary antibodies described in the tables above and in the figures.
  • antibody fragments and antibody portions comprise a portion of a full-length antibody, generally at least the antigen binding portion/domain or the variable region thereof.
  • antibody fragments include diabodies, single-chain antibody molecules, immunotoxins, and multispecific antibodies formed from antibody fragments.
  • antibody fragments comprise single chain polypeptides having the characteristics of a VH chain binding S protein and/or RBD, namely being able to assemble together with a VL chain or of a VL chain binding to S protein or RBD domain, namely being able to assemble together with a VH chain to form a functional antigen binding pocket and thereby providing the property of binding to S protein or RBD domain.
  • the terms also comprise fragments that per se are not able to provide effector functions (e.g., ADCC/CDC) but provide this function after being combined with the appropriate antibody constant domain(s).
  • a suitable antibody, antibody portion, fragment, or variant can bind to at least one of epitope in S protein or RBD domain.
  • the term “antibody” refers to antibody digestion fragments, specified antibody portions and variants thereof, including antibody mimetics, or portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof.
  • Functional fragments include antigen-binding fragments that bind to one or more therapeutic epitopes.
  • antibody fragments capable of binding to a therapeutic epitope include, but are not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab')2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and re-aggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are provided by the present disclosure. See also, William E.
  • Antibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site.
  • a combination gene encoding an F(ab')2 heavy chain portion can be designed to include DNA sequences encoding the CHI domain and/or hinge region of the heavy chain.
  • the various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using routine genetic engineering techniques
  • chimeric antibody refers to a monoclonal antibody comprising a variable region, i.e., binding region, from one source or species and at least a portion of a constant region derived from a different source or species, usually prepared by recombinant DNA techniques. Chimeric antibodies comprising a murine variable region and a human constant region are especially preferred. Such murine/human chimeric antibodies are the product of expressed immunoglobulin genes comprising DNA segments encoding murine immunoglobulin variable regions and DNA segments encoding human immunoglobulin constant regions.
  • Other forms of "chimeric antibodies" encompassed by the present disclosure are those in which the class or subclass has been modified or changed from that of the original antibody.
  • Such “chimeric” antibodies are also referred to as "class-switched antibodies.”
  • Methods for producing chimeric antibodies involve conventional recombinant DNA and gene transfection techniques now known in the art. See, e.g., Morrison, S. L., etal., Proc. Natl. Acad. Sci. USA 81 (1984) 6851-6855; U.S. Pat. Nos. 5,202,238 and 5,204,244.
  • the disclosure provides humanized antibodies and human antibodies.
  • humanized antibody refers to antibodies in which the framework regions (FR) and/or the complementarity determining regions (CDR) have been modified to comprise the CDR of an immunoglobulin of different specificity as compared to that of the parent immunoglobulin.
  • a murine CDR is grafted into the framework region of a human antibody to prepare the "humanized antibody.” See, e.g., Riechmann, L., etal., Nature 332 (1988) 323-327; and Neuberger, M. S., et al., Nature 314 (1985) 268-270.
  • Particularly preferred CDRs correspond to the underlined in Table 2.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the constant regions of the antibody can be, for example, constant regions of human IgGl type. Such regions can be allotypic and are described by, e.g., Johnson, G, and Wu, T. T., Nucleic Acids Res. 28 (2000) 214-218 and the databases referenced therein, and are preferentially useful for some embodiments, as long as the properties of induction of ADCC and for example CDC according to the disclosure are retained.
  • the disclosure provides bispecific antibodies that bind S protein or RBD and at least one other antigen.
  • a bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites.
  • Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Brinkmann U. and Kontermann RE. “The making of bispecific antibodies”, MABS, 2017 and Songsivilai & Lachmann Clin. Exp. Immunol. 79: 315-321 (1990), Kostelny etal. J. Immunol. 148:1547-1553 (1992).
  • an antibody can be engineered that has dual Fc regions and can thereby have enhanced complement lysis and ADCC capabilities.
  • the antibody of the present disclosure has modifications of the Fc region, such that the Fc region does not bind to the Fc receptors.
  • the Fc receptor is Fey receptor.
  • Particularly preferred are antibodies with modification of the Fc region such that the Fc region does not bind to Fey, but still binds to neonatal Fc receptor.
  • the antibody comprises a humanized IgA version of the antibodies of Tables 1 through 3. Binding of IgA Fc region has been described for two IgA receptors: Fc ⁇ / ⁇ R (binds both IgA and IgM) and Fc alpha receptor I ( Fc ⁇ RI).
  • plgR receptor is present at the basolateral side of epithelial cells in mucosal surfaces and transports dimeric IgA to the luminal side (mucosal surface) in a form of secretory IgA.
  • Variants in the Fc region can enhance or diminish effector function of the antibody and may alter the pharmacokinetic properties, for example, the half-life, of the antibody.
  • the antibodies include an altered Fc region (also referred to herein as “variant Fc region”) in which one or more alterations have been made in the Fc region in order to change functional and/or pharmacokinetic properties of the antibodies. Such alterations may result in a decrease or increase of Clq binding and CDC or of FcyR binding, for IgG, and ADCC, or ADCP.
  • the present disclosure encompasses the antibodies described herein with variant Fc regions wherein changes have been made to fine tune the effector function, enhancing or diminishing, or providing a desired effector function.
  • Antibodies that include a variant Fc region are also referred to here as “Fc variant antibodies.”
  • native refers to the unmodified parental sequence and the antibody that includes a native Fc region is herein referred to as a “native Fc antibody”.
  • Fc variant antibodies can be generated by methods well known to one skilled in the art. Non-limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region. Alternatively, the antigen-binding portion or variable region of an antibody may be sub-cloned into a vector encoding a variant Fc region.
  • the variant Fc region exhibits a similar level of inducing effector function as compared to the native Fc region. In another embodiment, the variant Fc region exhibits a higher induction of effector function as compared to the native Fc.
  • Methods for measuring effector function are well known in the art Modification of the Fc region, includes, but is not limited to, amino acid substitutions, amino acid additions, amino acid deletions and changes in post- translational modifications to Fc amino acids (e.g glycosylation) and may be used to fine tune the effector function.
  • the Fc region includes the constant region of an antibody excluding the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and and the flexible hinge N-terminal to these domains.
  • Fc may include the J chain.
  • Fc includes immunoglobulin domains Cgamma2 and Cgamma3 (Cy2 and Cy3) and the hinge between Cgammal (Cyl) and Cgamma2 (Cy2).
  • the human IgG heavy chain Fc region is usually defined to include residues C226 or P230 to its carboxyl-terminus, wherein the numbering is according to the EU index as set forth in Rabat Fc may refer to this region in isolation, or this region in the context of an antibody, antibody fragment, or Fc fusion protein.
  • Fc variant antibodies exhibit altered binding affinity for one or more Fc receptors including, but not limited to FcRn, FcyRl (CD64) including isoforms FcyRIA, FcyRIB, and FcyRIC; FcyRII (CD32 including isoforms FcyRIIIA, FcyRIIIB, and FcyRUC); andFc ⁇ RIII (CD16, including isoforms FcyRIIIA and FcyRLLLB) as compared to an native Fc antibody.
  • FcRn FcRn
  • FcyRl CD64
  • FcyRII CD32 including isoforms FcyRIIIA, FcyRIIIB, and FcyRUC
  • Fc ⁇ RIII CD16, including isoforms FcyRIIIA and FcyRLLLB
  • Antibody effector functions include ADCC, a form of cytotoxicity in which secreted Ig bound onto Fc receptors (FcRs) present on certain cytotoxic cells (e.g., Natural Killer (NK) cells, neutrophils, and macrophages) enables these cytotoxic effector cells to bind specifically to an antigen-bearing cells and subsequently kill the cells with cytotoxins.
  • FcRs Fc receptors
  • Specific high-affinity IgG antibodies directed to the surface of cells “arm” the cytotoxic cells and are required for such killing. Lysis of the cell is extracellular, requires direct cell-to-cell contact, and does not involve complement.
  • CDC Another antibody effector function is CDC, which refers to a biochemical event of cell destruction by the complement system.
  • the complement system is a complex system of proteins found in normal blood plasma that combines with antibodies to destroy pathogenic bacteria and other foreign cells.
  • antibody effector function is ADCP, which refers to a cell-mediated reaction wherein nonspecific cytotoxic cells that express one or more effector ligands recognize bound antibody on a cell and subsequently cause phagocytosis of the cell.
  • the antibodies of the disclosure may induce cell death via antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cell- mediated cytotoxicity (CDC) and/or antibody dependent cell-mediated phagocytosis (ADCP).
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cell- mediated cytotoxicity
  • ADCP antibody dependent cell-mediated phagocytosis
  • Fc ⁇ R receptor for an antibody
  • C ⁇ 2 domain the second domain of C region of the antibody
  • C ⁇ 2 domain several amino acid residues in the hinge region and the second domain of C region (hereinafter referred to as “C ⁇ 2 domain”) of the antibody are important (Eur. J. Immunol., 23, 1098 (1993), Immunology, 86, 319 (1995), Chemical Immunology, 65, 88 (1997)) and that a sugar chain in the Cy2 domain (Chemical Immunology, 65, 88 (1997)) is also important.
  • an in vitro ADCC assay can be used, such as that described in U.S. Pat. No. 5,500,362.
  • the assay may also be performed using a commercially available kit, e.g. CytoTox 96 ® (Promega).
  • NK cell lines expressing a transgenic Fc receptor (e.g. CD16) and associated signaling polypeptide (e.g. FC ⁇ RI-y) may also serve as effector cells (WO 2006/023148).
  • Fc receptor e.g. CD16
  • FC ⁇ RI-y associated signaling polypeptide
  • FC ⁇ RI-y FC ⁇ RI-y
  • the antibody can also be tested for complement activation.
  • cytolysis is detected by the release of label from the lysed cells.
  • the extent of cell lysis may also be determined by detecting the release of cytoplasmic proteins (e.g. LDH) into the supernatant.
  • antibodies can be screened using the patient's own serum as a source of complement and/or immune cells.
  • Antibodies that are capable of mediating human ADCC in the in vitro test can then be used therapeutically in that particular patient ADCC activity of the molecule of interest may also be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Set. (USA) 95:652-656 (1998).
  • ADCC ADCC
  • CDC CDC-activated cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclopentase cyclos, and cyclopentaptotic mechanisms.
  • methods including assays utilizing viable dyes, methods of detecting and analysing caspases, and assays measuring DNA breaks can be used to assess the apoptotic activity of cells cultured in vitro with an antibody of interest
  • the serum half-life of proteins that include Fc regions may be increased by increasing the binding affinity of the Fc region for FcRn.
  • antibody half-life as used herein means a pharmacokinetic property of an antibody that is a measure of the mean survival time of antibody molecules following their administration. Antibody half-life can be expressed as the time required to eliminate 50 percent of a known quantity of immunoglobulin from the patient's body or a specific compartment thereof, for example, as measured in serum, i.e., circulating half- life, or in other tissues. Half-life may vary from one immunoglobulin or class of immunoglobulin to another. In general, an increase in antibody half-life results in an increase in mean residence time (MRT) in circulation for the antibody administered.
  • MRT mean residence time
  • a salvage receptor binding epitope refers to an epitope of the Fc region of an IgG molecule (e.g.; IgGl, IgG2; IgG3, or IgG4) that is responsible for increasing the in vivo serum half-life of the IgG molecule.
  • IgGl IgG2
  • antibodies with increased half-lives may be generated by modifying amino acid residues identified as involved in the interaction between the Fc and the FcRn receptor.
  • the half-life of antibodies described herein may be increased by conjugation to PEG or Albumin by techniques widely utilized in the art.
  • the present disclosure provides Fc variants, wherein the Fc region includes a modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from 221, 225, 228, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 247, 250, 251, 252, 254, 255, 256, 257, 262, 263, 264, 265, 266, 267, 268, 269, 279, 280, 284, 292, 296, 297, 298, 299, 305, 308, 313, 316, 318, 320, 322, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 339, 341, 343, 370, 373, 378, 392, 416, 419, 421, 428, 433, 4
  • the Fc region may include a modification at additional and/or alternative positions known to one skilled in the art.
  • the present disclosure provides an Fc variant, wherein the Fc region includes at least one substitution selected from 221K, 221 Y, 225E, 225K, 225W, 228P, 234D, 234E, 234N, 234Q, 234T, 234H, 234Y, 2341, 234V, 234F, 235A, 235D, 235R, 235W, 235P, 235S, 235N, 235Q, 235T, 235H, 235Y, 2351, 235V, 235E, 235F, 236E, 237L, 237M, 237P, 239D, 239E, 239N, 239Q, 239F, 239T, 239H, 239Y, 2401, 240A, 240T, 240M, 241W, 241L, 241Y, 241
  • the Fc region may include additional and/or alternative amino acid substitutions known to one skilled in the art.
  • the present disclosure provides an Fc variant antibody, wherein the Fc region includes aatt least one modification (e.g., amino acid substitutions, amino acid insertions, amino acid deletions) at one or more positions selected from 228, 234, 235 and 331 as numbered by the EU index as set forth in Rabat.
  • the modification is at least one substitution selected from 228P, 234F, 235E, 235F, 235Y, and 331S as numbered by the EU index as set forth in Rabat.
  • the present disclosure provides an Fc variant antibody, wherein the Fc region is an IgG4 Fc region and includes at least one modification at one or more positions selected from 228 and 235 as numbered by the EU index as set forth in Rabat.
  • the Fc region is an IgG4 Fc region and the non- naturally occurring amino acids are selected from 228P, 235E and 235 Y as numbered by the EU index as set forth in Rabat.
  • the present disclosure provides an Fc variant, wherein the Fc region includes at least one non-naturally occurring amino acid at one or more positions selected from 239, 330 and 332 as numbered by the EU index as set forth in Rabat.
  • the modification is at least one substitution selected from 239D, 330L, 330Y, and 332E as numbered by the EU index as set forth in Rabat
  • the present disclosure provides an Fc variant antibody, wherein the Fc region includes at least one non- naturally occurring amino acid at one or more positions selected from 252, 254, and 256 as numbered by the EU index as set forth in Rabat.
  • the modification is at least one substitution selected from 252Y, 254T and 256E as numbered by the EU index as set forth in Rabat.
  • the modification includes three substitutions 252Y, 254T and 256E as numbered by the EU index as set forth in Rabat (known as “YTE”).
  • the effector functions elicited by IgG antibodies depend on the carbohydrate moiety linked to the Fc region of the protein (Claudia Ferrara et al, (2006) Biotechnology and Bioengineering 93:851-861).
  • glycosylation of the Fc region can be modified to increase or decrease effector function.
  • the Fc regions of antibodies include altered glycosylation of amino acid residues.
  • the altered glycosylation of the amino acid residues results in lowered effector function.
  • the altered glycosylation of the amino acid residues results in increased effector function.
  • the Fc region has reduced fucosylation.
  • the Fc region is afucosylated.
  • an antibody can be modified with an appropriate sialylation profile for a particular therapeutic application.
  • the Fc regions of an antibody includes an altered sialylation profile compared to the native Fc region.
  • the Fc regions of antibodies include an increased sialylation profile compared to the native Fc region. In another embodiment, the Fc regions of antibodies include a decreased sialylation profile compared to the native Fc region. Other modifications and/or substitutions and/or additions and/or deletions of the Fc domain will be readily apparent to one skilled in the art.
  • the glycosylation pattern in the variable region of the present antibodies is modified to alter the affinity of the antibody for antigen.
  • the antibody is agly coslated (i.e., the antibody lacks glycosylation). Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • aglycosylated antibodies may be produced in bacterial cells which lack the necessary glycosylation machinery.
  • the antibody is of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), subisotype (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or allotype (e.g., Gm, e.g., G1 m(f, z, a or x), G2m(n), G3m(g, b, or c), Am, Em, and Km(l, 2 or 3)).
  • an antibody of the disclosure comprises a human IgG constant domain having one or more amino acid substitutions relative to a wild-type human IgG constant domain.
  • An antibody of the disclosure may comprise a human IgG constant domain having the M252Y, S254T, and T256E (“YTE”) amino acid substitutions, wherein amino acid residues are numbered according to the EU index as in Rabat
  • the disclosure does not relate to antibodies in natural form, i.e., they are not taken from their natural environment but are isolated and obtained by purification from natural sources, or obtained by genetic recombination or chemical synthesis, and thus they can carry unnatural amino acids.
  • the twenty conventional amino acids and their abbreviations follow conventional usage. See Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference.
  • Stereoisomers e.g., D-amino acids, Nle, Nva, Cha, Om, Hie, Chg, Hch, or Har
  • unnatural amino acids such as .alpha.-, .alpha.-disubstituted amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids
  • unconventional amino acids include (i.e., are not limited to): 4-hydroxyproline, gamma. -carboxyglutamate, epsilon.- N,N,N-trimethyllysine, . epsilon.
  • -N-acetyllysine O-phosphoserine, N-acetylserine, N- formylmethionine, 3 -methylhistidine, 5-hydroxy lysine, . sigma.
  • -N-methylarginine and other similar amino acids and imino acids (e.g., 4-hydroxyproline).
  • the left-hand direction is the amino terminal direction and the right-hand direction is the carboxy-terminal direction, in accordance with standard usage and convention.
  • the present disclosure does not relate to nucleotide sequences in their natural chromosomal environment, i.e., in a natural state.
  • the sequences of the present disclosure have been isolated and purified, i.e., they were sampled directly or indirectly, for example by a copy, their environment having been at least partially modified.
  • Isolated nucleic acids obtained by recombinant genetics, by means, for example, of host cells, or obtained by chemical synthesis are also provided.
  • the “percentage identity” between two sequences of nucleic acids or amino acids means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the differences between the two sequences being distributed randomly along their length.
  • the comparison of two nucleic acid or amino acid sequences is traditionally carried out by comparing the sequences after having optimally aligned them, said comparison being able to be conducted by segment or by using an “alignment window”.
  • Optimal alignment of the sequences for comparison can be carried out, in addition to comparison by hand, by means of the local homology algorithm of Smith and Waterman (1981) [Ad. App. Math.
  • the percentage identity between two nucleic acid or amino acid sequences is determined by comparing the two optimally-aligned sequences in which the nucleic acid or amino acid sequence to compare can have additions or deletions compared to the reference sequence for optimal alignment between the two sequences. Percentage identity is calculated by determining the number of positions at which the amino acid nucleotide or residue is identical between the two sequences, for example between the two complete sequences, dividing the number of identical positions by the total number of positions in the alignment window and multiplying the result by 100 to obtain the percentage identity between the two sequences.
  • the BLAST program “BLAST 2 sequences” (Tatusova et al., “Blast 2 sequences - a new tool for comparing protein and nucleotide sequences”, FEMS Microbiol, 1999, Lett. 174:247-250) available on the site http://www.ncbi.nlm.nih.gov/gorfQjl2.html, can be used with the default parameters (notably for the parameters “open gap penalty”: 5, and “extension gap penalty”: 2; the selected matrix being for example the “BLOSUM 62” matrix proposed by the program); the percentage identity between the two sequences to compare is calculated directly by the program.
  • a reference amino acid sequence e.g., CDR, heavy chain variable region, light chain variable region
  • preferred examples include those containing the reference sequence, certain modifications, notably a deletion, addition or substitution of at least one amino acid, truncation or extension.
  • substitutions are preferred in which the substituted amino acids are replaced by “equivalent” amino acids.
  • the expression “equivalent amino acids” is meant to indicate any amino acids likely to be substituted for one of the structural amino acids without however modifying the biological activities of the corresponding antibodies and of those specific examples defined below.
  • Equivalent amino acids can be determined either on their structural homology with the amino acids for which they are substituted or on the results of comparative tests of biological activity between the various antibodies likely to be generated.
  • One of ordinary skill in the art is familiar with the possible substitutions likely to be carried out without resulting in a significant modification of the biological activity of the corresponding modified antibody; inverse substitutions are naturally possible under the same conditions.
  • the disclosure also provides hybridomas producing the antibodies of the disclosure.
  • Figure 3 shows inhibition activity of monoclonal antibodies specific to SARS-CoV-2 S protein and its RBD in competitive ELISA. Results revealed 13 monoclonal antibodies (MAb 12, 96, 290, 266, 352, 677, 68, 322, 97, 99, 462, 175 and 67) that effectively inhibited interaction of RBD domain with ACE2. Interaction between S protein and ACE2 overexpressed on HEK 293T/17 cells was blocked by 12 antibodies (MAb 12, 96, 290, 266, 352, 677, 68, 322, 97, 99, 462 and 175). The lowest inhibition activity (25%) was shown by the monoclonal antibody 677.
  • a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line) with one of a variety of antibody- producing cells.
  • suitable immortal cell lines include, but not limited to, Sp2/0, Sp2/0-AG14, P3/NSl/Ag4-l, NSO, P3X63Ag8.653, MCP-11, S-194, heteromyelomas, fusion products thereof, or any cell or fusion cell derived therefrom, or any other suitable cell line as known in the art, and/or commercially available for this purpose (e.g., ATCC).
  • Suitable antibody producing cells include, but are not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination of the same. See, e.g., Ausubel, supra, and Colligan, Immunology, supra, Chapter 2, entirely incorporated herein by reference.
  • the antibodies are optimized full-length antibodies, chimeric or humanized, which can be produced by anyone or a combination of known techniques, as listed and exemplified in, for example, Chapters 3, 4, and 5, of “Business Insights, Preclinical Development of Monoclonal Antibodies and Related Biologicals-Emerging technologies and new therapeutic candidates, by James Shirvill, 2010,” the entire contents of which is incorporated by reference, such as: CDR grafting, such as UBC’s SLAM technology, PDL’s SMART technology, Arana Therapeutics pic’s Superhumanization, Framework patching, techniques for making composit human antibodies, BioAtla LLC’s ATLAb platform, humaneering, Mutational Lineage Guided (MLG) strategies, deimmunisation strategies, humanation strategies, human engineering (e.g., XOMA’s HE technology), FcX, Biolex Therapeutics Inc
  • XmAb XmAb, Sugar Engineered Antibodies (e.g, Seattle Genetics Inc (Bothell, WA, US)), “Wox” (tryptophan oxidized) antibodies (e.g., InNexus Biotechnology Inc (Vancouver, BC, Canada)); and the like.
  • Sugar Engineered Antibodies e.g, Seattle Genetics Inc (Bothell, WA, US)
  • Wix tryptophan oxidized antibodies
  • InNexus Biotechnology Inc Vancouver, BC, Canada
  • the antibodies are fully human monoclonal antibodies, and can be produced by one or a combination of technology platforms, as listed and exemplified in, for example, Chapter 4 of “Business Insights, Preclinical Development of Monoclonal Antibodies and Related Biologicals-Emerging technologies and new therapeutic candidates, by James Shirvill, 2010,” and including, but not limited to: phage display (e.g., PDL, Dyax Corp; Cambridge, MA, US); Molecule Based Antibody Screening (MBAS) (e.g., Affitech A/S, described in, e.g., EP0547201 and US 6,730,483); cell based antibody selection (CBAS) platforms; Human Combinatorial Antibody Libraries (HuCAL; e.g., MorphoSys AG); MAbstract platforms (e.g., Crucell NV), including those with the PERC6 cell line; Adimab platforms; XenoMouse; UltiMAb platforms;
  • the antibodies are modified by linking them to non-antibody agents, using one or more of the technology platforms and methods as described in Chapter 5 of “Business Insights, Preclinical Development of Monoclonal Antibodies and Related Biologicals-Emerging technologies and new therapeutic candidates, by James Shirvill, 2010,” including: antibody drug conjugate (e.g., ADC, Seattle Genetics); targeted antibody payload (TAP; Immunogen Inc); Probodies (e.g., CytomX Therapeutics); antibody cloaking (e.g., BioTransformations); targeted photodynamic therapy (e.g., PhotoBiotics; AlbudAb (e.g., GSK); hyFc (e.g., Genexine); Ligand traps (e.g., BioLogix); CovX-Body (e.g., CovX); Dynamic Cross- Linking (e.g., InNexus Biotechnology); LEC Technology (e.g., Pivotal BioSciences, Morphotek);
  • antibody drug conjugate e
  • the antibodies whose amino acid sequences are described herein are modified or serve as the basis for making binding molecules with one or more of the antigen-binding properties described for these antibodies.
  • binding proteins can be made by one or more of the techniques listed and exemplified in, for example, Chapter 6 of “Business Insights, Preclinical Development of Monoclonal Antibodies and Related Biologicals-Emerging technologies and new therapeutic candidates, by James Shirvill, 2010,” including:Fab, TetraMABs (e.g., Galileo Oncologies); scFv; Immuna (e.g., ESBA Tech AG); [scFv]2, including binding molecules that bind any epitope in S protein or RBD; BiTE (Affitech, Micromet AG); Avibodies (e.g., Avipep Pty); TandAb (e.g., Affimed Therapeutics); Flexibody (e.g., Affimed); V-NAR (e.g.,
  • GSK US Patent No. 6,248,516 and EP0368684
  • Heteropolymer e.g., Elusys Therapeutics Inc.
  • Unibody e.g., GenMab A/S
  • Domain Exchanged Antibodies e.g., Calmune Corporation, Science. 2003 Jim 27;300(5628):2065-71
  • Small Modular ImmunoPharmaceuticals SMTP
  • SCORPION molecules e.g., Trubion Pharmaceuticals
  • Dual Variable Domain Immunoglobulin, DVD-Ig (Abbott Laboratories); and the like.
  • the antibodies of the present disclosure or their corresponding immunoglobulin chain(s) can be further modified using conventional techniques known in the art, for example, by using amino acid deletion(s), insertions), substitution(s), addition(s), and/or recombination(s) and/or any other modification ⁇ ) known in the art either alone or in combination. See, e.g., the EXAMPLES provided further below. Methods for introducing such modifications in the DNA sequence underlying the amino acid sequence of an immunoglobulin chain are known to the person skilled in the art. See, e.g., Sambrook (supra) and Ausubel (Supra).
  • Modifications of the antibody of the disclosure include chemical and/or enzymatic derivatizations at one or more constituent amino acids, including side chain modifications, backbone modifications, and N- and C -terminal modifications including acetylation, hydroxylation, methylation, amidation, and the attachment or removal of carbohydrate or lipid moieties, cofactors, and the like.
  • the present disclosure encompasses the production of chimeric proteins which comprise the described antibody or some fragment thereof at the amino terminus fused to heterologous molecule such as an immunostimulatory ligand at the carboxyl terminus. See, e.g., international application WO00/30680 for corresponding technical details, incorporated herein by reference in its entirety.
  • the disclosure also provides antibodies coupled to other moieties for purposes such as drug targeting and imaging applications.
  • moieties for purposes such as drug targeting and imaging applications.
  • Those of ordinary skill in the art will recognize that a large variety of possible moieties can be coupled to the resultant antibodies or to other molecules of the disclosure. (See, for example, “Conjugate Vaccines”, Contributions to Microbiology and Immunology, J. M Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).
  • Moieties suitable for attachment to the antibodies include, but are not limited to, proteins, peptides, drugs, labels, and cytotoxins and can be conjugated to the antibody or fragment thereof to alter or fine tune one or more characteristics (e.g., biochemical, binding and/or functional) of the antibody or fragment.
  • Methods for forming conjugates, making amino acid and/or polypeptide changes and post-translational modifications are well known in the art. Exemplary moieties are described elsewhere in this application.
  • Such coupling can be conducted chemically after expression of the antibody to site of attachment or the coupling product can be engineered into the antibody of the disclosure at the DNA level. The DNAs are then expressed in a suitable host system, and the expressed proteins are collected and renatured, if necessary.
  • the present disclosure also relates to polynucleotides encoding one or more of the antibody-based agents provided by the disclosure.
  • the nucleotide for example encodes at least the binding domain or variable region of an immunoglobulin chain of the antibodies described above.
  • said variable region encoded by the polynucleotide comprises at least one complementarity determining region (CDR) of the VH and/or VL of the variable region of the said antibody.
  • CDR complementarity determining region
  • each variable domain (the heavy chain VH and light chain VL) of an antibody comprises three hypervariable regions, sometimes called complementarity determining regions or "CDRs" flanked by four relatively conserved framework regions or "FRs" and refer to the amino acid residues of an antibody which are responsible for antigen-binding.
  • CDRs complementarity determining regions
  • FRs relatively conserved framework regions
  • the hypervariable regions or CDRs of the human IgG subtype of antibody comprise amino acid residues from residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31-35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain as described by Rabat et al., Sequences of Proteins of Immunological Interest, Sth Ed Public Health Service, National Institutes of Health, Bethesda, Md. (1991) and/or those residues from a hypervariable loop, i.e.
  • the IMGT unique numbering provides a standardized delimitation of the framework regions (FR1- IMGT: positions 1 to 26, FR2-IMGT: 39 to 55, FR3-IMGT: 66 to 104 and FR4-IMGT: 118 to 128) and of the complementarity determining regions: CDR1-IMGT: 27 to 38, CDR2-IMGT: 56 to 65 and CDR3-IMGT: 105 to 117.
  • the IMGT unique numbering is used in 2D graphical representations, designated as IMGT Colliers de Perles. See, e.g., Ruiz, M and Lefranc, M-P., Immunogenetics, 53, 857-883 (2002); Raas, Q.
  • the disclosure also relates to an isolated nucleic acid characterized in that it is selected among the following nucleic acids (including any degenerate genetic code): [00173] a nucleic acid, DNA or RNA, coding for an antibody or antibody fragment (e.g., CDR, heavy chain variable, light chain variable region) according to the disclosure;
  • nucleic acid complementary to a nucleic acid as defined in a [00175] a nucleic acid of at least 18 nucleotides capable of hybridizing under highly stringent conditions with at least one of the CDRs chosen from the CDRs underlined in Table 3; and
  • nucleic acid of at least 18 nucleotides capable of hybridizing under highly stringent conditions with at least one of the heavy chain of nucleic acid sequence of Table 1 and/or at least one of the light chain of nucleic acid sequences of Table 2, or a sequence with at least 80%, for example 85%, 90%, 95%, 98% and 99% identity after optimal alignment with those sequences, for example with at least one of the CDRs therefrom according to the IMGT numbering.
  • Nucleic sequences exhibiting a percentage identity of at least 80%, for example 85%, 90%, 95%, 98% and 99% after optimal alignment with a preferred sequence means nucleic sequences exhibiting, with respect to the reference nucleic sequence, certain modifications such as, in particular, a deletion, a truncation, an extension, a chimeric fusion and/or a substitution, notably punctual.
  • these are sequences which code for the same amino acid sequences as the reference sequence, this being related to the degeneration of the genetic code, or complementarity sequences that are likely to hybridize specifically with the reference sequences, for example under highly stringent conditions, notably those defined below.
  • Hybridization under highly stringent conditions means that conditions related to temperature and ionic strength are selected in such a way that they allow hybridization to be maintained between two complementarity DNA fragments.
  • the highly stringent conditions of the hybridization step for the purpose of defining the polynucleotide fragments described above are advantageously as follows.
  • DNA-DNA or DNA-RNA hybridization is carried out in two steps: (1) prehybridization at 42°C for three hours in phosphate buffer (20 mM, pH 7.5) containing 5X SSC (IX SSC corresponds to a solution of 0.15 M NaCl + 0.015 M sodium citrate), 50% formamide, 7% sodium dodecyl sulfate (SDS), 10X Denhardt’s, 5% dextran sulfate and 1% salmon sperm DNA; (2) primary hybridization for 20 hours at a temperature depending on the length of the probe (i.e.: 42°C for a probe >100 nucleotides in length) followed by two 20-minute washings at 20°C in 2X SSC + 2% SDS, one 20- ⁇ ninute washing at 20°C in 0.1X SSC + 0.1% SDS.
  • IX SSC corresponds to a solution of 0.15 M NaCl + 0.015 M sodium citrate
  • SDS sodium dodecyl sulf
  • the last washing is carried out in 0. IX SSC + 0.1% SDS for 30 minutes at 60°C for a probe >100 nucleotides in length.
  • the highly stringent hybridization conditions described above for a polynucleotide of defined size can be adapted by a person skilled in the art for longer or shorter oligonucleotides, according to the procedures described in Sambrook, et al. (Molecular cloning: a laboratory manual, Cold Spring Harbor Laboratory; 3rd edition, 2001.
  • Substantially identical sequences may be polymorphic sequences, i.e., alternative sequences or alleles in a population.
  • An allelic difference may be as small as one base pair.
  • Substantially identical sequences may also comprise mutagenized sequences, including sequences comprising silent mutations.
  • a mutation may comprise one or more residue changes, a deletion of one or more residues, or an insertion of one or more additional residues.
  • the polynucleotides/nucleic acids may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may also be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
  • a suitable source e.g., an antibody cDNA library, or
  • Amplified nucleic acids generated by PCR may then be cloned into replicable cloning vectors using any method well known in the art.
  • the nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • variable domain of the antibody having the above-described variable domain can be used for the construction of other polypeptides or antibodies of desired specificity and biological function.
  • present disclosure also encompasses polypeptides and antibodies comprising at least one CDR of the above-described variable domain and which advantageously have substantially the same or similar binding properties as the antibody described in the appended examples.
  • the person skilled in the art will appreciate that using the variable domains or CDRs described herein antibodies can be constructed according to methods known in the art, e.g., as described in European patent applications EP 0451 216 Al andEP 0549581 Al.
  • the present disclosure also relates to antibodies wherein one or more of the mentioned CDRs comprise one or more, for example not more than two amino acid substitutions.
  • the antibody of the disclosure comprises in one or both of its immunoglobulin chains two or all three CDRs of the variable regions as set forth in Table 2.
  • the antibody of the disclosure comprises in one or both of its immunoglobulin chains two or all three CDRs as set forth in Table 2.
  • the polynucleotides or nucleic acids encoding the above-described antibodies can be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides either alone or in combination.
  • the polynucleotide is part of a vector. Such vectors can comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • the polynucleotide is operatively linked to one or more expression control sequences, allowing expression in prokaryotic or eukaryotic cells.
  • Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells, for example mammalian cells, are known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly- A signals ensuring termination of transcription and stabilization of the transcript Additional regulatory elements can include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions.
  • the polynucleotides encoding at least the variable domain of the light and/or heavy chain can encode the variable domains of both immunoglobulin chains or only one.
  • said polynucleotides can be under the control of the same promoter or can be separately controlled for expression.
  • Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the A0X1 or GALI promoter in yeast or the CMV-, SV40-, RSV- promoter, CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements that are responsible for the initiation of transcription can also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • transcription termination signals such as the SV40-poly-A site or the tk-poly-A site
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium can be added to the coding sequence of the polynucleotides and are known in the art
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and optionally, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product
  • suitable expression vectors include, without limitation, the Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDMS, pRc/CMV, pcDNAl, pcDNA3 (Invitrogen), and pSPORTl (GIBCO BRL).
  • the expression control sequences can be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts can also be used.
  • the vector Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the immunoglobulin light chains, heavy chains, light/heavy chain dimers or intact antibodies, binding fragments or other immunoglobulin forms can follow. See, e.g., Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N.Y., (1979).
  • the disclosure provides vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise a polynucleotide encoding a variable domain of an immunoglobulin chain of an antibody of the disclosure; optionally in combination with a polynucleotide of the disclosure that encodes the variable domain of the other immunoglobulin chain of an antibody of the disclosure.
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, can be used for delivery of the polynucleotides or vector of the disclosure into targeted cell population. Any methods that are known to those skilled in the art can be used to construct recombinant viral vectors. See, for example, the techniques described in Sambrook (supra) and Ausubel (supra). Alternatively, the polynucleotides and vectors provided by the disclosure can be reconstituted into liposomes for delivery to target cells.
  • the vectors containing the polynucleotides provided by the disclosure can be transferred into the host cell by known methods, which vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment or electroporation can be used for other cellular hosts.
  • the present disclosure furthermore relates to host cells transformed with a polynucleotide or vector provided by the disclosure.
  • the host cell can be a prokaryotic or eukaryotic cell.
  • the polynucleotide or vector that is present in the host cell can either be integrated into the genome of the host cell or it can be maintained extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species S. cerevisiae.
  • the antibodies or immunoglobulin chains encoded by the polynucleotide of the present disclosure can be glycosylated or can be non-glycosylated. Certain antibodies provided by the disclosure, or the corresponding immunoglobulin chains, can also include an initial methionine amino acid residue.
  • a polynucleotide of the disclosure can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art. Furthermore, methods for preparing fused, operably linked genes and expressing them in, e.g., mammalian cells and bacteria are well-known in the art. See, e.g., Sambrook.
  • the genetic constructs and methods described therein can be utilized for expression of the antibodies provided by the disclosure, or their corresponding immunoglobulin chains, in eukaryotic or prokaryotic hosts.
  • expression vectors containing promoter sequences which facilitate the efficient transcription of the inserted polynucleotide are used in connection with the host.
  • the expression vector typically contains an origin of replication, a promoter, and a terminator, as well as specific genes which are capable of providing phenotypic selection of the transformed cells.
  • Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas," Fifth edition (1985) Manassas, VA, U.S.A., and other available version, incorporated herein by reference).
  • transgenic animals for example mammals, comprising cells of the disclosure can be used for the large scale production of the antibodies of the disclosure.
  • the present disclosure includes recombinant methods for making an anti- SARS coronavirus S protein antigen-binding protein, such as an antibody or antigen-binding fragment thereof of the present disclosure, or an immunoglobulin chain thereof, comprising (i) introducing one or more polynucleotides (e.g., including the nucleotide sequence of any one or more of the sequences of Table 3) encoding light and/or heavy immunoglobulin chains, or CDRs, of the antigen-binding protein, e.g., of Table 3, for example, wherein the polynucleotide is in a vector; and/or integrated into a host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., CHO or Pichia or Pichia pastoris) under condition favorable to expression of the polynucleotide and, (iii) optionally, isolating the antigen-binding protein, (
  • a polynucleotide can be integrated into a host cell chromosome through targeted insertion with a vector such as adeno-associated virus (AAV), e.g., after cleavage of the chromosome using a gene editing system (e.g., CRISPR (for example, CRISPR-Cas9), TALEN, megaTAL, zinc finger, or Argonaute).
  • AAV adeno-associated virus
  • CRISPR for example, CRISPR-Cas9
  • TALEN for example, CRISPR-Cas9
  • TALEN megaTAL
  • zinc finger or Argonaute
  • Targeted insertions can take place, for example, at host cell loci such as an albumin or immunoglopbulin genomic locus.
  • insertion can be at a random locus, e.g., using a vector such as lentivirus.
  • an antigen-binding protein e.g., antibody or antigen-binding fragment
  • an immunoglobulin chain e.g., an antibody that comprises two heavy immunoglobulin chains and two light immunoglobulin chains
  • co-expression of the chains in a single host cell leads to association of the chains, e.g., in the cell or on the cell surface or outside the cell if such chains are secreted, so as to form the antigen-binding protein (e.g., antibody or antigen-binding fragment).
  • the methods include those wherein only a heavy immunoglobulin chain or only a light immunoglobulin chain (e.g., any of those discussed herein including mature fragments and/or variable domains thereof) is expressed.
  • the present invention also includes anti- SARS coronavirus S protein antigen-binding proteins, such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence set forth in Table 3 and a light chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a nucleotide sequence set forth in Table 3 which are the product of such production methods, and, optionally, the purification methods set forth herein.
  • anti- SARS coronavirus S protein antigen-binding proteins such as antibodies and antigen-binding fragments thereof, comprising a heavy chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encoded by a polynucleotide comprising a nucleotide sequence set forth in Table 3 and a light chain immunoglobulin (or variable domain thereof or comprising the CDRs thereof) encode
  • the product of the method is an anti-CoV-S antigen-binding protein which is an antibody or fragment comprising an HCVR comprising an amino acid sequence set forth in Table 3 and an LCVR comprising an amino acid sequence set forth in Table 3, wherein the HCVR and LCVR sequences are selected from a single antibody listed in Table 3.
  • the product of the method is an anti- SARS coronavirus S protein antigen-binding protein which is an antibody or fragment comprising HCDR1, HCDR2, and HCDR3 comprising amino acid sequences set forth in Table 3 and LCDR1 , LCDR2, and LCDR3 comprising amino acid sequences set forth in Table 3, wherein the six CDR sequences are selected from a single antibody listed in Table 3.
  • the product of the method is an anti- SARS coronavirus S protein antigen-binding protein which is an antibody or fragment comprising a heavy chain comprising an HC amino acid sequence set forth in Table 3 and a light chain comprising an LC amino acid sequence set forth in Table 3.
  • Mammalian cell lines available as hosts for expression of recombinant antibodies are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), human epithelial kidney 293 cells, and a number of other cell lines.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • human epithelial kidney 293 cells e.g., Hep G2
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the antibody or portion thereof
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, W138, BT483, Hs578T, HTB2, BT20 and T47D, NSO (a murine myeloma cell line that does not endogenously produce any functional immunoglobulin chains), SP20, CRL7O3O and HsS78Bst cells.
  • Human cell lines developed by immortalizing human lymphocytes can be used to recombinantly produce monoclonal antibodies.
  • Additional cell lines which may be used as hosts for expression of recombinant antibodies include, but are not limited to, insect cells (e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5bl-4) or yeast cells (e.g. 8. cerevisiae, Pichia, U.S. Pat No. 7,326,681; etc.), plants cells (US20080066200); and chicken cells (WO2008142124).
  • insect cells e.g. Sf21/Sf9, Trichoplusia ni Bti-Tn5bl-4
  • yeast cells e.g. 8. cerevisiae, Pichia, U.S. Pat No. 7,326,681; etc.
  • plants cells e.g. 8. cerevisiae, Pichia, U.S. Pat No. 7,326,681; etc.
  • plants cells e.g. 8. cerevisiae, Pichia, U.S. Pat No. 7,326,681; etc.
  • antibodies of the disclosure are expressed in a cell line with stable expression of the antibody.
  • Stable expression can be used for long-term, high-yield production of recombinant proteins.
  • cell lines which stably express the antibody molecule may be generated.
  • Host cells can be transformed with an appropriately engineered vector comprising expression control elements (e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.), and a selectable marker gene. Following the introduction of the foreign DNA, cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • expression control elements e.g., promoter, enhancer, transcription terminators, polyadenylation sites, etc.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells that stably integrated the plasmid into their chromosomes to grow and form foci which in turn can be cloned and expanded into cell lines.
  • Methods for producing stable cell lines with a high yield are well known in the art and reagents are generally available commercially.
  • antibodies of the disclosure are expressed in a cell line with transient expression of the antibody. Transient transfection is a process in which the nucleic acid introduced into a cell does not integrate into the genome or chromosomal DNA of that cell. It is in fact maintained as an extra-chromosomal element, e.g. as an episome, in the cell.
  • the mammalian cell culture media is based on commercially available media formulations, including, for example, DMEM or Ham's F12. In other embodiments, the cell culture media is modified to support increases in both cell growth and biologic protein expression.
  • cell culture medium As used herein, the terms “cell culture medium,” “culture medium,” and “medium formulation” refer to a nutritive solution for the maintenance, growth, propagation, or expansion of cells in an artificial in vitro environment outside of a multicellular organism or tissue.
  • Cell culture medium may be optimized for a specific cell culture use, including, for example, cell culture growth medium which is formulated to promote cellular growth, or cell culture production medium which is formulated to promote recombinant protein production.
  • the terms nutrient, ingredient, and component are used interchangeably herein to refer to the constituents that make up a cell culture medium.
  • the cell lines are maintained using a fed batch method.
  • fed batch method refers to a method by which a fed batch cell culture is supplied with additional nutrients after first being incubated with a basal medium.
  • a fed batch method may comprise adding supplemental media according to a determined feeding schedule within a given time period.
  • a “fed batch cell culture” refers to a cell culture wherein the cells, typically mammalian, and culture medium are supplied to the culturing vessel initially and additional culture nutrients are fed, continuously or in discrete increments, to the culture during culturing, with or without periodic cell and/or product harvest before termination of culture.
  • the cell culture medium used and the nutrients contained therein are known to one of skill in the art
  • the cell culture medium comprises a basal medium and at least one hydrolysate, e.g., soy-based hydrolysate, a yeast-based hydrolysate, or a combination of the two types of hydrolysates resulting in a modified basal medium.
  • the additional nutrients may include only a basal medium, such as a concentrated basal medium, or may include only hydrolysates, or concentrated hydrolysates.
  • Suitable basal media include, but are not limited to Dulbecco's Modified Eagle's Medium (DMEM), DME/F12, Minimal Essential Medium (MEM), Basal Medium Eagle (BME), RPMI 1640, F-10, F-12, a-Minimal Essential Medium (a-MEM), Glasgow's Minimal Essential Medium (G-MEM), PF CHO (see, e.g., CHO protein free medium (Sigma) or EX-CELLTM 325 PF CHO Serum-Free Medium for CHO Cells Protein-Free (SAFC Bioscience), and Iscove's Modified Dulbecco's Medium.
  • DMEM Dulbecco's Modified Eagle's Medium
  • MEM Minimal Essential Medium
  • BME Basal Medium Eagle
  • RPMI 1640 F-10, F-12
  • a-MEM a-Minimal Essential Medium
  • G-MEM Glasgow's Minimal Essential Medium
  • PF CHO see, e.g., CHO protein free medium
  • basal media examples include BME Basal Medium (Gibco-Invitrogen; see also Eagle, H (1965) Proc. Soc. Exp. Biol. Med. 89, 36); Dulbecco's Modified Eagle Medium (DMEM, powder) (Gibco-Invitrogen (#31600); sseeee also Dulbecco and Freeman (1959) Virology 8, 396; Smith et al. (1960) Virology 12, 185. Tissue Culture Standards Committee, In Vitro 6:2, 93); CMRL 1066 Medium (Gibco-Invitrogen (#11530); see also Parker R. C. et al (1957) Special Publications, N.Y.
  • the basal medium may be serum-free, meaning that the medium contains no serum (e.g., fetal bovine serum (FBS), horse serum, goat serum, or any other animal-derived serum known to one skilled in the art) or animal protein free media or chemically defined media.
  • the basal medium may be modified in order to remove certain non-nutritional components found in standard basal medium, such as various inorganic and organic buffers, surfactants), and sodium chloride. Removing such components from basal cell medium allows an increased concentration of the remaining nutritional components, and may improve overall cell growth and protein expression.
  • omitted components may be added back into the cell culture medium containing the modified basal cell medium according to the requirements of the cell culture conditions.
  • the cell culture medium contains a modified basal cell medium, and at least one of the following nutrients, an iron source, a recombinant growth factor; a buffer; a surfactant; an osmolarity regulator; an energy source; and non-animal hydrolysates.
  • the modified basal cell medium may optionally contain amino acids, vitamins, or a combination of both amino acids and vitamins.
  • the modified basal medium further contains glutamine, e.g, L- glutamine, and/or methotrexate.
  • Antibody production can be conducted in large quantity by a bioreactor process using fed-batch, batch, perfusion or continuous feed bioreactor methods known in the art.
  • Large- scale bioreactors have at least 1000 liters of capacity, preferably about 1,000 to 100,000 liters of capacity. These bioreactors may use agitator impellers to distribute oxygen and nutrients.
  • Small scale bioreactors refers generally to cell culturing in no more than approximately 100 liters in volumetric capacity, and can range from about 1 liter to about 100 liters.
  • single-use bioreactors SUV may be used for either large-scale or small-scale culturing.
  • Temperature, pH, agitation, aeration and inoculum density will vary depending upon the host cells used and the recombinant protein to be expressed.
  • a recombinant protein cell culture may be maintained at a temperature between 30 and 45° C.
  • the pH of the culture medium may be monitored during the culture process such that the pH stays at an optimum level, which may be for certain host cells, within a pH range of 6.0 to 8.0.
  • An impellor driven mixing may be used for such culture methods for agitation.
  • the rotational speed of the impellor may be approximately 50 to 200 cm/sec tip speed, but other airlift or other mixing/aeration systems known in the art may be used, depending on the type of host cell being cultured.
  • aeration is provided to maintain a dissolved oxygen concentration of approximately 20% to 80% air saturation in the culture, again, depending upon the selected host cell being cultured.
  • a bioreactor may sparge air or oxygen directly into the culture medium.
  • Other methods of oxygen supply exist, including bubble- free aeration systems employing hollow fiber membrane aerators.
  • an antibody molecule may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigens Protein A or Protein G, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present disclosure or fragments thereof may be fused to heterologous polypeptide sequences (referred to herein as “tags”) to facilitate purification.
  • tags heterologous polypeptide sequences
  • the particulate debris either host cells or lysed fragments, is removed, for example, by centrifugation or ultrafiltration.
  • Carter et al, Bio/Technology, 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted into the periplasmic space of E. coli.
  • supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit
  • a protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.
  • the antibody composition prepared from the cells can be purified using, for example, hydroxylapatite chromatography, hydrophobic interaction chromatography, ion exchange chromatography, gel electrophoresis, dialysis, and/or affinity chromatography either alone or in combination with other purification steps.
  • the suitability of protein A as an affinity ligand depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody and will be understood by one of skill in the art
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • the Bakerbond ABX resin (J. T. Baker, Phillipsburg, N.J.) is useful for purification.
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin, SEPHAROSE chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS- PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • antibodies of the disclosure that are substantially purified/isolated.
  • these isolated/purified recombinantly expressed antibodies may be administered to a patient to mediate a prophylactic or therapeutic effect
  • a prophylactic is a medication or a treatment designed and used to prevent a disease, disorder or infection from occurring.
  • a therapeutic is concerned specifically with the treatment of a particular disease, disorder or infection.
  • a therapeutic dose is the amount needed to treat a particular disease, disorder or infection.
  • these isolated/purified antibodies may be used to diagnose SARS-CoV virus infection.
  • the present disclosure encompasses small peptides including those containing a binding molecule as described above, for example containing the CDR3 region of the variable region of any one of the mentioned antibodies, in particular CDR3 of the heavy chain since it has frequently been observed that, for certain antibodies, the heavy chain CDR3 (HCDR3) is the region having a greater degree of variability and a predominant participation in antigen- antibody interaction.
  • Such peptides may be synthesized or produced by recombinant means to produce a binding agent useful according to the disclosure. Such methods are known to those of ordinary skill in the art Peptides can be synthesized for example, using automated peptide synthesizers which are commercially available.
  • the peptides can also be produced by recombinant techniques by incorporating the DNA expressing the peptide into an expression vector and transforming cells with the expression vector to produce the peptide.
  • the above-described fusion proteins can further comprise a cleavable linker or cleavage site for proteinases, which can be called spacer moieties. These spacer moieties, in turn, can be either insoluble or soluble (Diener et al., Science 231 (1986), 148) and can be selected to enable drug release from the antibody at the target site.
  • therapeutic agents which can be coupled to the antibodies of the present disclosure for immunotherapy are drugs, radioisotopes, lectins, and toxins.
  • the drugs that can be conjugated to the antibodies and antigens of the present disclosure include compounds which are classically referred to as drugs such as mitomycin C, daunorubicin, and vinblastine.
  • drugs such as mitomycin C, daunorubicin, and vinblastine.
  • certain isotopes can be more preferable than others depending on such factors as leukocyte distribution as well as isotype stability and emission.
  • some emitters can be preferable to others.
  • alpha and beta particle emitting radioisotopes are preferred in immunotherapy.
  • the radioisotopes are short range, high energy alpha emitters such as 212 Bi.
  • radioisotopes which can be bound to the antibodies or antigens of the disclosure for therapeutic purposes are 125 I, 131 I, 90 Y, 67 Cu, 212 Bi, 212 At, 211 Pb, 47 Sc, 109 Pd and 188 Re. In certain cases, the radiolabel is 64 Cu .
  • Molecules and particles with an antibody, peptide, or binding molecule/protein of the disclosure also have diagnostic utility.
  • Antibodies directed against a SARS-CoV-2 spike protein may be used in methods known within the art relating to the localization and/or quantitation of SARS-CoV-2 (e.g., for use in measuring levels of the SARS-CoV-2 protein within appropriate physiological samples, for use in diagnostic methods, for use in imaging the protein, and the like).
  • the disclosure provides antibodies that recognize S protein and its RBD both in vitro and in vivo.
  • the antibodies can bind these proteins/peptides in a variety of assays, including biochemical, immunoprecipitation, ELISA, Western blotting, and immunohistochemistry assays (e.g., fresh, fixed, frozen, paraffin-embedded), as well as in vivo imaging using, e.g., radiolabeled antibodies (including antibody fragments such as single chain antibodies derived from those of Tables 1 and Table 2). They are capable of doing so in both solid and fluid (e.g., blood, plasma, CSF, homogenates) animal (e.g., rodents, humans) samples and biopsies.
  • assays including biochemical, immunoprecipitation, ELISA, Western blotting, and immunohistochemistry assays (e.g., fresh, fixed, frozen, paraffin-embedded), as well as in vivo imaging using, e.g., radiolabeled antibodies (including antibody fragments such as single chain antibodies derived from those of Tables 1 and Table 2). They
  • the antibodies of the present disclosure can be labeled (e.g., fluorescent, radioactive, enzyme, nuclear magnetic, heavy metal) and used to detect specific targets in vivo or in vitro including immunochemistry-like assays in vitro. Also, in vivo, they could be used in a manner similar to nuclear medicine imaging techniques to detect tissues, cells, or other material having S protein or RBD. Targeting S protein and RBD with diagnostic imaging probes detectable by MRI or PET would provide a biological marker for viral presence.
  • the disclosure provides for the use of the antibodies described herein for the preparation of a composition for, and in methods of, SARS-CoV-2 detection and/or targeting a diagnostic agent to S protein or RBD for diagnosis.
  • the disclosure provides antibodies suitable for use in immunoassays in which they can be utilized in liquid phase or bound to a solid phase carrier.
  • immunoassays which can utilize the antibodies of the disclosure are competitive and non-competitive immunoassays in either a direct or indirect format.
  • examples of such immunoassays are the radioimmunoassay (RIA), the sandwich (immunometric assay), flow cytometry and the Western blot assay.
  • RIA radioimmunoassay
  • sandwich immunometric assay
  • flow cytometry flow cytometry
  • Western blot assay The antibodies of the disclosure can be bound to one of many different carriers and used to isolate cells specifically bound thereto.
  • Examples of known carriers include glass, polystyrene, polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran, nylon, amyloses, natural and modified celluloses, polyacrylamides, agaroses, and magnetite.
  • the carrier can be either soluble or insoluble for the purposes of the disclosure. There are many different labels and methods of labeling known to those of ordinary skill in the art.
  • labels examples include enzymes, radioisotopes and radionuclides, colloidal metals, fluorescent compounds, chemiluminescent compounds, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and chemi/electrochemi/bioluminescent compounds.
  • a secondary reporter e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags
  • chemi/electrochemi/bioluminescent compounds examples include enzymes, radioisotopes and radionuclides, colloidal metals, fluorescent compounds, chemiluminescent compounds, biotinyl groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), and chemi/electrochemi/bio
  • the enzymes include peroxidase (e.g, HRP), luciferase, alkaline phosphatase, a-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetyl-cholinesterase, lysozyme, malate dehydrogenase or glucose-6 phosphate dehydrogenase.
  • HRP peroxidase
  • luciferase alkaline phosphatase
  • alkaline phosphatase e.g., a-D-galactosidase
  • glucose oxidase oxidase
  • glucose amylase e.g., glucose amylase
  • carbonic anhydrase e.g., acetyl-cholinesterase
  • lysozyme malate dehydrogenase or glucose-6 phosphate dehydrogenase.
  • the label is biotin, digoxigenin
  • Fluorescent labels can be also combined with the antibodies and S protein and/or RBD-binding proteins provided by the disclosure, including rhodamine, lanthanide phosphors, fluorescein and its derivatives, fluorochromes, rhodamine and its derivatives, green fluorescent protein (GFP), Red Fluorescent Protein (RFP) and others, dansyl, umbelliferone.
  • rhodamine lanthanide phosphors
  • fluorescein and its derivatives fluorescein and its derivatives
  • fluorochromes rhodamine and its derivatives
  • GFP green fluorescent protein
  • RFP Red Fluorescent Protein
  • the antibodies/binding proteins of the disclosure can be prepared by methods known to a person skilled in the art They can then be bound with enzymes or fluorescent labels directly; via a spacer group or a linkage group such as polyaldehyde, glutaraldehyde, ethylenediaminetetraacetic acid (EDTA) or diethylenetriaminepentaacetic acid (DPTA); or in the presence of other binding agents such as those routinely known in the art
  • Conjugates carrying fluorescein labels can be prepared by, for example, reaction with an isothiocyanate. In certain situations, the label or marker can also be therapeutic.
  • Others conjugates can include chemiluminescent labels such as luminol and dioxetane, bioluminescent labels such as luciferase and luciferin, or radioactive labels such as iodine 123 , iodine 125 , iodine 126 , iodine 133 - 131 , bromine 77 , technetium 99 TM, indium 111 , indium 113 TM, gallium 67 , gallium 68 , ruthenium 95 , ruthenium 97 , ruthenium 103 , ruthenium 105 , mercury 107 , mercury 203 , rhenium 99 TM, rhenium 101 , rhenium 105 , scandium 47 , tellurium 121 TM, tellurium 122 TM, tellurium 125 TM, thulium 165 , thulium 167 , thulium 168 , fluorine 18
  • the disclosure also provides antibodies and other S protein/RBD-binding molecules that can also be used in a method for the diagnosis of a viral disorder in an individual by obtaining a body fluid sample from the individual, which can be a blood sample, a lymph sample or any other body fluid sample and contacting the body fluid sample with an antibody of the instant disclosure under conditions enabling the formation of antibody-antigen complexes.
  • the samples may be tissue samples taken from, for example, nasal passages, sinus cavities, salivary glands, lung, liver, pancreas, kidney, ear, eye, placenta, alimentary tract, heart, ovaries, pituitary, adrenals, thyroid, brain or skin.
  • the presence and/or amount of such complexes is then determined by methods known in the art, a level significantly higher than that formed in a control sample indicating the presence of disease in the tested individual.
  • the present disclosure relates to an in vitro immunoassay comprising an antibody of the disclosure.
  • the present disclosure relates to in vivo imaging techniques employing any one of the S protein and/or RBD-binding molecules of the present disclosure.
  • the medical imaging technique Positron emission tomography (PET) which produces a three- dimensional image of body parts is based on the detection of radiation from the emission of positrons.
  • PET Positron emission tomography
  • a biomolecule is radioactively labeled, e.g. it incorporates a radioactive tracer isotope.
  • the radioactively labeled biomolecule becomes concentrated in tissues of interest.
  • the subject is then placed in the imaging scanner, which detects the emission of positrons.
  • a labeled, for example 64 Cu labeled binding molecule such as an antibody is administered to a subject and detection of the binding molecule and thus SARS-CoV-2 is performed by placing the subject in an imaging scanner and detecting the emission of positrons, thereby indicating an infection if emission is detected.
  • the present disclosure thus encompasses a method for PET imagining, comprising the step of administering a ⁇ Cu-labelled or equivalent labeled binding molecule of the present disclosure to a subject.
  • the present disclosure also provides an article of manufacture, such as pharmaceutical and diagnostic packs or kits comprising one or more containers filled with one or more of the above described ingredients, i.e. binding molecule, antibody or binding fragment thereof, polynucleotide, vector or cell, as provided by the disclosure.
  • Associated with such containers can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the kit comprises reagents and/or instructions for use in appropriate diagnostic assays.
  • the composition or kit of the present disclosure is suitable for the diagnosis, prophylaxis, prevention, and treatment of SARS-CoV-2 -related diseases or disorders.
  • an embodiment of the present disclosure pertains to a diagnostic kit comprising the antibody or antigen-binding fragment thereof of the disclosure.
  • the antibody of the present disclosure used in the diagnostic kit may be delectably labeled.
  • Various methods that may be used to label biomolecules are well known to those skilled in the art and are considered to fall within the scope of the present disclosure.
  • Examples of labels useful in the present disclosure may include enzymes, radioisotopes, colloidal metals, fluorescent compounds, chemiluminescent compounds and bioluminescent compounds.
  • Commonly used labels include fluorescent substances (e.g., fluorescein, rhodamine, Texas red, etc.), enzymes (such as horseradish peroxidase, 0-galactosidase, or alkaline phosphatase), radioisotopes (e.g. 32P or 1251), biotin, digoxigenin, colloidal metals, or chemiluminescent or bioluminescent compounds (such as dioxetane, luminol or acridinium). Labeling methods such as covalent bonding, iodination, phosphorylation, biotinylation, etc.
  • fluorescent substances e.g., fluorescein, rhodamine, Texas red, etc.
  • enzymes such as horseradish peroxidase, 0-galactosidase, or alkaline phosphatase
  • radioisotopes e.g. 32P or 1251
  • biotin digoxigenin
  • colloidal metals
  • Detection methods include, but are not limited to, autoradiography, fluorescence microscopy, direct and indirect enzyme reactions, and the like.
  • a commonly used detection assay is the radioisotope or non-radioisotope method. Particularly useful are western blotting, overlay analysis, RIA (radioimmunoassay), IRMA (immunoradioimmunometric assay), EIA (enzyme immunoassay), ELISA (enzyme-linked immunosorbent assay), FIA (fluorescent immunoassay) and CLIA (chemiluminescent immunoassay).
  • the diagnostic kit of the present disclosure may be used to detect the presence or absence of SARS-CoV-2 by contacting a sample with the antibody and observing the reaction.
  • the sample may be, but is not limited to, any one selected from the group consisting of sputum, saliva, blood, sweat, lung cells, mucus of lung tissue, respiratory tissue and spit of a subject, and the sample may be prepared using a process typically known to those skilled in the art.
  • the biological activity of the binding molecules e.g., antibodies provided by the disclosure suggests that they have sufficient affinity to make them candidates for drug localization/drug delivery to cells or tissue.
  • the targeting and binding to S protein and RBD could be useful for the delivery of therapeutically or diagnostically active agents and gene therapy/gene delivery.
  • the disclosure provides for the use of the antibodies described herein for the preparation of a composition for, and in methods of, detection and/or targeting a therapeutic or diagnostic agent to S protein and RBD. These compositions and methods can be used as part of a treatment protocol for SARS-CoV-2 -related diseases or disorders.
  • compositions comprising one or more of the aforementioned compounds, including binding molecules, antibodies, binding fragments; chemical derivatives thereof; polynucleotides, vectors, and cells.
  • Certain compositions can further comprise one or more pharmaceutically acceptable carriers and one or more pharmaceutically acceptable diluents.
  • Certain chemical derivatives comprise chemical moieties that are not normally a part of the base molecule or cell (e.g., of the antibody, binding molecule, polynucleotides, vectors, and cells) but are linked to them by routine methods. Such moieties can function to, for example, improve the solubility, half-life, visualization, detectability, and/or absorption, of the base molecule or cell. Alternatively, the moieties can attenuate undesirable side effects of the base molecule or decrease the toxicity of the base molecule.
  • compositions typically comprise the antibody or agent and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non- aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
  • a pharmaceutical composition of the disclosure is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
  • the pharmaceutical composition can be formulated without blood, plasma or a major component of blood or plasma (e.g., blood cells, fibrin, hemoglobin, albumin, etc.).
  • the pharmaceutical composition can comprise from about 0.001 to about 99.99 wt% of the antibody or antigen-binding fragment according to the total weight of the composition.
  • the pharmaceutical composition can comprise from about 0.001 to about 1%, about 0.001 to about 5%, about 0.001 to about 10%, about 0.001 to about 15%, about 0.001 to about 20%, about 0.001 to about 25%, about 0.001 to about 30%, about 1 to about 10%, about 1 to about 20%, about 1 to about 30%, about 10 to about 20%, about 10 to about 30%, about 10 to about 40%, about 10 to about 50%, about 20 to about 30%, about 20 to about 40%, about 20 to about 50%, about 20 to about 60%, about 20 to about 70%, about 20 to about 80%, about 20 to about 90%, about 30 to about 40%, about 30 to about 50%, about 30 to about 60%, about 30 to about 70%, about 30 to about 80%, about 30 to about 90%, about 40 to about 50%, about 40 to about 60%, about 40 to about 70%, about 40 to about 80%, about 40 to about 90%, about 50 to about 99.99%, about 50 to about 99%, about 60 to about 99%, about 70 to about 99%, about 80 to about 99%
  • compositions described herein can also comprise oonnee or more pharmaceutically acceptable excipients and/or carriers.
  • the pharmaceutically acceptable excipients and/or carriers for use in the compositions of the present invention can be selected based upon a number of factors including the particular compound used, and its concentration, stability and intended bioavailability; the subject, its age, size and general condition; and the route of administration.
  • Some examples of materials which can serve as pharmaceutically acceptable carriers in the compositions described herein are sugars such as lactose, glucose, and sucrose; starches such as com starch and potato starch; cellulose and its derivatives such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as cocoa butter and suppository waxes; oils such as peanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; com oil; and soybean oil; glycols such as propylene glycol; esters such as ethyl oleate and ethyl laurate; agar; detergents such as Tween 80; buffering agents such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; artificial cerebral spinal fluid (CSF),
  • compositions of the invention are identified, for example, in The Handbook of Pharmaceutical Excipients, (American Pharmaceutical Association, Washington, D.C., and The Pharmaceutical Society of Great Britain, London, England, 1968). Additional excipients can be included in the pharmaceutical compositions of the invention for a variety of purposes. These excipients can impart properties which enhance retention of the compound at the site of administration, protect the stability of the composition, control the pH, facilitate processing of the compound into pharmaceutical compositions, and so on.
  • compositions further comprises at least one other therapeutic, prophylactic and/or diagnostic agent.
  • the therapeutic and/or prophylactic agents are capable of preventing and/or treating an coronavirus infection and/or a condition/symptom resulting from such an infection.
  • Therapeutic and/or prophylactic agents include, but are not limited to, anti-viral agents. Such agents can be binding molecules, small molecules (dexamethasone), organic or inorganic compounds, enzymes, polynucleotide sequences, anti- viral peptides, etc.
  • the therapeutic and/or prophylactic agent can comprise an M2 inhibitor (e.g., amantadine, rimantadine) and/or a neuraminidase inhibitor (e.g., zanamivir, oseltamivir).
  • the anti-viral agent can comprise paxlovid, molnupiravir, baricitinib, baloxavir, oseltamivir, zanamivir, peramivir, remdesivir, tixagevimab, cilgavimab, or any combination thereof such as sotrovimab, a combination of bamlanivimab and etesevimab, and a combination of two antibodies called casirivimab and imdevimab.
  • the therapeutic and/or prophylactic agent can also include various anti-malarial such as chloroquine, hydroxychloroquine, and analogues thereof.
  • the additional antibodies or therapeutic/prophylactic and/or diagnostic agents may be used in combination with the antibodies and antigen-binding fragments of the present invention.
  • “In combination” herein means simultaneously, as separate formulations (e.g., co- administered), or as one single combined formulation or according to a sequential administration regiment as separate formulations, in any order.
  • Agents capable of preventing and/or treating an infection with coronavirus (e.g., SARS-CoV-2) and/or a condition resulting from such an infection that are in the experimental phase might also be used as other therapeutic and/or prophylactic agents useful in the present invention.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • the disclosure also provides pharmaceutical compositions comprising combinations of the antibodies provided herein with further agents.
  • the antibodies can be combined with other agents but administered as separate compositions, before, during, or after each other.
  • the antibody compositions may be administered in conjunction with ancillary immunoregulatory agents.
  • cytokines, lymphokines, and chemokines including, but not limited to, IL-2, modified IL-2 (Cys 125— >Serl25), GM-CSF, IL-12, y-interferon, IP-10, MIP1P, and RANTES.
  • the antibodies are administered in combination with one or more drugs that are standard of care for viral diseases.
  • the agents are selected from anti-inflammatory therapy such as tocilizumab and favipiravir, tocilizumab, sarilumab, leronlimab, rintatolimod, BPI-002, REGN3048 and REGN 3051, another monoclonal antibody designed to bind SARS-CoV-2; Anti-Viral therapy such as remdesivir, lopinavir and ritonavir, danoprevir and ritonavir, favipiravir, darunavir and cobicistat, umifenovir, galidesivir, linebacker and equivir, compounds that inhibit the virus interaction with the receptor ACE2; immunomodulators, antimalarial drugs, glucocorticoids, convalescent plasma or antibody therapy; immunotherapy such as RNA vaccines (e.g., targeting Spike protein), DNA vaccines (e.g., targeting Spike protein), recombinant protein vaccines (e.g., targeting Spike protein),
  • the disclosure provides methods (also referred to herein as “screening assays”) for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that modulate or otherwise interfere with the fusion of a SARS-CoV-2 to the cell membrane. Also provided are methods of identifying compounds useful to treat SARS-CoV-2 infection. The disclosure also encompasses compounds identified using the screening assays described herein. For example, the disclosure provides assays for screening candidate or test compounds which modulate the interaction between the SARS- CoV-2 and the cell membrane.
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs) that modulate or otherwise interfere with the fusion of a SARS-CoV-2 to the cell membrane.
  • agents e.g., peptides, peptidomimetics, small molecules or other drugs
  • a candidate compound is introduced to an antibody-antigen complex and determining whether the candidate compound disrupts the antibody-antigen complex, wherein a disruption of this complex indicates that the candidate compound modulates the interaction between a SARS-CoV-2 and the cell membrane.
  • at least one SARS-CoV-2 protein is provided, which is exposed to at least one neutralizing monoclonal antibody of the disclosure. Formation of an antibody-antigen complex is detected, and one or more candidate compounds are introduced to the complex. If the antibody- antigen complex is disrupted following introduction of the one or more candidate compounds, the candidate compounds is useful to treat a SARS-CoV-2 -related disease or disorder.
  • the at least one SARS-CoV-2 protein may be provided as a SARS-CoV-2 molecule.
  • Determining the ability of the test compound to interfere with or disrupt the antibody-antigen complex can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the antigen or biologically-active portion thereof can be determined by detecting the labeled compound in a complex.
  • test compounds can be labeled with 1251, 35S, 14C, or 3H, either directly or indirectly, and the radioisotope detected by direct counting of radioemission or by scintillation counting.
  • test compounds can be enzymatically-labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting an antibody-antigen complex with a test compound, and determining the ability of the test compound to interact with the antigen or otherwise disrupt the existing antibody-antigen complex.
  • determining the ability of the test compound to interact with the antigen and/or disrupt the antibody-antigen complex comprises determining the ability of the test compound to preferentially bind to the antigen or a biologically-active portion thereof, as compared to the antibody.
  • the assay comprises contacting an antibody-antigen complex with a test compound and determining the ability of the test compound to modulate the antibody-antigen complex. Determining the ability of the test compound to modulate the antibody-antigen complex can be accomplished, for example, by determining the ability of the antigen to bind to or interact with the antibody, in the presence of the test compound.
  • EXAMPLE 1 PREPARATION OF RECOMBINANT SARS-COV-2 SPIKE
  • PROTEIN S
  • SPIKE RECEPTOR BINDING DOMAIN RDB
  • the plasmid DNA was isolated using PureLink® HiPure Plasmid Filter Maxiprep Kit (Invitrogen) with the last ethanol washes done in a sterile laminar-flow hood. After overnight incubation in sterile water at 4°C, DNA concentration was measured using NanoPhotometer and the DNA was aliquoted and stored at -20°C.
  • ACE2 protein Expression of ACE2 protein.
  • modified ACE2 angiotensin- converting enzyme 2 gene sequence, containing tags for purification, was synthesized at BioCat Co. (BioCat GmbH, Germany) and cloned into a bacterial pUC-based vector.
  • the ACE2 coding sequence was then sub-cloned into a pCMV-based eukaryotic expression vector under a control of the CMV promoter, amplified in a DH5alpha maxi-culture and plasmid DNA was extracted with maxiprep kit from Invitrogene (ThermoFisher).
  • the purified ACE2 expression plasmid was electroporated into Expi293 cells using MaxCyte STX apparatus (MaxCyte, MD, USA). After the 4 days of culture the culture medium was harvested by centrifugation at 300xg, for 12 min and then frozen at -20°C until purification.
  • the column was washed with 20 ml of the Tris-buffer and a His-tagged protein was eluted by a step gradient of 0.5 M imidazole.
  • the fractions containing the target protein were pooled and further purified on the Strep-Tactin® media (IB A GmbH, Germany) as described by the manufacturer, using 10 mM desthiobiotin as an elution reagent.
  • Purified proteins were concentrated by ultrafiltration and buffer-exchanged into PBS on a 5 ml HiTrap Desalting column (GE Healthcare). The concentration was determined from UV absorbance at 280 run. The proteins were sterile-filtered and stored at -20°C.
  • Extracellular part of the human ACE2 protein fused to human IgG Fc fragment, preceded by a HRV3C protease cleavage site was purified by His-Trap and Strep-Tactin® media as above.
  • the Fc fragment was cleaved off by an overnight incubation with HRV3C protease at +6 °C.
  • ACE2 was afterwards polished by size-exclusion chromatography on a Superdex 75 16/60 column (GE Healthcare), concentrated by ultrafiltration with 10 kDa MW cut off filter device, sterile-filtered and stored at +6°C.
  • EXAMPLE 2 PREPARATION OF HYBRIDOMA CELL LINES PRODUCING
  • mice Six-week-old Balb/c mice were primed subcutaneously either with 30 ⁇ g of recombinant SARS-Cov-2 Spike protein (S) or Spike-RBD (RBD), prepared as described in Example 1, in the complete Freund’s adjuvant (SIGMA- ALDRICH) and boosted three times at three-week intervals with 20 ⁇ g of the same antigen in the incomplete Freund's adjuvant.
  • SIGMA- ALDRICH complete Freund’s adjuvant
  • mice were injected intravenously with 20 ⁇ g of the same antigens in PBS.
  • Spleen cells from immunized mice were fused with NS/0 myeloma cells according to the method of Kontsekova et al. (1988).
  • Splenocytes were mixed with NS/0 myeloma cells (ratio 5: 1) and fused for 1 minute in 1 ml of 50% polyethylene glycol (PEG) 1550 (Serva) in serum free Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% dimethyl sulfoxide (DMSO).
  • PEG polyethylene glycol
  • DMEM serum free Dulbecco's modified Eagle's medium
  • DMSO dimethyl sulfoxide
  • the fused cells were resuspended in DMEM containing 20% horse serum, L-glutamine (2 mM), hypoxanthine (0.1 mM), aminopterin (0.04 mM), thymidine (0.016 mM), and gentamycin (40 U/ml), at a density of 2.5 x 10 5 spleen cells per well in 96- well plates.
  • the cells were incubated for 10 days at 37°C and growing hybridomas were screened for the production of monoclonal antibodies specific to the S protein and RBD by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Microtiter plates were coated overnight with the particular protein (2 ⁇ g/ml, 50 pl/well) at 37°C in PBS. After blocking with with PBS-0.1% Tween 20 to reduce nonspecific binding, the plates were washed with PBS-0.05% Tween 20 and incubated with 50 pl/well of hybridoma culture supernatant for 1 hr at 37°C.
  • Bound monoclonal antibodies were detected with sheep anti-mouse immunoglobulin (Ig) conjugated with horse radish peroxidase (HRP, DakoCytomation, Denmark).
  • the reaction was developed with TMB one (Kementec Solutions A/S, Demark) as a peroxidase substrate and stopped with 50 pl of 0.25 M H2SO4.
  • Absorbance at 450 NM was measured using a Powerwave HT (Bio-Tek). Readouts with an absorbance value of at least twice the value of the negative controls (PBS) were considered positive.
  • Positive hybridoma cultures were further subcloned in soft agar according to the procedure described in Kontsekova et al. (1991). After subcloning of hybridoma, the antibodies isotypes were determined by ELISA using a mouse Ig isotyping kit (ISO-2, SIGMA).
  • 10 000 RU (response units) of polyclonal anti-mouse antibody (No. Z 0420; DakoCytomation, Denmark) was coupled at pH 5.0 via primary amines simultaneously in four flow cells. One of them was used as a reference cell in kinetic measurements. In each analysis cycle, three individually diluted hybridoma supernatants were captured in three analytical flow cells for 1 min, to reach an immobilization level of 100-600 RU.
  • 100 nM RBD protein purified as described above in Example 1
  • PBS-P running buffer
  • Figure 1 shows the results of the immunoreactivity of hybridoma culture supernatants to Spike protein of SARS-CoV-2 in ELISA. All selected hybridoma clones produced high levels of antibodies with binding activity to the S protein. Moreover, most tested antibodies recognized also RBD of the S protein, except for the antibody produced by the hybridoma clone number 3. The binding site of this antibody lies outside of the S protein RBD and would not inhibit binding of SARS-CoV-2 to the target cell, consistent with previous findings (Zeng, F. et al. 2006; Premkumar, L. et al., 2020; Robbiani, D. F. et al. , 2020).
  • the epitopes of the selected antibodies are localized in RBD of the S protein, which is essential for the interaction of the SARS-CoV-2 virus with its receptor on permissive human cells, the ACE2 protein. Therefore, it is very likely that antibodies targeting the S protein in RBD can interfere with its binding to the receptor ACE2, and thus may block the virus entry into the host cell.
  • the epitope or epitopes recognized by the antibody or binding fragment thereof of the disclosure may have a number of uses.
  • the epitope in purified or synthetic form can be used to raise immune responses (i.e., as a vaccine, or for the production of antibodies for other uses) or for screening sera for antibodies that immunoreact with the epitope.
  • an epitope recognized by the antibody or binding fragment thereof of the disclosure, or an antigen having such an epitope may be used as a vaccine for raising an immune response.
  • the antibodies and binding fragments of the disclosure can be used to monitor the quality of vaccines, for example, by determining whether the antigen in a vaccine contains the correct immunogenic epitope in the correct conformation.
  • Figure 2 shows the results of the immunoreactivity of hybridomas producing antibodies specific to the RBD protein of SARS-CoV-2 Spike in ELISA. All selected hybridoma clones produced high levels of monoclonal antibodies specific to RBD domain and most of them recognized also the recombinant S protein of SARS-CoV-2. Antibodies with weak binding to S protein probably will have no capability to inhibit virus sufficiently effectively.
  • EXAMPLE 3 ANALYSIS OF the INHIBITION ACTIVITY OF
  • ACE2 recombinant protein at 350 ng per well in 50 pl of phosphate-buffered saline (PBS) overnight at 4°C, followed by blocking with PBS supplemented with 0.1% Tween-20 (PBS-T).
  • HRP horseradish peroxidase
  • HRB horseradish peroxidase
  • a colorimetric signal was developed by the enzymatic reaction of HRP with a chromogenic substrate TMB one (Kementec Solutions A/S, Denmark). An equal volume of stop solution (0.25 M H2SO4) was added to stop the reaction, and the absorbance of the product at 450 NM was measured using a PowerWave HT microplate reader (BioTek, USA). Inhibition activity of sample was determined as follows:
  • Cell-based S-ACE2 interaction inhibition assay utilizes the HEK 293T/17 cells stably expressing human ACE2 protein (HEK 293T/17-hACE2).
  • the cells were seeded at 60-70% plating density in 48-well plate and cultivated O/N at 37°C, 5% CO2 in a humidified incubator in DMEM supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin/streptomycin (all from Life Technologies Invitrogen, Carlsbad, CA, USA) and 100 ⁇ g/ml hygromycin (ThermoFisher Scientific).
  • Recombinant Spike protein was labelled with Alexa FluorTM546 (ThermoFisher Scientific) according to the manufacturer’s recommendations.
  • Labelled S protein (40 ng/ml) was preincubated with tested hybridoma culture supernatants producing MAbs (diluted 1:50 in DMEM) for 30 min at 37°C. Then the preincubation mixtures were added to HEK 293T/17-hACE2 cells and incubated for 2 hrs at 37 °C, 5% COz in a humidified incubator. Subsequently cells were gently re-suspended in 0.5 ml PBS, transferred into flow cytometry tubes and immediately evaluated for S protein internalization by flow cytometry.
  • Results from the cell-based S-ACE2 interaction inhibition assay are shown in Figure 4. Interaction between S protein and ACE2 overexpressed on HEK 293T/17 cells was blocked by 12 antibodies (MAb 12, 96, 290, 266, 352, 677, 68, 322, 97, 99, 462 and 175). The lowest inhibition activity (25%) was shown by the monoclonal antibody 677.
  • results obtained from the ELISA and cell-based inhibition assay demonstrate that the correlation between these assays is high.
  • the assays are suitable for the rapid screening and selection of antibodies with inhibition activity on in vitro level.
  • EXAMPLE 4 NEUTRALIZATION OF SARS-CoV-2 S-TYPED
  • S-PVPs S protein typed pseudoviral particles
  • S-PVPs were prepared in human embryonic kidney cells according to the modified protocol of Millet et al. (DOI: 10.3791/59010).
  • HEK-293T/17 cell lines ATCC, Cat. no.: CRL- 112678 constitutively expressing SARS-CoV-2 Spike protein was prepared.
  • Mammalian expression plasmid with codon-optimized gene for the entire SARS-CoV-2 S protein (Creative Biolabs, USA) under a CMV promoter was linearized and transfected into HEK-293T/17 cells.
  • the cell lines stably expressing S protein were established under hygromycin selection pressure.
  • the expression profile of several established cell lines was determined by western blotting for S protein and the highest expressing cells (clones S/3; S/7) were used for generation of S-PVPs.
  • the cell lines S/3 and S/7 were transfected with gag-pol and luciferase encoding plasmids and S-PVPs were harvested after 72 hrs. After the transient transfection, aliquoted and stored at -80°C. Each batch of S-PVPs was characterized and specific infection dose of the S-PVPs was used for infection of recipient cells (HEK293/17) stably expressing ACE2 and TMPRSS2.
  • the neutralization assay was performed as follows: the serially diluted selected monoclonal antibodies were mixed with a defined amount of S-PVPs. The mix was incubated at 37°C in a CO2 incubator for 1 hr. Then the mix was added to the cells in a 96 well-plate (50 ⁇ l of mix per well) in triplicates. After 48 hrs of incubation of the recipient cells with the S-PVP mix the luciferase activity was measured using a luminometer (Fluoroskan Ascent® FL, Labsystems) and half maximal effective concentration was calculated for the measured monoclonal antibodies.
  • a luminometer Fluoroskan Ascent® FL, Labsystems
  • RLU relative luminescent units
  • Figure 5 documents the neutralization capacity of pseudoviral particles by monoclonal antibodies. The results have shown that antibodies block or inhibit the entry of the pseudovirus into the cells in a dose-depended manner. The neutralization effectivity of the tested antibodies differs from one another. The highest inhibition activity is shown by MAb68 and then group of four antibodies (MAb96, MAb290, MA266 and MAb352). [00237] Although the precise mechanism of inhibition needs further investigation, the results indicate that these antibodies target the critical epitopes on SARS-CoV-2 S protein that are essential for the infection of the recipient cells.
  • the plaque reduction neutralization test (PRNT) with the live SARS-CoV-2 virus (performed in a containment laboratory of Biosafety level 3 at the Department of Virus Ecology at the Biomedical Research Center of the Slovak Academy of Sciences, Slovak Republic) was used as a test for determination of neutralization capacity for the coronavirus of MAbs elicited by immunization with the Spike protein or its RBD. Serial dilutions of supernatants from the hybridoma clones producing the selected MAbs were incubated with 100 plaque-forming units of SARS-CoV-2 at 37°C for 2 hrs.
  • MAb-virus mixtures were then added to Vero E6 cell monolayer in 24- well plates and incubated at 37°C for additional 1 h. After incubation, cells were overlaid with 2% (w/v) carboxymethylcellulose in Eagle's minimal essential medium (EMEM) supplemented with 5% foetal calf serum (FCS). Plates were incubated at 37°C for 72 hrs. Then, the cells were fixed for 30 min with 4% formaldehyde in phosphate-buffered saline (PBS). After incubation the SARS-CoV-2 plaques were visualized by staining with 0.5% crystal violet at room temperature for 10 min. After washing the wells with water, the number of plaques was counted. The antibody neutralization activity was determined as the reciprocal of the highest dilution resulting in an infection reduction of >50% (PRNT50).
  • EMEM Eagle's minimal essential medium
  • FCS foetal calf serum
  • Figure 6 shows the neutralization activity of two isolates of SARS-CoV-2 infectious virus (BMC5 and BMC6, deposited in GISAID.org under the accession IDs EPI ISL 417879 and EPI ISL 417880, respectively) from Slovakia. Importantly, selected antibodies are able to neutralize both virus isolates in a dose-depended manner.
  • MAb96 Five of the MAbs (MAb96, MAb290, MAb266, MAb677 and MAb68) displayed strong neutralizing activity against SARS-CoV-2.
  • MAb68 displayed the most potent neutralizing activity against SARS-CoV-2, with a remarkable neutralization titer.
  • the nucleotide sequence of variable regions was determined by DNA sequencing of cDNA synthesized using total RNA extracted from the identified and isolated mouse hybridoma cell lines, which express the monoclonal antibodies. Total RNA was extracted using RNeasy Mini kit with RNAse-free DNase Set (Qiagen). Synthesis of the first strand cDNA was carried out using the High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific).
  • composition of the reagents for the 2x reverse transcription master-mix was as follows (quantities per 20 pL reaction): 2 pl of 10x RT buffer; 0.8 pl of 25x dNTP Mix (100 rnM); 2 pl of 10x RT Random Primers (50 pM); 1 pl of each 20x RT specific primers; 1 pl of MultiScribeTM Reverse Transcriptase (50 U/pl); 0.2 pl of nuclease-free H2O.
  • KC 5’-CAGGAAACAGCTATGACCACTGGATGGTGGGAAGATGG-3’
  • CArevl 5’-CAGGAAACAGCTATGACCGTAGATGGTGGGATTTCTCGC-3’
  • CArev2 5’-CAGGAAACAGCTATGACCACATCCAATTCTTGGACGGGG-3’ (SEQ ID NO: 20).
  • RNA sample 1 ⁇ g/10 pl
  • cDNA was synthesized under the following conditions: 10 min at 25°C, 120 min at 37°C, 5 min at 85°C, and final cooling to 4°C.
  • PCR polymerase chain reaction
  • the forward primers used for individual antibodies are in the Table 4.
  • the reverse primers for the light and heavy chains are in the Table 4.
  • CArevl 5’-CAGGAAACAGCTATGACCGTAGATGGTGGGATTTCTCGC-3’
  • [00259] were derived from kappa, IgGl and IgA chains constant regions, respectively.
  • EXAMPLE 6 IDENTIFICATION OF ADDITIONAL MONOCLONAL ANTIBODIES
  • Monoclonal antibodies targeting the viral protein S have enormous potential to prevent SARS-CoV-2 infection and treat patients with mild to moderate COVID-19 (Jiang et al., 2020).
  • Several antibodies targeting RBD of the Spike protein have already been authorised for emergency use, including REGN10933/REGN10987, LY-CoV555 and JS016/LyCoV016 (Yang et al, 2020).
  • SARS-CoV-2 variants B.1.351 and P.l were partially (REGN10933) or completely (LY-CoV555) resistant against antibodies used for COVID-19 treatment (Hoffmann et al., 2021). This is in concordance with a study showing that the interaction between the mutant RBD containing three mutations N417/K484/Y501 and LY-CoV555/Bamlanivimab was completely abolished (Liu et al, 2021).
  • CoV-2 or its RBD was further studied in terms of their ability to inhibit Spike- ACE2 interaction and neutralization of live SRS-CoV-2 virus.
  • the next several examples revealed that two mouse monoclonal antibodies (AX290, AX677) and their chimeric counterparts with non-overlapping epitopes demonstrated subnanomolar or nanomolar affinity to RBD of Spike carrying several mutations found in the naturally existing variants of SARS-CoV-2 that have appeared in the population.
  • Their excellent neutralization potency was confirmed in the plaque reduction neutralizing test against live viruses including the variants of concern B.l.1.7 and B.1.351.
  • AX96, AX290, AX677, AX266 and AX99 displayed high neutralization potency of the live SARS-CoV-2 virus in PRNT (FIG. 17F).
  • the neutralizing antibodies bind RBD with nanomolar or sub-nanomolar affinities.
  • Affinity measurements by SPR revealed that antibodies exhibited a large distribution of dissociation kinetic rates, spanning two orders of magnitude (1.3x10"* - 1.8x10 -2 s -1 ; FIG17H, I; FIG. 20). Association kinetics also varied, but to a lesser extent (4.6x10 5 - 1.7x10 6 M -1 s -1 ).
  • Four antibodies with the highest affinities (AX12, AX96, AX290 and AX677) have very slow dissociation rates below 10' 3 s' 1 .
  • EXAMPLE 7 EPITOPE BINNING IDENTIFIED TWO IMMUNODOMINANT
  • AX290 and AX677 showed the highest binding affinities to RBD of all members of the epitope group I and n, respectively, and showed the highest neutralizing activities against the live authentic virus.
  • Hydrogen-deuterium exchange coupled to mass spectrometry (HDX) was used to identify their binding sites on RBD. Deuterium uptake was monitored on 288 unique peptides, covering 89% of the RBD sequence (FIG. 23).
  • the regions protected by AX677 antibody include peptides 42-47, 42-48, 118-125, 118-128, 126-139, 126-135, 129-135, 129-133 and 129- 139 (RBD numbering) (FIG.
  • the region protected by the AX290 antibody includes the peptides 151-157, 156-170, 156-172, 158-170 and 171-179 (FIG. 21B), which correspond to peptides 467-473, 472-486, 472-488, 474-486 and 487- 495, respectively, of the S protein.
  • the peptides protected by AX677 antibody included amino acids 42-48 and 118-139 (RBD numbering), which correspond to amino acids 358-364 and 434-455, respectively, of the S protein.
  • the RBD peptides protected by the AX290 antibody encompassed amino acids 151-179, which correspond to peptides 467-495, respectively, of the S protein.
  • the published model of ACE2-bound RBD from PDB 6M0J was used to highlight the peptides identified in HDX experiments to show the approximate location of the epitopes of AX290 and AX677 (FIG. 21 C, D). They appear on the opposite parts of the RBD structure.
  • the epitope of AX290 is located in the region of RBD that comprises some of the contacts to ACE2, is often targeted by neutralizing Abs, (Barnes et al, 2020) and overlaps with epitopes of REGN10933 and LY-CoV555.(Hansen et al., 2020; Hoffmann, M et al., 2021b).
  • the epitope of AX677 is located outside of the ACE2-binding interface, which explains why it poorly inhibits ACE2-RBD interaction in the competition ELISA and cellular inhibition assays.
  • the deuterium uptake data suggest that the binding of AX677 partially overlaps with that of REGN10987 and COV2-2130. (Dong et al., 2021; Hansen et al., 2020)
  • the AX677 mode of neutralization might be allosteric or via interference with attachment to other cell surface molecules.
  • EXAMPLE 8 EFFECT OF MUTATIONS IN RBD OF SPIKE PROTEIN ON BINDING
  • AX290 and AX677 performed best overall in the in vitro and in vivo assays within members of their respective epitope groups. Since the SARS-CoV-2 appears to pick up mutations during the pandemic, this has resulted in compromised activities of some vaccines and therapeutic antibodies (Hoffmann et al., 2021; Liu et al, 2021). Since treatment with a single monoclonal antibody might further accelerate genetic modifications of the virus, a combination of two or more antibodies, preferentially with non-overlapping epitopes, is a better choice for therapy. The synergistic effect of combining AX290 and AX677 in a virus escape assay was evaluated (FIG. 26).
  • Antibodies were serially diluted and incubated, either alone or in combination, with live authentic SARS-CoV-2 virus (Slovakia/SK-BMC5/2020, wild type with D614G) at MOI of 0.5 and added to the VERO E6 cells. After 3-4 days, when the cytopathic effect of the escaping viruses became evident, culture supernatants from the first wells with cytopathic effect (CPE) were mixed with antibody dilutions and added to cells again for another round of mutant virus amplification to select for the escape viral mutants (FIG. 26A).
  • CPE cytopathic effect
  • EXAMPLE 9 BINDING AND NEUTRALIZATION CHARACTERISTICS OF
  • the heavy chain contains the full mouse heavy chain variable regions of AX290 (or AX677) (Table 3) fused to the IgG4 constant region UniProtKB/Swiss-Prot: P01861.1 with S228P substitution that stabilizes the antibody by preventing Fab-arm exchange (Angal et al. 1993).
  • the light chain contains the full mouse light chain variable region of AX290 (or AX677) (Table 3) fused to the human kappa chain constant region (UniProtKB/Swiss-Prot: P01834.2.
  • Both chimeric antibodies recognised all tested mutants with nanomolar or sub- nanomolar affinities, which guarantees their unmodified neutralizing capacity (FIG. 27C, FIG.28).
  • AX290ch maintained its picomolar affinity to all mutants except those containing the K417N mutation alone or in the combination N501Y/E484K/K417N (3-fold and 2-fold decrease in affinity, respectively).
  • AX677ch bound the RBD with N501Y mutation with 1.4-fold higher affinity than wild type RBD, reaching picomolar values (FIG. 27C, FIG.28).
  • Hybridoma technology was used to generate a pair of virus neutralizing antibodies
  • AX290 and AX677 which are suitable for COVID-19 therapy since they are oblivious to the recently emerged mutations in the RBD of the viral Spike protein, as shown by ELISA and SPR assays.
  • the antibodies neutralized live wild type SARS-CoV-2 (with D614G) and two fast- spreading variants of concern B.l.1.7 (South East of England) and B.1.351 (South Africa).
  • the combination of these antibodies exhibited a strong synergistic neutralizing effect confirmed in a PRNT assay with authentic SARS-CoV-2 virus, which showed that cytopathic (virus escape) effect of the live authentic SARS-CoV-2 virus appeared only at more than 600-fold lower concentration of the AX290+AX677 mixture than it appeared with each antibody alone.
  • the mutation T345N that appeared in the escape mutant with AX677 is a very rare mutation, only 1 T-N substitution was identified in the USA so far, and only 25 substitutions of T345 overall (GISAID database). The reason might be because this region of Spike RBD is not much targeted by the immune system, which preferentially selects neutralizing antibodies that interfere with the Spike-ACE2 binding. This makes the AX677 antibody a promising candidate for a long-term efficacy against SARS- CoV-2.
  • AX290ch and AX677ch that have distinct and non-overlapping epitopes on the RBD, will maintain the antibodies’ ability to neutralize current SARS-CoV-2 variants of concern and thus they can efficiently prevent viral mutational escape.
  • the AX677 chimera showed very consistent, slightly higher activities (approx. 2-fold) against all three VOCs compared to the original (WT) virus.
  • AX290 chimera showed exceptionally high activity against the variant B.1.351 , and slightly reduced (two- and three-fold) activities against WT and B.1.1.7. Its neutralization efficiency was reduced (approx.
  • Table 5 Summary table with characteristics of mAbs specific to Spike protein/RBD of SARS-CoV-2 with neutralization activity.
  • ELISA inhibition test (1:50); PRNT (1:25).
  • the mAb concentrations were adjusted to the same starting concentration (by diluting with clean cell culture medium) prior to the dilution of the culture supernatants for the experiments.
  • NT not tested; ND, value could not be determined due to low activity
  • EXAMPLE 10 MATERIALS AND METHODS FOR EXAMPLES 6 THROUGH 9
  • Dulbecco s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum (FBS), 2 mM L-glutamine (GIBCO), 5 ⁇ g/ml gentamicin (SIGMA) and 10O ⁇ g/ml hygromycin B (Invitrogen) at 37°C in 5%CO2.
  • FBS heat-inactivated fetal bovine serum
  • GOBCO 2 mM L-glutamine
  • SIGMA 5 ⁇ g/ml gentamicin
  • 10O ⁇ g/ml hygromycin B Invitrogen
  • ECACC Cat# 85020206, RRID:CVCL_0574 were cultured in Eagle's minimal essential medium (EMEM, Lonza) supplemented with 5% FBS (GIBCO), Penicillin-Streptomycin-
  • Amphotericin B Solution (10ml/1, Lonza).
  • NS0 mouse myeloma cells (ECACC Cat# 85110503,
  • RRID:CVCL_3940 are routinely used for hybridoma preparation. All cell lines were purchased from certified suppliers, upon delivery they were thawed, amplified and frozen in working aliquots according to the suggestion of the suppliers. Upon thawing they are verified for consistency in morphology. The cells have not yet been genetically authenticated
  • the prefusion-stabilized SARS-CoV-2 S protein ectodomain (residues 1-1208 from GenBank: MN908947, with proline substitutions at residues 986 and 987, a “GSAS” (SEQ ID NO: 21) substitution at the furin cleavage site residues 682-685, a C-terminal T4 fibritin trimerization motif, an HRV3C protease cleavage site, a TwinStrepTag and an SXHisTag (SEQ ID NO: 22)), the receptor binding domain (RBD) fragment of the S protein (amino acids 319-591), containing the His-tag and Twin-Strep-tag and modified ACE2 (angiotensin-converting enzyme 2), containing tags for purification were synthesized at BioCat Co.
  • VIRUSES Three cell culture isolates of SARS-CoV-2 were used in the in vitro characterization and validation of the anti-Spike antibodies. They were all isolated from clinical samples of COVID-19 patients in Slovakia. All three isolates were deposited in the European virus archive GLOBAL. The strain Slovakia/SK-BMC5/2020 (available at https://www.european-virus- archive.com/virus/sars-cov-2-strain-slovakiask-bmc52020) represents strains circulating in Europe in spring 2020 and carries the Spike D614G mutation (lineage B.l).
  • strain Slovakia/SK-BMC-P1A/2021 (available at httDs://www.european-virus-archive.com/virus/sars- cov-2-strain-slovakiask-bmc-pla2021 -bl 17-variant-voc-20201201) was isolated in January 2021 and belongs to the B.l.1.7 lineage (VOC 202012/01, Alpha).
  • strain Slovakia/SK-BMC- BA11/2021 (available at ht ://www.european-virus-archive.com/virus/sars-cov-2- in- slovakiask-bmc-bal 12021 -bl 351 -variant-aka-20h501w2-or-south-afri can-variant) was isolated in March 2021 and belongs to the B.1.351 lineage (20H/501 Y.V2, Beta).
  • the strain Slovakia/SK- BMC-B Al 5/2021 (available at https://www.european-virus-archive.com/virus/sars-cov-2-strain- slovakiask-bmc-bal 52021 -bl 6172-variant-aka-delta-voc-or-indian- variant) was isolated in June 2021 and belongs to the lineage B.l.617.2 (Delta).
  • the complete genome sequences of all four viruses were deposited in the GiSAID.org database under the accession IDs EPI ISL 417879, EPI_ISL_77965.1, EPI_ISL_1234458 and EPI_ISL_2657324, respectively.
  • the strain SARS- CoV-2/human/Czech Republic/951/2020 was used for infection of ACE2 humanized mice in the course of the in vivo testing of the antibodies.
  • the virus was isolated from a clinical sample at the National Institute of Public Health, Prague, Czech Republic, and passaged in Vero E6 cells five times before its use in this study. .
  • the synthetic genes coding for the Spike protein (S), receptor binding domain (RBD) or ACE-2 weree cloned under the CMV promoter in a pCMV-based mammalian expression vector immediately after a signal peptide accomplishing secretion of recombinant proteins from the cells into medium.
  • the plasmids were amplified in DH5a bacterial cells.
  • the cells carrying the plasmid were inoculated into 5 ml LB medium with kanamycin for 8 hrs, transferred into 250 ml cultures and incubated overnight with shaking at 37°C.
  • the plasmid DNA was isolated using PureLink® HiPure Plasmid Filter Maxiprep Kit (Invitrogen) with the last ethanol washes done in a sterile laminar-flow hood. After overnight incubation in sterile water at 4°C, DNA concentration was measured using NanoPhotometer and the DNA was aliquoted and stored at -20°C. Insertion of the desired point mutations in S-RBD was done using QuickChange II site directed mutagenesis kit (Agilent Technologies). Primers for mutagenesis were designed using The QuickChange® Primer Design Program provided by manufacturer. Prepared mutations, verified by DNA sequencing were transformed in DH5a bacterial cells for further amplifications.
  • TAGTTATAATCCGCAATATTGCCGGTCTGGCCCG SEQ ID NO: 30; T345N,
  • Proteins were produced in human cell line Expi293 (A14635, ThermoFisher Scientific, RRID:CVCL_D615). Plasmids that drive protein expression from the CMV promoter were either transfected with polyethyleneimine (PEI) or by electroporation into Expi293 cells that were in exponential growth phase in Expi293 expression medium (A14331-1, ThermoFisher Scientific). For PEI transfections, 3.75ml PEI (PEI MAX®, 24765-1, Polysciences Europe GmbH, Germany) and 1.25mg DNA was added to a suspension of 10 9 cells in a total volume of 50 ml, shaken at 37°C for 3 hrs and diluted into working concentrations.
  • PEI polyethyleneimine
  • the proteins were purified from cell media using Akta FPLC (GE Healthcare) as follows.
  • the culture medium was clarified by centrifugation at 20,000xg, NaCl was added to a final concentration of 0.5 M and the solution was filtered through a 0.2 ⁇ m membrane filter.
  • a 5 ml His-Trap affinity column (#17524802, Cytiva) charged with nickel ions was equilibrated in a Tris buffer, pH 7.4, 0.5 M NaCl and the culture medium was loaded onto the column. Subsequently, the column was washed with 20 ml of the Tris-buffer and His-tagged protein was eluted by 0.5 M imidazole in Tris buffer.
  • the fractions containing the target protein were pooled and further purified on the Strep-Tactin® media (#2-1206-025, IB A GmbH, Germany) as described by the manufacturer, using 10 rnM desthiobiotin (#2-1000-005, IB A GmbH, Germany) as an elution reagent.
  • Purified proteins were concentrated by ultrafiltration and buffer-exchanged into PBS on a 5 ml HiTrap Desalting column (GE Healthcare). The concentration was determined from UV absorbance at 280 nm. The proteins were sterile-filtered and stored at -20°C.
  • Extracellular part of the human ACE2 protein fused to human IgG Fc fragment, preceded by a HRV3C protease cleavage site was purified by His-Trap and Strep-Tactin® media as above. The Fc fragment was cleaved out by an overnight incubation with HRV3C protease at +6°C.
  • ACE2 was afterwards polished by size-exclusion chromatography on a Superdex 75 16/60 column (GE Healthcare), concentrated by ultrafiltration with a Amicon Ultra centrifugal filter of 10 kDa MW cut off (#UFC901096, Millipore), sterile-filtered and stored at +6°C. [00295] PREPARATION OF HYBRIDOMA CELL LINES PRODUCING
  • mice Six-week-old Balb/c AnNCrl mice (RRID:IMSR_CRL:028, Charles River Laboratories, delivered by Velaz s.r.o., , Czech Republic) were primed subcutaneously either with 30 ⁇ g of recombinant SARS-Cov-2 Spike protein (S) or Spike-RBD (RBD) in complete Freund’s adjuvant (#F 5881, SIGMA- ALDRICH) and boosted three times at three-week intervals with 20 ⁇ g of the same antigen in incomplete Freund's adjuvant Three days before the spleen extraction, mice were injected intraperitoneally with 20 ⁇ g of the same antigens in PBS.
  • S SARS-Cov-2 Spike protein
  • RBD Spike-RBD
  • complete Freund’s adjuvant #F 5881, SIGMA- ALDRICH
  • Spleen cells from immunized mice were fused with NS/0 myeloma cells according to the method of Kontsekova et al. (1988).
  • Splenocytes were mixed with NS/0 myeloma cells (ratio 5:1, ECACC Cat# 85110503, RRID:CVCL_3940) and fused for 1 minute in 1 ml of 50% polyethylene glycol (PEG) 1550 (Serva) in serum free Dulbecco’s modified Eagle’s medium (DMEM , 11960-044, ThermoFisher Scientific) supplemented with 10% dimethyl sulfoxide (DMSO).
  • PEG polyethylene glycol
  • DMEM serum free Dulbecco’s modified Eagle’s medium
  • DMSO dimethyl sulfoxide
  • the fused cells were resuspended in DMEM containing 20% horse serum, L-glutamine (2 mM), hypoxanthine (0.1 mM), aminopterin (0.04 mM), thymidine (0.016 mM), and gentamycin (40 U/ml) HAT media supplement Hybri-MaxTM and gentamycin (40 U/ml) (both from Sigma- Aldrich), at a density of 2.5 x 10 5 spleen cells per well in 96-well plates and incubated for 10 days at 37°C.
  • the growing hybridomas were screened for the production of monoclonal antibodies specific to the S protein and RBD by an enzyme-linked immunosorbent assay (ELISA).
  • ELISA enzyme-linked immunosorbent assay
  • Microtiter plates were coated overnight with one of the proteins (2 ⁇ g/ml, 50 pl/well) at 37°C in PBS. After blocking with PBS-0.1% Tween 20 to reduce nonspecific binding, the plates were washed with PBS-0.05% Tween 20 and incubated with 50 pl/well of hybridoma culture supernatant for 1 hr at 37°C.
  • Bound monoclonal antibodies were detected with goat anti-mouse immunoglobulin (Ig) conjugated with horse radish peroxidase (HRP, P0447, DakoCytomation, Denmark).
  • the reaction was developed with TMB one (4380A, Kementec Solutions A/S, Demark) as a peroxidase substrate and stopped with 50 pl of 0.25 M H2SO4.
  • Absorbance at 450 nm was measured using a Powerwave HT (Bio-Tek). Readouts with an absorbance value of at least twice the value of the negative controls (PBS) were considered positive.
  • ELISA positive hybridoma cultures were further subcloned in soft agar according to the procedure described in Kontsekova et al. (1991). After purified hybridoma clones were obtained, the antibodies isotypes were determined by ELISA using a mouse Ig isotyping kit (ISO-2, SIGMA).
  • Fab fragment of anti-mouse IgG (0.5 ⁇ g/ml, 50 pl/well in PBS) was immobilized overnight on microtiter ELISA plate at 37°C. After blocking with PBS-0.1% Tween 20 to reduce nonspecific binding, the plates were washed with PBS-0.05% Tween 20 and incubated with 50 pl/well of mAb 1 (0.6 ⁇ g/ml) for lhr at 37°C.
  • HRP horseradish peroxidase
  • HRP horseradish peroxidase
  • HEK 293T/17 cells ATCC Cat# ACS-4500, RRID:CVCL_4V93 stably expressing human ACE2 protein (HEK 293T/17-hACE2) were seeded at 60-70% plating density in 48-well plates and cultivated O/N at 37°C, 5% CO2 in a humidified incubator in DMEM supplemented with 10% (v/v) fetal calf serum, 2 mM L-glutamine, 100 units/mL penicillin/streptomycin (all from Life Technologies Invitrogen, Carlsbad, CA, USA) and 100 ⁇ g/ml hygromycin (ThermoFisher Scientific).
  • Recombinant Spike protein was labelled with Alexa FluorTM546 (ThermoFisher Scientific) according to the manufacturer’s recommendations.
  • Labelled S protein 40 ng/ml was preincubated with tested hybridoma culture supernatants producing mAbs (diluted 1:50 in DMEM) for 30min at 37°C. Then the preincubation mixtures were added to HEK 293T/17-hACE2 cells and incubated for 2 hrs at 37 °C, 5% CO2 in a humidified incubator.
  • PVPs concentration of the material was > 1x107 (determined from a TEM scan by the manufacturer). PVPs were aliquoted and stored at -80°C. Optimal dilution of the original PVPs stock for testing of the antibody neutralization efficiency was set in the range from 1:100 to 1:200, since this dose results in 50% of recipient cells infected with S-PVPs.
  • the neutralization assay was performed as follows: the serially diluted selected monoclonal antibodies were mixed in DMEM with a defined amount of S-PVPs.
  • the mix was incubated at 37°C in a CO2 incubator for 1 hr, then added to the cells in a 96 well-plate (50 pl of mix per well) in triplicates. After 48 hrs of incubation of the recipient cells with the S-PVP mix the luciferase activity was measured using a himinometer (Fluoroskan Ascent® FL, Labsystems) and half maximal effective concentration was calculated for the measured monoclonal antibodies. The measurement of relative luminescent units (RLU) and comparison to the positive control (a control antibody not recognizing S protein) and a negative control (cell extracts with no S-PVPs or extracts from the cells infected with S-deficient PVPs) was performed in all experiments.
  • RLU relative luminescent units
  • SARS-COV-2 PLAQUE RBDUCTION NEUTRALIZATION ASSAY [00310] The plaque reduction neutralization test (PRNT) with the live SARS-CoV-2 virus (performed in a containment laboratory of Biosafety level 3 by the Department of Virus Ecology at the Biomedical Research Center of the Slovak Academy of Sciences, Slovak Republic) was used as a test for determination of neutralization capacity for the coronavirus of mAbs elicited by immunization with the Spike protein or its RBD. Serial dilutions of supernatants from the hybridoma clones producing the selected mAbs were incubated with 100 plaque-forming units of SARS-CoV-2 at 37°C for 2 hrs.
  • PRNT plaque reduction neutralization test
  • MAb-virus mixtures were then added to Vero E6 cell (ECACC Cat# 85020206, RRID:CVCL_0574) monolayer in 24-well plates and incubated at 37°C for additional 1 h. After incubation, cells were overlaid with 2% (w/v) carboxymethylcellulose (9004- 32-4, Sigma Aldrich) in Eagle's minimal essential medium (EMEM) or (DMEM Low Glucose w/ Stable Glutamine w/ Sodium Pyruvate; LM-D 1102/500 ; Biosera) supplemented with 5% FBS(SUPERIOR Supplemented FBS, EU approved, S0615-500ML, Sigma Aldrich). Plates were incubated at 37°C for 72 hrs.
  • EMEM Eagle's minimal essential medium
  • DMEM Low Glucose w/ Stable Glutamine w/ Sodium Pyruvate LM-D 1102/500 ; Biosera
  • the mixed viral stocks were then further diluted in DMEM and used for infection of Vero E6 cells with the MOI of 0-001 (100 PFU per 100,000 cells).
  • the experiment was performed in 24-well plates in triplicates.
  • the virus inoculum of 100 ⁇ l was incubated with the cells (100,000 cells per well) for 60 minutes at 37°C.
  • the innoculum was then removed and ImL/well of fresh cultivation medium was added to the cells. After 3 days of incubation at 37°C, the cell culture supernatants were harvested. From each replicate, 140 ul of the supernatant was used for RNA extraction with the Viral RNA Minikit (52904, QIAGEN) according to manufacturer’s instructions.
  • Obtained RNA was then subjected to the nanopore sequencing analysis as described below. Changes in the proportion of the mutant virus before the infection and after three days of cultivation in the absence of the monoclonal antibodies were considered as a read-out indicator of improved or reduced viral fitness of the mutant viruses in comparison to the parental virus.
  • the sequencing libraries were constructed essentially as described in the Eco PCR tiling of SARS-CoV-2 virus with native barcoding protocol (Oxford Nanopore Technologies), except the amplicons spanning the SARS-CoV-2 genome sequence were generated using the ⁇ 2.5- kbp primer panel (J. S. Eden, E. Sim, SARS-CoV-2 Genome Sequencing Using Long Pooled Amplicons on Illumina Platforms, https://www.protocols.io/view/sars-cov-2-genome-sequencing- using-long-pooled-amp-befyjbpw (2020)) in which the rightmost primer pair was replaced by corresponding pair from the ⁇ 2.0-kbp panel (P.C.
  • RNA 8 ⁇ l was converted into cDNA using a LunaScript RT SuperMix Kit (New England Biolabs) and used as a template in two separate amplification reactions generating odd- and even-numbered tiled amplicons.
  • the PCR was performed using a Q5 Hot Start High-Fidelity 2X Master Mix (New England Biolabs) and the cycling conditions were: 30 sec at 98°C (initial denaturation), followed by 30 cycles of 15 sec at 98oC (denaturation) and 5 min at 65°C (combined annealing and polymerization).
  • the overlapping amplicons were pooled and purified using 0.5 volume of AMPure XP magnetic beads (Beckman Coulter).
  • the ends of amplicons were treated with a NEBNext Ultra II End repair / dA-tailing Module (E7442L, New England Biolabs) and barcoded using an EXP-NBD196 kit (Oxford Nanopore Technologies) and a Blunt/TA Ligation Master Mix (New England Biolabs). Barcoded samples (96) were pooled and purified using 0.5 volume of AMPure XP magnetic beads.
  • the AMU sequencing adapter (EXP-AMH001, Oxford Nanopore Technologies) was ligated to about 300 ng of barcoded pools using Quick T4 DNA ligase (New England Biolabs) and the library was purified using 0.5 volume of AMPure XP magnetic beads.
  • Variants were called using the ARTIC pipeline (https://artic.network/ncov-2019/ncov2019-bioinformatics-sop.html), which internally filters reads based on quality and length, aligns them to the reference (Wuhan-Hu- 1 isolate, NC_045512) using minimap2 (https://github.com/lh3/minimap2), trims primers, and calls variants using Nanopolish (https://github.com/jts/nanopolish). All variants reported in Table SI were determined based on read coverage of at least 380. All new nonsynonymous mutations were supported by at least 90% of the analyzed reads overlapping a particular site. Note that due to higher error rates of nanopore sequencing, even fixed variants typically do not achieve 100% support. [00318] IMMUNOCYTOCHEMISTRY.
  • HEK293T/17, hACE2 cells were plated on cover glass pre-coated with rat-tail collagen, type I (Sigma-Aldrich, St. Louis, Missouri, United States) and cultivated for 24 h in DMEM with 10% FCS. Cells were incubated with S protein alone (labelled with Alexa546) or with S-antibody complexes (antibodies AX290 and AX68), washed with PBS and fixed with 4% PFA-PHEM, pH 6.9 (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCh, PFA) for 12 min.
  • S protein alone labelled with Alexa546
  • S-antibody complexes antibodies AX290 and AX68
  • cells were fixed with 4% paraformaldehyde and labelled with anti-human ACE2 antibody at 10 ⁇ g/ml (cat # 600-401-X59; Rockland Immunochemicals), followed by goat anti-rabbit IgG secondary antibody at 1/500 dilution (Alexa Fluor 488; #A-11008; Invitrogen) (EXAMPLES 11 onwards).
  • the samples were mounted in FluoroshieldTM medium with DAPI (Sigma- Aldrich). Images were captured by LSM 710 confocal microscope (Zeiss, Jena, Germany).
  • the deuterated mixture was then digested online by using the same nepenthesin-2 column and the resultant peptides were desalted with flow through on a trap column (Phenomenex UHPLC fully Porous Polar Cl 8, 2.1mm, Torrance, CA, USA) with 0.4% formic acid in water.
  • the eluted peptides were further separated on an analytical column (Lima® Omega 1.6 pm Polar C18 100 A, 100 x 1.0 mm, Torrance, CA, USA), by using a gradient of acetonitrile and water with 0.1% formic acid.
  • Peptides were introduced to a timsTOF (Bruker, Bmo, Czech Republic) mass spectrometer for mass analysis.
  • AAV humanized mice were moved to BSL3 animal housing facility on day eight after transduction with AAV -hACE2 and after the necessary acclimatization (three days) the treatment study was started. The mice were randomly assigned to cages and the cages were then randomized into groups.
  • AX290 and AX677 antibodies, their mixture and an isotype antibody control (DC8E8 antibody against pathological neuronal protein tau (Kontsekova E., 2014)) were administered subcutaneously (high-dose experiment; 1 -25 mg/0-2 ml PBS, 10 animals per group) or intraperitoneally (low-dose experiment; 0-5 mg/0-2 ml PBS, 10 animals per group) twelve days after applying the AAV-hACE2 virus.
  • mice 24 hrs later the mice were infected intranasally with SARS-CoV-2 (1 x 10 4 plaque- forming units, strain SARS-CoV-2/human/Czech Republic/951/2020, isolated from a clinical sample at the National Institute of Health, Prague, Czech Republic, and passaged in Vero E6 cells) in a total volume of 50 pl DMEM under isoflurane anesthesia as described previously. (De Gasparo, K, 2021). This day was marked as the Day zero and used as a reference day for all analyses of the treatment efficacy. Mice were monitored for health status and weighed daily for the remainder of the experiment.
  • mice On the day three and seven post-infection selected groups of mice (five animals per group per collection day) were sacrificed and lung tissues collected for analyses. One lobe was homogenized in sterile PBS using 30 Hz sonication for 3 min, centrifuged at 10,000 xg for 10 min at 4°C, and resulting suspension used for viral plaque titration analysis (day three post infection) and for viral RNA analysis (days three and seven post infection; high-dose experiment only). The other lung lobe was fixed in 4% paraformaldehyde in PBS and stored in a fridge at 2-8°C till further histopathological processing (high-dose experiment only).
  • Plaque assays were performed in Vero E6 cells (ATCC CRL-1586; mycoplasma- free) grown at 37 °C and 5% CO2 in DMEM supplemented with 10% FBS, and 100 U/ml penicillin, 100 ⁇ g/ml streptomycin, and 1% L-glutamine using a modified version of a previously published protocol. (De Madrid, AT, 1969). In brief, serial dilutions of virus were prepared in 24- well tissue culture plates and cells were added to each well (0-6 x 10 5 — 1 -5 x 10 5 cells per well).
  • RNA was eluted in 50 pl of the elution buffer. Viral RNA was quantified using EliGene COVID19 Basic a RT (#90077-RT-A; Elisabeth Pharmacon) according to the manufacturer’s instructions. A calibration curve was constructed from four dilutions of a sample quantified using Quanty COVID-19 kit (#RT-25; Clonit) according to the recommendations from the manufacturer. All real-time PCR reactions were performed using a LightCycler 480 (Roche).
  • the fixed lung tissues (collected from the high-dose experiment) were processed on Leica ASP6025 automatic vacuum tissue processor and embedded in Leica EG1150 H+C embedding station. For sectioning, Leica RM2255 rotary microtome (2 ⁇ m sections) was used. Samples were stained with Leica ST5020 + CV5030 Stainer and coverslipper. Hematoxylin/eosin staining was used as a standard descriptive histopathological staining according to an internal SOP. To assess the presence of macrophages a rabbit anti-mouse F4/80 monoclonal antibody (Catno.70076, Cell Signaling Technology) was used as primary antibody. This is a well- established marker for mouse macrophages.
  • the lung sections were deparafinized in Multistainer Leica ST5020 (Leica Biosystems). Antigens were retrieved by heating the slides in a citrate buffer pH 6 (Zytomed - Systems, Germany). Endogenous peroxidase was neutralised with 3% H2O2. Sections were incubated for 1 hour at RT with a 1:800 dilution of the primary antibody. After washing they were incubated with anti-rabbit secondary antibody conjugated with HRP (Zytomed - Systems, Germany). Staining of the sections was developed with a diaminobenzidine substrate kit (DAKO - Agilent) and sections were counterstained with Harris Hematoxylin (Sigma Aldrich - Merck) in Multistainer Leica.
  • DAKO - Agilent diaminobenzidine substrate kit
  • EXAMPLE 12 COMBINATION OF ANTIBODIES WITH NON-
  • the S477R substitution that allowed escape of the virus from AX290 is located in the region of RBD that was identified by HDX as the contact site of the antibody (FIG. 30). It was found in 0.048% of the sequenced SARS-CoV-2 isolates so far (https://www.gisaid.org/hcovl9- mutation-dashboard/, database version as of Aug 15, 2021, 2701750 sequences analysed). The alternative substitution S477N that is more widespread in circulating viruses (2.396%) was not found in the sequenced viral genomes.
  • the mutation T345N present in the escape virus mutant against AX677 is positioned close to peptides protected by the antibody, but not directly within their sequence (FIG. 30). It is a very rare mutation, only one T-N substitution at the position 345 of Spike has been found in circulating viruses, and only 82 substitutions of T345 were detected in the GISAID database until now. It is possible that this region of Spike RBD is less targeted by human immune repertoire resulting in a low selection pressure, or mutations there compromise the viral fitness. The latter is supported by data from deep mutational scanning of the Spike protein, which showed that amino acid exchange T-N at the position 345 reduced expression levels of the protein. (Starr TN, 2020).
  • the AX290 escape virus (Mutant Al , containing the S477R mutation in the Spike protein) showed only slight decline in viral fitness indicated by slight reduction in the proportion of the mutant in the mixture after the cultivation. A similar drop in the fitness was observed for the virus passaged in the presence of both AX677 and AX290 (FIG. 31, Mutant C25). These two escape viruses (Al and C25) share the insertion 13IACLV (SEQ ID NO: 35) in the Envelope protein, which might account for the observed reduction in the viral fitness.
  • the T345N escape mutation has also been identified previously using an S-typed VSV pseudovirus for antibody 2H04, which also did not compete with ACE2 for binding to the S protein. (Liu Z, et al., 2021).
  • AX290 and AX677 mAbs were tested in an ACE2 humanized mouse model that expresses human ACE2 (hACE2) in the cells of the upper and lower respiratory tract following inhalation of a modified adeno-associated virus (AAV-hACE2). De Gasparo R et al., 2021). These ACE2 humanized mice, upon infection with SARS-CoV-2, suffer from respiratory pathology and disease, progressive weight loss and require culling on day 8 post infection.
  • hACE2 human ACE2
  • AAV-hACE2 modified adeno-associated virus
  • mice were injected subcutaneously with 1 -25 mg of either antibody alone (approx. 50 mg/kg), their equimolar combination, or an isotype control antibody, 24 hours before infection with SARS-CoV-2. After intranasal inoculation of 10 4 pfu of SARS- CoV-2, mice were weighed daily, and terminal lung tissue sampling was done three and seven days post infection (dpi) to assess viral load, viral RNA and tissue histopathology (FIG. 32A).
  • dpi terminal lung tissue sampling was done three and seven days post infection (dpi) to assess viral load, viral RNA and tissue histopathology (FIG. 32A).
  • mice treated with the isotype control antibody experienced deterioration of their overall health status reflected in weight loss starting at day three post-infection (dpi) (FIG. 32B). By seven dpi, most mice treated with the isotype control had lost approximately 25-30% of their body weight. In contrast, prophylactic administration of AX290 and AX677, either individually or in a combination, neutralized the effect of the virus and fully prevented the weight loss in all mice, which appeared indistinguishable from the control non-infected mice (FIG. 32B).
  • the same prophylactic effect could also be seen and quantified using histopathology assessment of lung tissues.
  • the lung tissues of ACE2 humanized mice infected with SARS-CoV-2 showed pathological changes typical for inflammation characterised by the presence of inflammatory infiltrate, mostly represented by activated macrophages, which filled and structurally damaged alveoli, consistent with what was previously reported. (De Gasparo, R et al., 2021) (see also, FIG. 33.
  • the lung alveoli were, beside macrophages, also accompanied by lymphocytes, neutrophils and fibroblasts, from which one could assume significant cytokine production and proinflammatory action leading to lung fibrosis.
  • lungs from the control animals exhibited virus titer of 5 -5-6- 3 logio pfu/g (FIG. 34A). None of the SARS-CoV-2-infected animals treated with AX677, AX290, or a combination AX677+AX290 showed any weight loss during the whole experiment, while SARS-CoV-2-infected mice treated with the isotype control showed a weight loss of about 20-25% by day seven post-infection (FIG. 34B).
  • Monoclonal antibodies targeting the viral protein S have enormous potential to prevent SARS-CoV-2 infection and treat patients with mild to moderate COVID-19.
  • Several antibodies targeting RBD of the Spike protein have already been authorised for emergency use, including REGN10933/REGN10987, LY-CoV555 and JS016/LyCoV016 (Yang et al, 2020) and VIR-7831 with others in the pipeline (TY027, CT-P59, BRII-196 and BRH-198, SCTA01 etc.).
  • SARS-CoV-2 variants B.1.351 and P.1 were partially or completely resistant against antibodies used for COVID-19 treatment (Hoffmann, M et al., 2021b). This is in concordance with a study showing that the interaction between the mutant RBD containing three mutations N417/K484/Y501 and LY-CoV555/Bamlanivimab was completely abolished (Liu, H. et al., 2021a). To avoid this therapeutic limitation, the inventors screened and selected the antibodies showing neutralization activity against several variants of SARS-CoV-2.
  • Binding of AX677ch was not affected at all, while binding of AX290ch was reduced. The data indicate that AX677ch could retain unaltered potency, however the neutralizing activities of the antibodies may be verified with the authentic Omicron SARS-CoV-2 virus in the PRNT assay.
  • the inventors’ authentic virus approach identified antibody-specific escape mutations in RBD of the Spike protein, S477R and T345N, that directly affect the recognition by the antibodies. It also allowed one to evaluate the cost the mutations pose on the fitness of the authentic virus.
  • the inventors found that the AX677 escape virus carrying the T345N mutation in Spike had greatly reduced fitness in comparison with the parental virus under antibody-free conditions (FIG. 31). This observation is supported by an extremely rare occurrence of mutations altering T345 in SARS-CoV-2 isolates and indicates an advantage of using AX677 for prophylaxis and treatment.
  • the use of the authentic SARS-CoV-2 virus it also allowed to identify mutations in other regions of the viral genome, the envelope (E) protein, Orf3a and Orfla, that might help the SARS-CoV-2 virus to adapt to and escape from the selection pressure imposed by the Spike protein-binding antibodies.
  • the AX290 selection pressure led to an insertion of three amino acids in the transmembrane region of the viroporin E protein, 13IV to 13IACLV (SEQ ID NO: 35). Remarkably, exactly the same insertion was also found in the virus isolated from the cells ‘protected’ by the cocktail of AX290+AX677, but not in the virus that escaped the KXfiTlI antibody.
  • the AX677 virus contained different mutation in the E protein, the V5F substitution.
  • Both E mutations are in the N-terminal part of the protein that is predicted to be on (V5F) or close to (13IACLV (SEQ ID NO: 35)) the surface of the virus and the luminal side of endoplasmic reticulum (ER)-Golgi intermediate compartment (Mandala et al., 2020).
  • 13IACLV SEQ ID NO: 35
  • the inventors found that the insertion 13IACLV (SEQ ID NO: 35) slightly reduced the fitness of the virus.
  • 13IACLV SEQ ID NO: 35
  • several large in-frame deletions were identified in the E protein in various SARS-CoV-2 viral lineages in India (Kumar et al., 2021). However, all were located in its C-terminal half oriented on the cytoplasmic side of the ER membrane.
  • the escape mutant virus from AX290 contained another mutation, deletion of two amino acids 13VTL to 13V in orf3a, another ion channel protein encoded by the SARS-CoV-2 virus (Barrantes, 2021).
  • the E and orf3a ion channels can potentially transport cations from the ER and thus activate the inflammasome and downregulate the type-I interferon, but mostly are implicated in the later stages of the virus cycle - assembly and release (Barrantes, 2021 ; Mandala et al., 2020).
  • the combination of the two antibodies exhibits a strong synergistic neutralizing effect against the authentic live SARS-CoV-2 virus observable at >600-fold lower concentration than each antibody alone. More importantly, the antibody combination also prevented emergence of escape mutations of the authentic SARS-CoV-2 virus. This is important for potential therapeutic application, where an antibody monotherapy might lead to the emergence of escape mutations in humans, as it was shown for bamlanivimab (Peiffer-Smadja et al., 2021).
  • the antibodies neutralised the virus in vivo in a novel mouse model in which human ACE2 (hACE2) is expressed by both upper and lower respiratory tract cells upon inhalation of Adeno Associated Virus (AAV-hACE2) (De Gasparo et al., 2021). Infection of these mice with SARS-CoV-2 results in progressive weight loss and respiratory pathology requiring euthanasia on day 8 (De Gasparo et al, 2021). Both antibodies AX290 and AX677, applied either individually or in combination, practically eliminated live viral loads, greatly reduced viral RNA and inflammation in the lungs and completely prevented systemic disease.
  • hACE2 human ACE2
  • AAV-hACE2 Adeno Associated Virus
  • the antibodies AX290 and AX677 recognize non-overlapping epitopes on functionally different portions of the S protein RBD and apparently different mechanisms of how they neutralize the virus. They protect hACE2-sensitized mice against lethal infection of SARS- CoV-2 and efficiently neutralize the currently spreading authentic SARS-CoV-2 variants of concern, B.l.1.7 (Alpha), B.l.351 (Beta) and B.l.617.2 (Delta), in cell assays, and AX677 might even retain its frill potency against B.l.1.529 (Omicron).
  • the cocktail of the two antibodies might thus provide an effective COVID-19 treatment and protection against antibody resistance.
  • the dose-sparing opportunity provided by topical administration (Halwe et al, 2021; Nambulli et al, 2021) would even allow a combination of more than two well-selected antibodies and provide further benefits preventing viral resistance.
  • Kaneko, N. et al. (2020) Loss of Bcl-6-Expressing T Follicular Helper Cells
  • S-variant SARS-CoV-2 lineage Bl.1.7 is associated with significantly higher viral loads in samples tested by ThermoFisher TaqPath RT-qPCR. J Infect Dis. 2021 Feb 13:jiab082. doi: 10.1093/infdis/jiab082.
  • Zhao S Lou J, Cao L, Zheng H, Chong MKC, Chen Z, Chan RWY, Zee BCY, Chan PKS, Wang MH. Quantifying the transmission advantage associated with N501Y substitution of SARS-CoV-2 in the UK: an early data-driven analysis. J Travel Med. 2021 Feb 23;28(2):taab011. doi: 10.1093/jtm/taab011.
  • Tan, C. W. et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2-spike protein-protein interaction. Nature Biotechnology 38, (2020).

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Abstract

La divulgation concerne, en partie, des anticorps contre la protéine de spicule du SARS-CoV-2 et/ou son RBD, des compositions comprenant ces anticorps ou acides nucléiques codant ceux-ci et leurs méthodes d'utilisation.
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